Review www.microbialcell.com Ser/Thr protein phosphatases in fungi: structure, regulation and function Joaquín Ariño1,*, Diego Velázquez1 and Antonio Casamayor1 1Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain. * Corresponding Author: Dr. Joaquín Ariño, Institut de Biotecnologia i Biomedicina (Ed. IBB), Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193, Barcelona, Spain; Phone: 34-93-5811315; E-mail: Joaquin.Arino@uab.es ABSTRACT Reversible phospho-dephosphorylation of proteins is a doi: 10.15698/mic2019.05.677 major mechanism for the control of cellular functions. By large, Ser Received originally: 01.02.2019; in revised form: 19.03.2019, and Thr are the most frequently residues phosphorylated in eukar- Accepted 21.03.2019, yotes. Removal of phosphate from these amino acids is catalyzed Published 24.04.2019. by a large family of well-conserved enzymes, collectively called Ser/Thr protein phosphatases. The activity of these enzymes has an enormous impact on cellular functioning. In this work we pre- Keywords: protein phosphorylation, protein sent the members of this family in S. cerevisiae and other fungal phosphatases, cell signaling, S. cerevisiae, fungi. species, and review the most recent findings concerning their regu- lation and the roles they play in the most diverse aspects of cell Abbreviations: biology. AID – autoinhibitory domain, CDRE – calcineurin- dependent response element, CFW – Calcofluor White, CLS – chronological lifespan, CNA – calcineurin A, CoQ – coenzyme Q, CWI – cell wall integrity, DSB – double strand break, eIF – eukaryotic translation initiation factor, GAP – GTPase-activating protein, HOG – high-osmolarity glycerol, MEN – Mitotic Exit Network, MTS – mitochondrial targeting sequence, NCR – nitrogen catabolite repression, PDC – pyruvate dehydrogenase complex, PPases – protein phosphatases, PPCDC – phosphopantothenoyl- cysteine decarboxylase, PTPA – Phosphotyrosyl Phosphatase Activator, RCAN – regulator of calcineurin, SAC – Spindle Assembly Checkpoint, SAM – S-adenosylmethionine, SAP – Sit4- associated protein, SIN – Septation Initiation Network, STRIPAK – STRiatin-Interacting Phosphatases And Kinases, TCA – tricarboxylic acid, TORC – Target of Rapamycin Complex, TPR – tetratricopeptides repeats, UPR – unfolded protein response. INTRODUCTION 43 protein phosphatases [1], in contrast to around 120 Phosphorylation of specific residues is a major mechanism bona fide protein kinases. Quite often, the subcellular lo- for the control of the function of proteins in eukaryotes. calization, substrate specificity, or activation state of Phosphorylation is catalyzed by protein kinases and these PPases is determined by their interaction with other pro- events are reverted by the action of protein phosphatases teins, collectively called regulatory subunits. While it has (PPases). As a common rule, the number of protein kinases been accepted in the past that activation of kinases is piv- exceeds in several fold the number of phosphatases. Thus, otal for regulation of signaling pathways, whereas PPases in the model yeast Saccharomyces cerevisiae there are only merely counteract the action of kinases, current evidence OPEN ACCESS | www.microbialcell.com 217 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review suggests that regulation of PPases could also play a key enzymes. These are, together with the type-2C proteins, role in signal transmission [2, 3]. what we define as "canonical" PPases. Besides, the S. cere- In eukaryotes, most phosphorylation events occur at visiae PPP family includes additional members that are specific Ser and Thr residues and have both structural and structurally closer to PP1 (Ppz1, Ppz2, Ppq1), PP2A (Pph3, signaling effects, while phosphorylation at Tyr, possibly Ppg1, Sit4), or PP2B (Ppt1). These proteins were identified accounting for only 1% of the phosphorylated sites, is by gene sequencing and are the ones we define here as mostly involved in signaling [4]. The genome of S. cere- "non-canonical". Experimental evidence for phosphatase visiae encodes 19 identified proteins with Ser/Thr protein activity has been obtained in most cases, at least for the phosphatase activity (Figure 1, see below). Twelve of these S. cerevisiae enzymes, whereas in other yeasts or fungi it is can be classified, based on their structure, into the PPP often assumed on the basis of conserved structural fea- group. This includes the homologs of the widely distributed tures. In this work we will review both canonical and non- type 1 (PP1), 2A (PP2A) and 2B (PP2B) phosphatases that canonical Ser/Thr PPases in the yeast S. cerevisiae, with were identified in the early 1980s by biochemical ap- eventual references to the equivalent proteins from other proaches, mostly based on substrate specificity and sensi- fungi. This review largely focuses on the function and regu- tivity to thermostable inhibitors [5]. The remaining seven lation of these enzymes, and therefore should effectively can be included into the PPM family and are likely homo- complement a very recent review by Offley and Schmidt [1], logs of the type-2C (PP2C) phosphatases, a kind of enzymes which is focused in S. cerevisiae only and emphasizes the also characterized biochemically many years ago. It must structural and catalytic aspects. To note that, in contrast to be noted that PPP and PPM members are an example of the mentioned review, we do not include Ppn2 because, in convergent evolution, sharing a similar catalytic mecha- spite of its somewhat distant relationship with PP2B pro- nism involving two metal ions at the catalytic site, but tein phosphatases, this enzyme has been recently reported largely differing in primary sequence and tertiary structure. to be a Zn2+-dependent polyphosphatase [6] and, as far as If the PPP family in S. cerevisiae is considered, we can we know, its protein phosphatase activity has not been identify the expected equivalents of the type 1 (Glc7), 2A proved. (Pph21 and Pph22), and 2B (Cna1 and Cna2) ubiquitous FIGURE 1: Phylogenetic analy- sis of protein phosphatase sequences from S. cerevisiae S288c (taxid:559292). The tree was constructed using the function "build" of Environ- ment for Tree Exploration (ETE) v3.0.0b32 as implemented on the GenomeNet site (https://www.genome.jp/tools /ete/). The output files were imported to the open source Dendoroscope 3 software (v. 3.5.9). Protein sequences are described in Supplemental Table S1 (identified with the prefix “Sc_”). OPEN ACCESS | www.microbialcell.com 218 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review PP1 AND PP1-LIKE PHOSPHATASES with two isoforms more (PP1γ1 and PP1γ2) which can be In addition to the ubiquitous catalytic subunits of PP1 en- generated by alternative splicing. The name GLC7 derives zymes (PP1c), fungi contain two PP1-related PPases, Ppq1 from the reduction in glycogen content identified in specif- and Ppz1, that are not found in other eukaryotes (Figure 2). ic mutant strains [10–12]. As its mammalian counterparts, the functions of Glc7 are regulated by the interaction with PP1 different regulatory subunits affecting their substrate spec- Protein phosphatase-1 (PP1) was among the first biochem- ificity and/or subcellular localization [8]. ically characterized Ser/Thr phosphatases and it is probably the most extensively studied. Because the existence of Structure relatively recent reviews [7, 8], we will provide here a gen- GLC7 encodes an essential protein of 312 amino acids that eral background and will then focus on the more recent is 85% identical to the four human PP1c proteins. The cen- findings. tral section of Glc7 is also shared with the related yeast In eukaryotes, PP1 is involved in many cellular func- protein phosphatases PP2A, PP2B and Ppz1,2. Orthology of tions including the regulation of glycogen metabolism, human PP1 isoenzymes with Glc7 has been verified by muscle physiology, RNA processing, protein synthesis, complementation of a glc7 mutant with human PP1c transmission of nerve signals, induction of apoptosis and cDNAs [13]. control of multiple checkpoints, and events that occur There are currently more than twenty 3D-structures throughout the cell cycle [8, 9]. To fulfill these roles, each available of the mammalian PP1 catalytic subunit. PP1c functional PP1 enzyme consists of a catalytic subunit (PP1c) adopts a compact α/ß fold, with a ß sandwich wedged be- which binds to different proteins called regulatory subunits. tween two α-helical domains, which are the C-terminus, These regulators are needed either to target the PP1 cata- and the extreme N-terminus of the protein. The ß sand- lytic subunit to specific subcellular localization, to modu- wich and the two helical domains form a “Y”-shaped cleft late substrate specificity or to serve as substrates them- where the active site is located. There, an invariant number selves. of residues (three His, two Asp and one Asn) coordinate PP1c is highly conserved among all eukaryotes, with two metal ions, Mn2+ and Fe2+, which are needed to con- approximately 70% or greater sequence identity. Most tribute to catalysis. These residues are highly conserved in fungal species contain one single gene coding for the PP1c, all members of the PPP family suggesting a common although in a few species, such as Schizosaccharomyces mechanism of metal-catalyzed reaction [14]. Through that pombe, two genes are present. In the yeast S. cerevisiae cleft, there are three grooves called hydrophobic, acidic this enzyme is encoded by a single gene, termed GLC7 (ali- and C-terminal. ases are DIS2S1 and CID1). For comparison, in mammals PP1c is encoded by three genes (PP1α, PP1β/δ and PP1γ), Regulation and binding motifs PP1c is a relatively small protein, which does not exist freely in the cell. It achieves its huge functional diversity by interacting with a large variety of structurally unrelated regulatory subunits, with distinct effects on the function of the phosphatase. More than 100 putative PP1 regulatory subunits have been described in mammals, whereas the yeast Glc7 phosphatase associates with around 30 of these proteins [1, 8]. Despite their apparent differences in sequence, most of these subunits bind to PP1c in the same manner. Binding to PP1c is mediated by docking motifs, that are short se- quences of about 4-8 residues present in the regulatory subunits that are combined to create a larger interaction surface for PP1c. Despite the conservation of motifs during evolution, they are somewhat degenerated, displaying variants of the consensus sequence that differ in affinity for PP1c. There are about ten known distinct PP1-docking motifs identified in the regulatory subunits in mammals, although not all of them are found in yeast. Most regulato- ry subunits bind to PP1c by the identifiable RVxF consensus sequence using the hydrophobic groove as PP1c interface FIGURE 2: Phylogenetic tree of PP1 and PP1-like phosphatases (see [9] for a review). Mutation of residues of this hydro- from various fungal species. The protein sequences of the as- phobic groove reduced affinity to some regulatory subunits, comycetes S. cerevisiae (Sc), S. pombe (Sp) and A. fumigatus (Af), resulting in phenotypic traits related to reduced Glc7 activ- as well as that of the basidiomycete Cryptococcus neoformans ity [15], and several of these variants could not restore (Cn, in red) were used. Analysis was performed as in Figure 1. The corresponding sequence codes can be found in Supple- viability in a glc7 deletion mutant [8]. Among the regulato- mental Table 1. ry subunits for which the RVxF motif is key for interaction OPEN ACCESS | www.microbialcell.com 219 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review are Ref2, Gip2, Afr1, Reg1, Reg2, Sla1, Bud14, Bni4 and al response. These effects are attributable to the inappro- Gac1. In some cases, more than a putative consensus is priate activation of Snf1 [29]. found (i. e. in Scd5, Gip1 or Fin1), although not necessarily As mentioned above, Glc7 is important in several cell all of them are required for interaction [16]. In contrast, cycle check-points. The lack of the essential regulatory Sds22 interacts with Glc7 at a region different from the subunit Ypi1, the yeast homologue of mammalian inhibitor hydrophobic groove and using a different motif that is 3, activates the morphogenetic checkpoint. Depletion of based, similarly to its mammalian counterpart, in the char- Ypi1 results in stabilization of the Pds1 securin, suggesting acteristic leucine-rich repeats [17, 18]. In the case of Pti1, the activation of a G2/M checkpoint [30]. Since under an essential component of the CPF (cleavage and polyad- normal conditions most of the Glc7 protein is found in the enylation factor) that interacts with Glc7, the interaction nucleus, in particular in the nucleolus, it is possible that motifs are unknown, but likely are not based on the RxVF these defects could be due to the alteration of its nuclear consensus [19]. Finally, certain regulatory subunits can localization previously reported for Ypi1-depleted cells [20]. associate to form larger complexes. For instance, for- Shp1, a protein involved in shmoo formation and bipolar mation of a trimeric complex involving Glc7 together with bud site selection, could also be a positive regulator of Glc7, Sds22 and Ypi1 has been reported as necessary for translo- working in a complex with the AAA-ATPase Cdc48 and cation of the phosphatase to the nucleus [20]. In this com- promoting cell cycle progression [31]. Nuclear localization plex both regulatory subunits interact with Glc7 at differ- of Glc7 requires the Cdc48-Sph1 complex, which possibly ent sites and the formation of the trimeric complex rein- functions as a molecular chaperone for the structural in- forces bipartite interactions. tegrity of the PP1 complex, and specifically promotes the assembly of Glc7-Sds22-Ypi1 for nuclear import [32] (Fig- Functions ure 3). Glc7 can play very diverse roles role in different cellular The subunits Bni4, Afr1 and Gip1 mediate different sep- localizations in yeast cells (Figure 3). Cytosolic functions for tin localization activities. Specifically, the Glc7-Bni4 holoen- Glc7 related to glucose repression, regulation of septins zyme regulates the targeting of chitin synthase III (Chs3) to assembly and chitin synthesis, bud site selection or endo- the incipient bud sites when Bni4 is phosphorylated by the cytosis and actin organization, as well as nuclear tasks, Pho85 kinase-Pcl1,2 cyclin complex [33]. It is worth noting related to transcriptional regulation (dependent and inde- that depletion of Ypi1 also causes depletion of the Cdc11 pendent of the CPF complex), microtubule attachment to septin, which possibly explains the failure to form properly kinetochores, and diverse cell cycle checkpoints have been assembled septin rings at the bud necks [30]. nicely reviewed in detail several years ago [8]. Therefore, Early work linked Glc7 to maintenance of monovalent we will only summarize here some of the more recent find- cation homeostasis [34]. More recently, two Glc7 subunits ings. have been found relevant for tolerance to toxic cations. In addition to Reg1, its paralog Reg2 has been found to Thus, lack of Ref2 is additive to blockage of the calcineurin assist Glc7 in the dephosphorylation of the protein kinase pathway and might disrupt multiple mechanisms control- Snf1 in the presence of high glucose concentrations [21, ling expression of the ENA1 Na+-ATPase-encoding gene in a 22]. Although Reg1-Glc7 regulates dephosphorylation of all way dependent on Glc7 but independent of its previously three Snf1 isoforms, it preferentially associates with the known function in the formation of mRNA via the APT (for one containing the Gal83 subunit [23], the only one capa- Associated with Pta1) subcomplex of the large CPF complex ble of nuclear localization upon glucose limitation. Moreo- [35]. Remarkably, partial depletion of the aforementioned ver, the tandem Glc7-Reg1 may also be important for inac- Ypi1 subunit renders cells sensitive to Li+ and this tivation of genes dispensable for growth in high glucose, phenotype is due, at least in part, to an increased such as HXK2, PDA1 and HSP60 [24]. Although Reg1-Glc7 expression of ENA1. It appears, however, to be plays the major role in the Snf1-mediated signaling path- independent of the role of Ypi1 as a Glc7 regulatory way, other phosphatases like Ptc1 or Sit4 contribute to subunit and, instead, the increase Li+ tolerance and ENA1 maintenance of the Snf1 activation loop in the expression is abolished in a cnb1 mutant, indicating that dephosphorylated state during growth on high glucose [25, the effect of Ypi1 on cation homeostasis is essentially 26]. A major target for Snf1 is the Mig1 repressor, whose mediated by calcineurin [36]. More recently, it has been phosphorylation promotes its eviction from the nucleus. A postulated that Ref2-Glc7 would be required for possible role for the Glc7-Reg1 phosphatase in the direct dephosphorylation of the formin Bni1, thus playing a role dephosphorylation of Mig1 was also hinted several years in defining the subcellular localization of formins during ago [27]. More recently, the possibility of the existence of cytokinesis [37]. an additional glucose and Glc7-Reg1 independent mecha- Recently, Rif1 was identified as a new possible regula- nisms for dephosphorylating Mig1, perhaps involving Tyr tory subunit of Glc7. The Rif1 protein, originally identified phospho-dephosphorylation, has been suggested [28]. as a telomere-binding factor in yeast, was also found to Glc7 also has relevance in the unfolded protein re- prevent premature activation of replication origins in late- sponse (UPR). It has been reported that lack of Reg1 causes replicating chromosomal domains. This is achieved by tar- hypersensitivity to UPR inducers, which is concomitant geting Glc7 to the sites of action to direct the dephosphor- with an augmented UPR element-dependent transcription- ylation of subunits of the Minichromosome Maintenance (MCM) complex [38] and could be important in the replica- OPEN ACCESS | www.microbialcell.com 220 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review FIGURE 3: Schematic depiction of the diverse associations of Glc7 with its regulatory subunits and the functions in which the complex- es are involved. See main text for details. tion of the highly repetitive and highly transcribed rDNA Glc7 was identified long ago as a major eIF2α phosphatase, locus [39]. It has been suggested that Rif1 interacts with in S. cerevisiae it was not evident which regulatory subunits Glc7 through two ‘SILK’ and two ‘RVxF’ putative consensus are relevant for targeting the phosphatase to eIF2α, as sequences located at its N-terminus [38]. Regulation of these were not known in mammalian cells. Only recently, replication by Rif1 through PP1c is an evolutionarily con- Rojas and coworkers [41] nicely demonstrated that, in fact, served mechanism. Recent work has provided evidence such subunit might not exist, and that it would be replaced that control of telomere length by Rif1 also involves target- by the eIF2γ component of the complex, by means of a ing of Glc7, and that this role is independent of its effect on RVxF-like motif (KKVAF) present in its N-terminal extension. replication timing (see [40] for a recent review). Such motif would be rather unique to certain yeast species, The eukaryotic translation initiation factor 2 (eIF2), is which would rely on the recruitment of PP1 in cis to the required for initiation of translation. eIF2 is composed of α, eIF2 complex to maintain eIF2α phosphorylation at the β, and γ subunits and translation initiation requires appropriate levels. dephosphorylation of the α subunit at Ser51. Although OPEN ACCESS | www.microbialcell.com 221 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review Role of Glc7 in virulence in Candida albicans have a general role in the assembly of PP1-like phospha- Virulence in Candida albicans, one of the most common tases containing Sds22 and Ypi1. fungal pathogen in humans, is largely linked to its ability to switch from yeast to hyphal forms [42]. Interestingly, some PPZ Phosphatases PP1c regulatory subunits or substrates have been related Structure to virulence in this organism. For instance, it has been The Ppz phosphatases are enzymes apparently restricted found that Sds22 plays an important role in Rad53 to fungal species and characterized by a well-conserved dephosphorylation and, therefore, in deactivation of the carboxy-terminal domain, related to type 1 PPases, and a DNA damage checkpoint, through inhibitory physical asso- N-terminal domain that largely differs in sequence and size ciation with Glc7 [43]. These same authors showed that among fungi. These enzymes were first identified in overexpression of SDS22 reduces C. albicans virulence in a S. cerevisiae, where two paralogs, PPZ1 and PPZ2 are found mouse model of systemic infection. [52, 53]. In this yeast Ppz enzymes display a C-terminal Deletion mutants for Cas5, encoding a transcriptional catalytic domain of about 300 residues, which is 75-90% regulator of genes involved in cell wall integrity that has no identical to other fungal Ppz enzymes and retains 60% orthologue in S. cerevisiae, display attenuated virulence identity with PP1 catalytic subunits. The N-terminal moie- and enhanced sensitivity to the antifungal fluconazole. ties of Ppz1 and Ppz2 are roughly of the same size ( 350 Recent work has shown that Cas5 is activated by Glc7 in residues), but they are more divergent in sequence. Still, response to cell wall stress, playing a role not only in cell they include a conserved Gly2 that is myristoylated in vivo wall homeostasis but also in regulating nuclear division [54], possibly due to the action of the Nmt1 N-myristoyl [44]. transferase. In addition, a relatively conserved sequence near the N-terminus of Ppz1 and Ppz2 (43SSRSRRSLPS52 and PPQ1 43SSRSLRSLRS52, respectively) can be found in many fungi in The gene PPQ1 encodes a type1-related phosphatase of the form of a SxRSxRxxS consensus [55]. Such sequence 549 residues in length. The C-terminal half contains the seems to have functional relevance (see below). Besides phosphatase domain, whereas its N-terminal extension is this, the N-terminal half of Ppz proteins exhibits low or rich in Ser and Asn residues (although unrelated in se- very low conservation among fungi and is often shorter quence to Ppz1/2 phosphatases, see below). The protein is than that found in S. cerevisiae. Ppz1 is recovered in par- not conserved in other eukaryotes and it is t even absent in ticulate fractions from yeast extracts [54] and different many fungal species. The gene was initially isolated (and studies have shown that, at least in part, is localized at the named SAL6) as an allosuppressor able to enhance the cell periphery [32, 56]. efficiency of omnipotent suppressors thought to be trans- lational ambiguity mutations [45], and a few years later Function cloned by sequence homology and by complementation of Deletion of S. cerevisiae PPZ1 results in many phenotypic the sal6-1 mutation. These initial studies (see [46] and ref- traits, whereas that of PPZ2 is hardly noticeable, suggesting erences therein) already prompted about a possible role of that the former enzyme has a more prominent cellular role. Ppq1/Sal6 in protein translation, still unknown, although However, deletion of PPZ2 in a ppz1 background usually subsequent studies showed that Sal6 does not potentiates the phenotypes. Ppz1 has a major role in salt dephosphorylate the eukaryotic release factor eRF1 [47]. tolerance, and strains lacking Ppz1 are hypertolerant to Little advance was made for quite a few years on the sodium or lithium cations, a phenotype enhanced by addi- functional role of Ppq1. Only recently, metabolomic studies tional deletion of PPZ2 [57]. Such tolerance, at least in part, using kinase and phosphatase mutants attributed a role of is the result of an increase in the expression of the ENA1 Ppq1 in metal homeostasis (mainly Mn+2) which would ATPase gene, whose levels are induced by salt stress and affect the activity of the tricarboxylic acid (TCA) cycle [48], alkaline pH and represents a major determinant for sodium although this issue has not been investigated further. In- tolerance in budding yeast. Thus, the effect of Ppz1 on terestingly, Ppq1 was also identified as a phosphatase able ENA1 expression is opposite (see below) to the effect de- to down-regulate the mating signaling pathway by target- scribed for the Ser/Thr phosphatase calcineurin, a positive ing at or upstream of the terminal MAP kinase Fus3 [49]. effector of the ENA1 gene [58]. In fact, it has been shown Such role was confirmed by an independent laboratory that the effect of the absence of Ppz1 on ENA1 expression using a phosphatase overexpression strategy, placing the requires an intact calcineurin pathway [59], thus suggest- MAPKK Ste7 and the MAPK Fus3 as possible targets [50]. ing that Ppz1 negatively regulates calcineurin activity. During the study of the role of the Cdc48–Shp1 complex in Nevertheless, Ppz1 also influences salt tolerance in an regulating nuclear targeting of Glc7 and promotion of the ENA1-independent way. Early evidence came from the assembly of the Glc7–Sds22–Ypi1 PP1 complex, it was observation that overexpression of the Sky1 protein kinase found that Ppq1 also associates with Shp1 and forms ag- increases sensitivity to LiCl in a way that requires the func- gregates in Shp1-depleted cells upon proteasome inhibi- tion of PPZ1 but not that of ENA1 [60]. Shortly afterwards, tion [32]. Ppq1 has been found to interact with Ypi1 and it was demonstrated that Ppz1 was a negative regulator of Sds22 as well [51] and, similarly to Glc7, the interaction potassium influx through the high-affinity potassium with Sds22 is necessary to permit the association between transport system encoded by Trk1 and Trk2 [61]. Indeed, Shp1 and Ppq1 [32]. This suggests that Cdc48–Shp1 might OPEN ACCESS | www.microbialcell.com 222 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review cells lacking PPZ1 and PPZ2 showed increased potassium Regulation uptake, leading to augmented intracellular turgor. This In S. cerevisiae, Ppz1 is regulated in vivo by Hal3 (Sis2), effect could explain the impact of Ppz1 on the cell wall encoded by a gene originally identified as a high-copy sup- integrity (CWI) pathway and provided the basis to under- pressor of the cell cycle-related growth defect of a strain stand earlier findings pointing to the involvement of Ppz1 lacking the Sit4 phosphatase [73] (also reviewed in this and Ppz2 in the maintenance of CWI, such as the fragility of work), and by its capacity to confer halotolerance [74]. ppz1 ppz2 mutants in the presence of caffeine, unless os- Hal3 binds to the carboxyl-terminal catalytic domain of motically stabilized [62], and the isolation of PPZ2 as a Ppz1 and strongly inhibits its phosphatase activity, thus high-copy suppressor of the lytic phenotype of slt2/mpk1 modulating its diverse physiological functions [75]. For and pkc1 mutants, lacking key components of the CWI instance, cells overexpressing Hal3 are salt-tolerant, pathway [53]. It is not known how Ppz phosphatases influ- whereas a hal3 strain is hypersensitive to sodium and lithi- ence Trk-mediated K+ transport, but it has been shown that um cations. Likewise, high-copy expression of HAL3 exac- Trk1 physically interacts with Ppz1, and that the in vivo erbates the lytic phenotype of a Slt2 MAP kinase mutant phosphorylation level of Trk1 increases in a Ppz-deficient whereas, in contrast, lack of HAL3 improves growth of this strain [56]. However, no experimental evidence for direct strain [75]. The effect of Hal3 overexpression on cell cycle dephosphorylation of Trk1 by Ppz1 has been obtained. The was also shown to depend on Ppz1 function, as deduced Ppz phosphatases also regulate potassium influx in a Trk- from the observation that mutation of PPZ1 rescues the independent way, which involves calcium signaling but not synthetic lethal phenotype of sit4 cln3 mutants [76]. calcineurin activation [63]. Interestingly, Ppz1 down- This general effect of the regulatory subunit Hal3 on regulated the contribution to K+ influx of an heterogously Ppz1 function appears rather different from the situation expressed barley HvHak1 transporter (a kind of K+ trans- described for Glc7. Deletion of GLC7 results in lethality [10, porter also present in some fungi but not in S. cerevisiae 11] whereas the absence of regulatory components yields [64]), thus raising the possibility that the regulatory net- less dramatic phenotypes (only three of them, Scd5, Sds22 work controlling K+ homeostasis in fungi could be con- and Ypi1 are also essential in S. cerevisiae), suggesting that served. The impact of Ppz-phosphatases on cation homeo- the diverse cellular roles attributed to Glc7 are the result of stasis likely lays on the basis of a number of reported phe- specific interactions of the catalytic subunit with different notypes: enhanced tolerance to toxic cations, such as Hy- regulatory subunits [8]. It must be noted, however, that gromycin B, tetramethylammonium or spermine [61, 63, Ppz1 and Glc7 might not be fully insulated with respect to 65], sensitivity to agents causing replicative stress or DNA some specific functions or to modulation by their counter- damage [66], formic acid susceptibility [67] or even modu- part regulators. For instance, PPZ1 and PPZ2 display genet- lation of flocculation and invasive growth phenotypes [68]. ic interactions with GLC7, as deduced from the different Recent evidences have linked Ppz phosphatases to the growth defects observed in cells carrying certain mutant regulation of ubiquitin homeostasis, possibly by controlling alleles of GLC7 in combination with null alleles of the PPZ the phosphorylation state of ubiquitin at Ser57, and it was phosphatases [77]. As mentioned above, many (about 2/3) porposd that the salt–related phenotypes of the ppz mu- of PP1c (and Glc7) regulatory subunits contain a RVxF con- tants are related to ubiquitin deficiency [69]. Even more sensus PP1c binding motif [78], which binds to a hydro- recently, the ubiquitin ligase adaptor Art1 has been recog- phobic groove strongly conserved in Ppz1. It is worth not- nized as a Ppz substrate. In this role, that would be distinct ing that in vivo interactions between Ppz1 and two Glc7 from that played on ubiquitin, Ppz would mediate the me- regulatory subunits displaying RVxF motifs (Glc8 and Ypi1), thionine-induced dephosphorylation of Art1. Such has been reported by 2-hybrid analysis [77]. Interaction dephosphorylation would promote cargo recognition, in between Ppz1 and Ypi1 has been also documented by pull- this case that of the methionine transporter Mup1, at the down assays (although Ypi1 barely affects Ppz1 activity), plasma membrane [70]. and it was shown that a W53A mutation in its RVxF motif The Ppz phosphatases are also likely influencing protein (48RHNVRW53) abolished binding to both the Glc7 and Ppz1 translation. Thus, it was demonstrated that Ppz1 interacts phosphatases [79]. In addition, both S. cerevisiae and in vivo with translation elongation factor 1Bα (Tef5), the C. albicans Ppz1 are sensitive in vitro to mammalian Inhibi- GTP/GDP exchanging factor for translation elongation fac- tor-2 [80, 81], a PP1c regulatory subunit that contains a 144RKLHY148 sequence functionally replacing the RVxF motif. tor 1, and that in ppz1 ppz2 cells the conserved Ser86 of Tef5 was hyperphosphorylated. Indeed, lack of Ppz phos- These observations suggested that the RVxF-binding motif phatases resulted in enhanced read-through at all three is also functionally conserved in Ppz1. nonsense codons, suggesting that translational fidelity The Ppz1 inhibitor Hal3 contains a 263KLHVLF268 se- might be affected [71]. A role of Ppz1 (and possibly Ppz2) quence alike to the RVxF motif. However, mutation of H 265 on protein translation accuracy has been reinforced by or F268 does not affect binding nor inhibitory capacity of evidences of its role in the regulation of read-through effi- Hal3 upon Ppz1 [82], suggesting that this RVxF-like motif is ciency and manifestation of non-Mendelian anti- not relevant for the interaction with Ppz1. Sequence com- suppressor determinant [ISP(+)] [72]. parisons and recent experimental evidence on the C. albi- cans Ppz1 C-terminal domain [81] indicate that diverse docking motifs found in PP1c, such as PNUTS or spinophilin, are likely not relevant for yeast Ppz1. The structural de- OPEN ACCESS | www.microbialcell.com 223 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review terminants for interaction between Ppz1 and Hal3 are still Ppz1 phosphatases in other fungi: relevance for virulence unknown, but they should differ substantially from those Ppz1 has been also characterized in diverse fungi, where used by PP1c-regulatory subunits to bind to PP1c, since usually only a single gene is found. The PZL-1 phosphatase Hal3 does not bind to Glc7 in vitro [75, 79]. In any case, from the filamentous fungus Neurospora crassa was able Ppz1 and Hal3 can be co-expressed in Escherichia coli and to replace S. cerevisiae Ppz1 in diverse phenotypic tests purified as a complex with an apparent 1:1 stoichiometry related to cation homeostasis and interaction with the CWI [83], and a recent study has suggested that inhibition of pathway [91]. C. albicans Ppz1 (CaPpz1) also behaves simi- Ppz1 by Hal3 could happen by occlusion of the catalytic site, larly to ScPpz1 albeit with some characteristic traits [92], in a way similar to that used by inhibitor-2 to inhibit PP1c whereas in the fission yeast S. pombe the single Pzh1 was [84]. In S. cerevisiae it has been postulated that the inter- shown to regulate cation homeostasis, but with distinct action between Ppz1 and Hal3 is dependent on the internal characteristics in comparison with budding yeast [93, 94]. pH and serves to maintain intracellular pH homeostasis In the halotolerant yeast Debaryomyces hansenii, DhPPZ1- [56]. deficient strains were salt tolerant, but the effect was The S. cerevisiae genome contains a paralog of Hal3, found related to the Na+/H+ antiporter [55]. named Vhs3, which was identified as a high-copy suppres- In the last few years, the focus has been placed on the sor of the synthetically lethal phenotype of the hal3 sit4 enzyme from pathogenic fungi. The Aspergillus fumigatus mutation [85]. Vhs3 also inhibits Ppz1 in vitro, although its ortholog phzA, when overexpressed in S. cerevisiae, mim- role regulating the phosphatase in vivo is far less important, icked in part the role of ScPpz1. In contrast, the A. fumiga- probably due to lower expression levels [86]. Remarkably, tus mutant did not display altered salt tolerance or CWI in S. cerevisiae the simultaneous deletion of HAL3 and defects, but exhibited sensitivity to oxidant agents [95]. VHS3 is synthetically lethal, and this is not due to hyperac- Further work confirmed the sensitivity to oxidative stress tivation of Ppz1 [86]. Such interaction was explained by the and found PhzA to be relevant for iron assimilation, conidi- discovery that Hal3 and Vhs3 are moonlighting proteins. ation and virulence [96, 97]. More recently, it has been Thus, Hal3 and/or Vhs3 associate with Cab3 (also a Hal3 reported that this mutant (named here ppzA) has de- and Vhs3 paralog) to form an active, heterotrimeric phos- creased production of diverse siderophores and other sec- phopantothenoylcysteine decarboxylase (PPCDC) enzyme ondary metabolites, which might be linked to the fact that [87]. PPCDC is an essential enzyme that catalyzes a key these mutants are avirulent in a murine infection model decarboxylation step in Coenzyme A (CoA) biosynthesis. [98]. While in most organisms PPCDC is an homotrimer with The enzyme from C. albicans was cloned, functionally three catalytic sites, each formed at the interface of two characterized, and found to be relevant for virulence [92, monomers, in budding yeast a single catalytic site is 95, 99]. The catalytic domain of CaPpz1 has been crystal- formed at the interface of Cab3 and either Hal3 or Vhs3, lized and its 3D-structure solved [81], providing insights thus explaining the essential nature of CAB3 and the syn- into unique Ppz1 features that could be useful for antifun- thetically lethal phenotype of the hal3 vhs3 mutations [87]. gal drug design. Recent evidence suggests that, as it was It has been proposed that Vhs3 has a higher tendency to demonstrated for ScPpz1, the N-terminal domain of form heterotrimers, whereas Hal3 can be easily released CaPpz1, although much shorter, is functionally relevant and undergo monomer exchange, thus becoming able to [100]. C. albicans contain two genes, orf19.3260 and interact with Ppz1 [83]. orf19.7378, encoding putative homologs of ScCab3 and The subunit composition of S. cerevisiae PPCDC is ra- ScHal3, respectively [88]. Remarkably, whereas both ther exceptional, not only because in most eukaryotic or- CaHal3 and CaCab3 retain their predicted PPCDC-related ganisms, such as humans and plants, PPCDC is an homo- functions (thus likely generating a heterotrimeric PPCDC), trimer, but also because this unique component subunit is only CaCab3 was able to regulate CaPpz1 in vivo. Therefore, a much shorter polypeptide ( 250 residues), lacking the CaCab3, but not CaHal3, acts as a moonlighting protein in N-terminal extension and the large acidic C-terminal tail C. albicans [88]. A recent proteomic analysis provided fur- also found in certain fungal orthologs, such as C. albicans ther support to the idea that Ppz phosphatases might be [88]. Previous studies have shown that this central domain, related to protein translation in fungi [101]. denoted as Hal3 PD, is required for Ppz1 binding and regu- Very recent work has characterized the functions of lation, although the acidic C-terminal tail also plays an im- Ppz1 in the pathogenic fungus Cryptococcus neoformans portant functional role [89]. Full-length Hal3 (as well as the and found that the phosphatase could only partially com- PD domain) can form trimers itself. This ability is altered by plement a S. cerevisiae ppz1 deletion mutant and was not mutation of L405 to Glu, which would disrupt a possible involved in virulence using a Galleria mellonela infection hydrophobic core in the trimer, although the change does system [102]. Remarkably, C. neoformans encodes two not abolish the ability to interact with Cab3 and to gener- similar Hal3-like proteins, CnHal3a and CnHal3b. Both of ate a functional PPCDC in vivo. Remarkably, this mutation them act as PPCDC, but none is able to regulate Ppz1 func- decreases binding with Ppz1 in vitro and causes partial loss tions in vivo nor inhibit the phosphatase in vitro [102], indi- of Ppz1-mediated functions in vivo [90]. cating that the inhibitory properties of Hal3-like proteins are not conserved across the fungal kingdom. Therefore, Hal3 proteins do not perform moonlighting tasks in C. neoformans. Deletion of the gene encoding CnHal3b OPEN ACCESS | www.microbialcell.com 224 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review renders cells less virulent [102]. No impact on virulence has been determined for the plant fungal pathogen Fusarium graminearum, the causative agent for wheat scab [103]. Therefore, involvement of Ppz1 in virulence seems not to be a general issue, but rather species-specific. PP2A AND PP2A-LIKE PHOSPHATASES The family of the catalytic subunits of PPases type 2A and 2A-like in fungi comprises the canonical PP2A and the non- canonical Sit4, Pph3 and Ppg proteins (Figure 4). The PP2A phosphatases The PP2A phosphatases are present in all organisms and their structure is conserved across eukaryotes. They are involved in many and essential processes, including cell growth, differentiation, apoptosis, cell motility, DNA dam- age response and cell cycle progression [104]. Structure PP2A can be found as a heterotrimeric complex, composed of a C catalytic subunit (PP2Ac), a scaffolding A subunit, and a B regulatory subunit which is thought to determine the substrate specificity, as extensively reviewed [105]. Although only one gene coding for PP2Ac is present in most fungi, this enzyme is encoded by two genes in S. cerevisiae: PPH21 and PPH22. These catalytic polypep- tides are highly conserved, as exemplified by the fact that FIGURE 4: Phylogenetic tree of PP2A and PP2A-like phosphatases the catalytic cores of the S. cerevisiae Pph21 (from amino- from various fungal species. Protein sequences correspond to acid 9 to the C-terminal) and human PP2Aβ (AAV38333.1, organisms described in the Figure 2. The analysis was performed from amino-acid 69 to the C-terminal) share 75.4% of their as described in Figure 1. residues, and 87.7% are similar. Deletion of both S. cere- visiae genes affect vegetative growth, and cells cannot survive if the yeast PP4 gene (PPH3) is also deleted [106]. transferase that catalyzes the methylation in S. cerevisiae Thus, Pph3 can perform, at least, the essential functions of of the carboxyl terminal Leu369 of PP2A, whereas Ppe1 Pph21/Pph22. The A subunit, encoded by the TPD3 gene in catalyzes its demethylation [110]. Tap42 (Two A Phospha- S. cerevisiae and by PAA1 in S. pombe, contains multiple tase Associated Protein) together with Tip41 (Tap42 Inter- HEAT repeats and is required for association to the catalyt- acting Protein of 41 kDa) acts as an inhibitor of the PP2A ic C subunit. Although mammals express a combination of proteins and, in the presence of a good nitrogen source, several splicing alternatives of diverse variable B regulatory TOR proteins promote the formation of the Tap42-PP2Ac subunits (classified in four main gene families), the alterna- complex. Binding of PP2Ac to Tap42 and Tpd3 is mutually tive regulatory subunits in yeasts are reduced to a 55 kDa exclusive [111]. The yeast PP6 protein Sit4 can also be regulatory B subunit (Cdc55 in S. cerevisiae, Pab1 in found as a complex with Tap42 (see below). Rrd1 and Rrd2, S. pombe), the 56 kDa B’ subunit (Rts1 in S. cerevisiae, Par1 also known as Ypa1 and Ypa2, are PP2A and PP2A-like posi- and Par2 in S. pombe), and a Saccharomycetales-specific tive regulators, and belong to the widely distributed phos- predicted B-subunit (Rts3 in S. cerevisiae). Pph21/Pph22 photyrosyl phosphatase activator (PTPA) family of proteins. regulatory proteins can also bind to non-canonical/atypical Rrd1,2 are involved in the regulation of the TOR pathway PP2Ac-like proteins, such as Sit4 (reviewed in [107]). [112, 113]. Phosphorylation of PP2A regulatory subunits is another Regulation mechanism for regulation of PP2A activity. Several exam- Several residues in Pph21 and Pph22 can be covalently ples are known in mammals, with different effects on PP2A modified by reversible phosphorylation and methylation, activity (recently reviewed in [114]). Multiple phospho- thus regulating the ability to form PP2A heterotrimers Ser/Thr residues have been identified in yeast Cdc55 and [108]. As other PP2A, it has been detected that yeast Rts1. For example, Rts1 is phosphorylated in its Thr242 by Pph21 is phosphorylated in the Tyr367 residue of the con- the Cdk Cdc28 [115]. served C-terminal sequence TPDYFL [108]. Mutagenesis PP2ACdc55 can be inhibited by the conserved Igo/ENSA studies determined that phosphorylation of either this Tyr endosulfine domain-containing proteins, frequently local- or Thr364 within the conserved motive, decreases the ized in the nucleus. In S. cerevisiae this family is represent- binding to Cdc55 [109]. Ppm1 was identified as the methyl- ed by the pair of paralogous Igo1 and Igo2, although in other fungi only one protein exists (see also below). The OPEN ACCESS | www.microbialcell.com 225 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review Saccharomycetales-specific Zds1 and Zds2, a pair of redun- Far3, Far7, Far10, Far11 and Vps64/Far9. No direct physical dant paralogs, localized in the cytoplasm and on the sites interaction has been detected between S. cerevisiae Far8 of cell polarity, are also negative modulators of PP2ACdc55 and any PP2Ac, according to the BioGrid database, but that can be considered as regulators of the PP2ACdc55 com- direct physical interactions of S. cerevisiae Far11 with plex localization. Zds2 protein directly binds to the Cdc55, Pph21, Pph22, Pph3 and Tpd3 have been identified [119]. Tpd3 and Pph21 subunits of PP2A but its direct binding to In S. pombe, Csc3 does not interact either to Ppa1 or Ppa2, Pph22, identified as part of the same complex, has not but it does with the PP2A-related Ppa3 (Ppg1 in S. cere- been detected [116]. No direct or indirect interactions visiae). A major biological role for the Far complex in have been identified between Zds proteins and the 56 kDa S. cerevisiae is the pheromone-induced cell cycle arrest, B’ regulatory subunits (Rts1 or Par1/Par2) neither in although other functions, such as regulation of spatial cell S. cerevisiae nor in S. pombe, according to the Biogrid da- growth by antagonizing TORC2, have been reported [119]. tabase (v. 3.5). The difference in localization suggests that The STRIPAK-like complex in S. pombe has been implicated Igo1/2 and Zds1/2 proteins control distinct functions of in the regulation of septation, being an inhibitor of the PP2ACdc55 and do so by different mechanisms. Zds proteins, Septation Initiation Network (SIN) [120]. however, play a major role in the inhibition of PP2A Cdc55 in PP2A could also be regulated by the type 1 protein early mitosis, when compared to the endosulfine proteins phosphatase, as was described in the fission yeast, where- [117]. by PP1 binds to and activates PP2APab1 through a conserved The recently characterized STRIPAK (STRiatin- RVXF motif present in the B55 subunits. Active PP2APab1 Interacting Phosphatases And Kinases), an eukaryotic pro- dephosphorylates Par1 and promotes PP1 recruitment to tein complex highly conserved in animal and fungal species, activate the PP2APar1 phosphatase. In this model, that could could also be considered as a regulatory mechanism for be valid for other organisms, PP1-induced activation of PP2A proteins [118]. First identified in human, striatin both PP2AB55 and PP2AB56 coordinates mitotic progression orthologs have been found in all fungi: Far8 in S. cerevisiae, and exit [121]. Csc3 in S. pombe or HAM-3 in N. crassa. The STRIPAK-like complexes in S. cerevisiae (also called yeast FAR complex) Functions of PP2A comprises, in addition to the Far8 striatin protein, PP2Ac PP2A activity has been found as a regulator of multiple and and its scaffolding regulatory subunit, Tdp3, together with essential cellular processes, such as the nutrient response, FIGURE 5: Multiple roles of PP2A in cellular functions. The cartoon shows the different combinations of PP2Ac with its regulatory subu- nits, and their role in different cell functions is depicted. Relevant substrates are also annotated and grouped with color codes denoting specific functions. OPEN ACCESS | www.microbialcell.com 226 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review polarized growth and cell division [107]. We summarize the phosphatase, required for dephosphorylation and nu- here most of the recent advances on PP2A functions (Fig- clear localization, of the transcription factors [124]. Siw14, ure 5). a tyrosine protein phosphatase necessary for the proper phosphorylation and localization of Gln3, has been identi- Nutrient-related functions. In fungi, as in other organisms, fied as a negative regulator of PP2A in response to caffeine intracellular energy level controls cell growth. The antago- [125]. PP2ACdc55 is also involved in the regulation of the nistic balance between the AMPK complex (SNF1 in fungi) nuclear accumulation and chromatin association of the and the Target of Rapamycin Complex 1 (TORC1) pathway environmental stress response transcription factors senses the cellular energetic status. Both systems are con- Msn2/4 [126]. served among eukaryotes and are susceptible of PP2A reg- Inhibition of the TORC1 complex by nutrient starvation ulation, as recently reviewed [122, 123]). induces the autophagic process, a catabolic response to PP2A participates in the nitrogen catabolite repression nutrient deprivation. These conditions cause dephosphory- (NCR), triggered in yeast cells growing in the presence of a lation of the TORC1 substrate Atg13, that binds to other preferred source of nitrogen. In these conditions active Atg proteins and form the Atg1 kinase complex, required TORC1 binds to and causes the phosphorylation of Tap42 for autophagosome formation. It has recently been shown that associates to PP2A (and to the PP2A-like phosphatase that both forms of PP2A (PP2ACdc55 and PP2ARts1) are need- Sit4), decreasing PP2A activity and leading to the transcrip- ed for the dephosphorylation of Atg13 and induction of tional repression of genes involved in the metabolism of autophagy after the inactivation of TORC1 [127]. PP2A is less preferred nitrogen sources by preventing the activity also required for a “non-nitrogen starvation” induced form of the downstream GATA transcription factors Gln3 and of autophagy, triggered by the lack of S- Gat1. The PP2A-Tap42 pathway regulates localization and adenosylmethionine (SAM). In this form of autophagy PP2A function of the GATA transcription factors, modulating the itself acts as a sensor of the SAM concentrations, since the NCR response. Inhibition of TORC1 by nitrogen depletion, ability of the methyltransferase Ppm1 to methylate the addition of rapamycin or caffeine, dissociates from the catalytic subunit of PP2A is conditioned by the concentra- Tap42–PP2A/PP2A-like complex from TORC1. This activates tion of SAM, the substrate of the transferase [128]. FIGURE 6: Role of PP2ACdc55 in mitotic entry. The cartoon shows the participation of PP1ACdc55 associated with Zds proteins (not shown) in the regulation of the phosphorylation state of the S. cerevisiae cyclin-dependent kinase Cdc28. See main text for details. OPEN ACCESS | www.microbialcell.com 227 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review Availability of nutrients modulates the cell size by a dosulfine-containing domain paralogs Igo1 and Igo2, alt- process that involves the PP2ARts1 complex and its role in hough in other fungi a unique protein might exists. ENSA modulation of the expression of G1 cyclins [129]. Rts1 is proteins are regulated by phosphorylation carried out by a also one of the ways to modulate the TORC2 signaling net- member of the conserved Greatwall family of protein ki- work via the dephosphorylation of the PI(4)P kinase Mss4 nases (Rim15 in S. cerevisiae and Ppk18 in S. pombe) [133]. when cells are shifted to a poor carbon source. PP2ARts1 Activation of this Greatwall-ENSA module is initiated with seems to transmit not only nutrient-dependent but also the phosphorylation of the Igo proteins by the Greatwall ceramide-dependent signals as a feedback regulatory protein kinase. Phosphorylated endosulfines are inhibitors mechanism of the TORC2 network [130]. of PP2ACdc55 activity, as recently reviewed [134]. Inhibition PP2A, together with PP1, are important regulators of of PP2ACdc55 in the budding yeast delays cell cycle progres- mitosis in most eukaryotic organisms, as recently reviewed sion into mitosis, and the progression towards the exit in [114]. In this regard, it is manifest that the roles of from mitosis requires the dephosphorylation of Igo pro- PP2ACdc55 and PP2ARts1 are not identical. PP2ACdc55 regulates teins in order to relieve the inhibition of PP2ACdc55. Activa- the G2/M transition and early mitotic exit. By contrast, tion of Greatwall depends on nutrient availability and, in PP2ARts1 is mostly required for controlling cell size and S. cerevisiae, requires the PKA and TORC1 kinases. Inhibi- spindle assembly checkpoints. tion of TORC1 and PKA by low nutrient availability results It is well established that entry into mitosis is triggered in activation of Rim15 that, once translocated to the nucle- by phosphorylation of hundreds of proteins, substrates of us, phosphorylates Igo1/2 (Figure 7). the cyclin B-Cyclin-dependent kinase 1 (Cdc28 in budding Modulation of the Greatwall-ENSA pathway in fission yeast; Cdc2 in S. pombe and other organisms). It has been yeast controls the cell-cycle machinery coupling the nutri- recognized during the last few years that progress into tional environment to cell size. Thus, growth in the pres- mitosis also requires the inhibition of PP2AB55, and that this ence of a rich nitrogen source activates PP2APab1, which is as important as the activation of Clb2-Cdc28 [131]. leads to subsequent activation of Wee1 that induces cell PP2AB55 regulates G2/M transition by dephosphorylating growth in G2 phase. On the contrary, inhibition of PP2APab1 and activating the Cdk1 phosphatase (Mih1 in budding under nitrogen deprivation releases the inhibitory effect of yeast and Cdc25 in S. pombe) during entry into mitosis by a Cdc25 on Cyclin B-Cdc2, allowing shorter cells entry into conserved mechanism identified in both S. cerevisiae and mitosis because the shortened G2 phase [135–137]. S. pombe. The Cdk1 phosphatase, as the Cdk1 kinase (Swe1 Components of the CWI pathway are involved in the in budding yeast, Wee1 in S. pombe) does, undergoes cy- PP2ACdc55-mediated regulation of cell cycle. Pkc1 has been cle-dependent changes in its phosphorylation state, being identified as a protein kinase that plays a critical role in phosphorylated by its substrate Cdk1 [132] (Figure 6). controlling phosphorylation of components of the PP2ACdc55-ENSA complex, being the phosphorylation of Cell cycle-related functions (via Greatwall-ENSA pathway). Cdc55 by Pkc1 important for the dissociation of the ENSA ENSA proteins negatively regulate PP2ACdc55 functions in protein from the complex [138]. In S. cerevisiae the Great- cell cycle in response to different cues [117]. In the yeast wall-ENSA-controlled PP2ACdc55 coordinates nutrient avail- S. cerevisiae this family is represented by the pair of en- ability with cell cycle progression by dephosphorylating FIGURE 7: Regula- tion of PP2ACdc55 functions by the Greatwall-endosul- fine pathway. The Rim15 protein kinase represents the Greatwall kinases in S. cerevisiae, where- as Igo1/2 are en- dosulfines. Prevalent species and process- es under each specif- ic condition (nutrient source) are denoted by bold font, where- as less active forms are in grey. See main text for details. OPEN ACCESS | www.microbialcell.com 228 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review Sic1, the inhibitor of the Cdc28-Clb complex, under TORC1 of PP2ACdc55, and inhibition of the phosphatase promotes regulation, releasing the inhibitory effect of Slt2- Gis1-driven transcription of specific nutrient-regulated phosphorylated Sic1 on G1/S transition (Figure 8). In fact, genes to coordinate entry into quiescence [142]. In the Slt2 and PP2ACdc55 activities are reciprocally controlled by same study, the mRNA-binding protein Vts1, a member of TORC1 [139]. the Smaug family of proteins, was also identified as a Whi5, the repressor of the budding yeast G1-specific PP2ACdc55 substrate. Since Vts1 regulates mRNA stability transcription, is dephosphorylated by PP2ACdc55, thus an- interfering with the 5’-3’ mRNA decay pathway, it could be tagonizing the phosphorylation exerted by Cdc28-Cln3. In possible that the Rim15-ENSA-PP2ACdc55 module regulates early G1 phase, when PP2ACdc55 is inhibited in response to not only transcription activation but also the protection low levels of Cdc28-Cln3, Whi5 is inactive, turning on the from degradation of the newly expressed mRNAs [133, SBF transcription factor involved in cell size homeostasis. 142]. In response to nutrient limitation, the Rim15-ENSA- This allows transcription of the late Cln1 and Cln2 [136, PP2ACdc55 module is also required for pre-meiotic autopha- 140]. PP2ACdc55 stabilizes Cln1 and Cln2 levels by direct gy, but the mechanisms that regulate entry into gameto- dephosphorylation of these cyclins, which prevents their genesis are independent on the transcription factors Msn2, SCF (E3 ubiquitin ligase complex)-dependent degradation Msn4 and Gis1, involved in gametogenesis [143]. until the end of G1 phase is reached [141]. Recent phosphoproteomics studies suggest additional Entry into gametogenesis and quiescence is also regu- roles for PP2ACdc55 in sensing several internal and environ- lated by the Rim15-ENSA-PP2ACdc55 module. Attenuation of mental cues [144] being important for the septin ring dy- the TORC1 and PKA pathways driven by limitation of nutri- namics and morphogenesis checkpoint (Figure 8). Thus, ents activate Rim15 leading to inhibition of PP2ACdc55. The PP2ACdc55 downregulates Swe1 after septin rings are transcription factor Gis1 has been identified as a substrate properly organized, promoting mitotic entry, and it is also FIGURE 8: Multiple roles of PP2A in cell cycle. The cartoon shows the different combinations of PP2Ac and its regulatory subunits and their role in different points of the cell cycle. Relevant substrates are also annotated with the same color code than that the correspond- ing phase of the cell cycle. SAC, Spindle Assembly Checkpoint; MGC, Morphogenetic checkpoint. OPEN ACCESS | www.microbialcell.com 229 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review involved in the regulation of the septin ring disassembly (APCCdc20). Activation of the APCCdc20 is required for the process [107]. ubiquitin-dependent degradation of the securin Pds1, an Mitotic exit requires the inactivation of the Greatwall inhibitor of the separase. Upon Pds1 degradation, active kinase Rim15 to reactivate PP2ACdc55 and initiate the separase promotes sister chromatids segregation, by cleav- dephosphorylation of the substrates previously phosphory- ing the cohesion complex, and triggers the FEAR (Cdc14 lated by the Cdc28-cyclin B. This yeast pathway is part of a Early Anaphase Release) pathway that leads to the exclu- regulatory module that, although conserved during evolu- sion of Cdc14 phosphatase from the nucleolus. Release of tion, has evolved in other organisms to meet specific mi- Cdc14 from the nucleolus promotes its role as a key effec- totic features [107, 137]. In S. pombe PP2APab1 determines tor of mitotic exit (see [149] and references therein). Acti- the fate of the cell (sexual differentiation or cellular prolif- vation of the MEN (Mitotic Exit Network) occurs upon eration) depending on the availability of nutrients, by regu- completion of chromosome segregation in late ana- lating the phosphorylation state of Taf12, a component of phase/telophase, where high levels of Cdc14 promote the the SAGA complex. The phosphorylation state of Taf12 destruction of the G2 cyclins and stabilization of the CDK results from the balance of the TORC1-activated PP2APab1 inhibitor Sic1, thus inactivating the CDK and leading to mi- and the TORC2-activated Gad8 protein kinase. Thus, under totic exit. The PP2ACdc55 module keeps dephosphorylated nitrogen-rich conditions TORC1-activated PP2APab1 several subunits of the APCCdc20 (such as Cdc27 and Cdc16) dephosphorylates and inactivates Gad8 and also directly upon damaged spindle [147, 151], being the dephosphory- dephosphorylates Taf12, preventing the expression of mat- lation of Cdc16 important for the adaptation to the meta- ing genes [135, 145]. phase arrest triggered by the SAC (Spindle Assembly Checkpoint) [152]. On SAC “satisfaction”, separase-driven Functions regulated by Zds proteins. Zds proteins (Zds1 down-regulation of Zds-PP2ACdc55 alters the Cdc14 phos- and Zds2 in S. cerevisiae) are important cell cycle regula- phatase nucleolar localization. This is caused by increased tors integrating multiple signals to control the activity of phosphorylation of Net1, a member of the FEAR complex, PP2ACdc55 on different substrates by regulating the nucleo- and by maintaining the phosphorylated form of Bfa1 and cytoplasmic distribution of PP2A-Cdc55 along the cell cycle Bub2, members of the MEN regulatory network [153–156]. [146, 147]. Zds proteins undergo changes in its phosphory- PP2ACdc55 also participate in meiotic chromosome segrega- lation state along the cell cycle and, in fact, binding of Zds1 tion since it is required for reductional chromosome segre- to PP2ACdc55 is required for the continuous dephosphoryla- gation during achiasmate meiosis by a FEAR-independent tion of Zds1 [146]. It has been proposed that during mitosis, mechanism [157, 158]. Zds proteins maintain PP2ACdc55 in a cytoplasmic localiza- Removal of active telomerase from telomers at the tion, excluding the phosphatase from performing nuclear G2/M transition is also regulated by PP2ACdc55. The function functions such as the inactivation of nuclear Cdc28 at mi- of Cdc13, a ssDNA binding protein that binds to the te- totic entry. Nuclear activation of both the APC-Cdc20 dur- lomerase subunit Est1 and interacts to Zds2, is regulated ing metaphase/anaphase transition and of the Cdc14 by phosphorylation, and it has been determined that phosphatase in the mitotic exit also require the nuclear Pph22-dependent dephosphorylation of Cdc13 negatively exclusion of PP2ACdc55 [147]. On the other hand, nuclear regulates the Cdc13-Est1 interaction and prevents te- Zds1 also interacts with the Esp1 separase in a Cdc55- lomerase recruitment during cell cycle progression [159]. independent way, and recruits PP2ACdc55 to the nucleolus, The PP2ACdc55–Zds1/2 complex has been identified as a where Zds proteins down-regulate the phosphatase activi- Rho1 effector promoting, in the absence of stress, polar- ty in early anaphase [148]. Zds proteins cannot be exclu- ized growth and cell wall synthesis by one side, and inhibit- sively considered inhibitors of PP2ACdc55 since, although ing the CWI pathway by the other. This is accomplished by they down-regulate PP2ACdc55 during mitotic exit, these inhibition of the Rho1 GTPase-activating protein (GAP) Lrg1 proteins promote PP2ACdc55 functions for mitotic entry [146, and by stabilization of Sac7, another Rho1 GAP. Under cell 149]. Furthermore, Zds proteins are necessary for the cor- wall stress the Slt2 MAPK pathway inhibits cortical tical anchoring of PP2ACdc55, which is important for the role PP2ACdc55 forcing Rho1 to activate the CWI pathway for cell of the phosphatase as a regulator of the GTP-binding pro- wall repair [150]. tein Rho1 [150]. Cell division has different characteristics in the fission Binding of Zds proteins to PP2ACdc55 is required to re- yeast, where PP2APab1 also plays important roles during vert the inhibitory phosphorylation of Cdc28 at G2/M. cytokinesis, cell morphology and cell wall morphogenesis There are evidences that this is accomplished by both inac- [160]. Thus, PP2APab1 regulates the SIN that, as the MEN in tivation of the Cdc28 phosphatase Mih1 and by budding yeast, is required for the coordination of the onset dephosphorylation and activation of Swe1, the Cdc28 inac- of cytokinesis. In fission yeast, PP2APab1 negatively regu- tivating kinase. In fact, it has been proposed a model in lates the Rho1 GTPase, which is required for synthesis of which the Zds-PP2ACdc55 module plays a negative role in the cell wall and septum polymer [161]. The fission yeast Swe1-driven G2/M morphogenesis checkpoint [116, 146] orthologue of Zds proteins, Zds1, contributes to sexual (Figure 6). differentiation, Ca2+ tolerance, maintenance of cell wall The PP2ACdc55 module is also a regulator of the ana- integrity, viability in the stationary phase and cell mor- phase onset (Figure 8), which is triggered by activation of phology. It remains to be determined if PP2A is involved in the Cdc20-dependent anaphase-promoting complex these processes in the fission yeast [162]. OPEN ACCESS | www.microbialcell.com 230 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review Other functions of PP2ACdc55. PP2ACdc55, one of the PP2ARts1-Sgo1 from centromers in a process mediated by ceramide-activated PPases, is involved in the rapid inhibi- the Cdc20-dependent degradation of Sgo1 [170]. tion of the signal triggered by heat stress that leads to Moreover, PP2ARts1 also controls cell cycle by regulating sphingolipid biosynthesis through phosphorylation of Orm a number of transcription factors. Rts1 is important for the proteins. This PP2ACdc55 function, that counteracts the early proper phosphorylation and localization of Ace2, a tran- Ypk1 kinase-mediated phosphorylation of Orm proteins in scription factor required for expression in late mitosis and response to the stress, ensures a transient sphingolipid early G1 of genes involved in transport, ribosome biosyn- production [163]. thesis, cell polarity, and septum destruction after cytokine- The lipolysis-dependent cell-cycle checkpoint, triggered sis, among other multiple functions [171]. Lack of Rts1 re- by the absence of enough lipid precursors derived from sults in higher than normal presence of Ace2 in the mother triglycerides breakdown for the synthesis of sphingolipids, cell nucleus, where it can activate ASH1, an inhibitor of the requires PP2ACdc55. A model has been proposed where transcription of the HO endonuclease, leading to the sphingolipids, identified long ago as effectors of PP2ACdc55 blockage of HO expression in mother cells [172]. function [164], are required to activate PP2ACdc55, resulting In the fission yeast, PP2A bound to the B56 subunits in attenuation of Swe1 kinase phosphorylation and inhibi- Par1 and Par2, inhibits the SIN in order to avoid multiple tory effect on Cdc28 [165]. rounds of septation, probably by regulating the localization of the SIN kinase Cdc7 [173]. Functions of PP2ARts1 A functional interaction of PP2ARts1 with the SAGA PP2A roles related to correct chromosome segregation complex has been recently identified. RTS1 has been de- during cell division (in both meiosis and mitosis) and septin scribed as high-copy number suppressor of several pheno- dynamics require the alternative B56 regulatory subunit types caused by the deletion of GCN5, probably by restor- (Rts1 in S. cerevisiae). The Rts1 subunit mediates the ing the low histone expression levels observed in the gcn5 dephosphorylation of cohesin, protecting it from destruc- mutant strain [174]. Gcn5 is a member of the SAGA that tion, thus maintaining cohesion between centromeric re- targets several lysine residues of histones 2 and 3. Curious- gions but allowing the sister chromatids to resolve along ly, another high-copy suppressor of gcn5 phenotypes the rest of the chromosome. PP2ARts1 needs to associate to found in the same study was ZDS1 [174]. the Shugoshin protein (Sgo1 in S. cerevisiae; Sgo1 and Sgo2 The multifaceted role of the various PP2A complexes in S. pombe). Sgo1 is a member of a functionally conserved during the different steps of the cell cycle is depicted in family of centromere-related proteins, involved in the ac- Figure 8. curate chromosome segregation during cell division by sensing the lack of tension between kinetochores and Other functions of PP2A spindle poles during the bipolar orientation [166]. Recent PP2A plays a role in the decreased recruitment of Pol I to studies have pointed out that Sgo1 directly interacts to and the 35S rDNA promoter induced by Cd2+, although the sub- is required for the recruitment of Rts1 to the centromere, unit required for this function is unknown [175]. configuring the PP2ARts1-Sgo1 complex that will protect A proteome-wide study performed in fission yeast has centromeric cohesin from premature removal [167]. defined multiple biological roles where PP2A could be in- PP2ARts1 is necessary for the timely dissociation of Sgo1 volved, including carbon and amino-acid metabolism, vit- from the pericentromere under spindle tension, as a part amin production, protein folding and the regulation of of a mechanism that ensures the proper sister chromatides glycerol levels during osmotic stress response [176]. bi-orientation of the mitotic spindle before the onset of anaphase. PP2ARts1antagonizes the Bub1-driven phosphory- PP2A phosphatase as potential virulence determinant lation of chromatin-associated substrates, which maintain PP2A, as a component of the STRIPAK-like complexes, plays Sgo1 bound to the pericentromere [167, 168]. In telophase, important roles in growth, sexual development, and viru- PP2ARts1 induces the septin ring disassembly, a process lence in filamentous fungi, as previously reviewed [118]. mediated, at least in part, by Gin4- and Cla4-dependent The PP2A regulatory subunit ParA from A. fumigatus, in- phosphorylation of the Shs1 septin. dispensable for hyphal extension, conidiation and normal PP2ARts1 also has important roles in meiosis I, protecting septation, seems not to be involved in virulence according Rec8, a protein that contributes to maintain cohesion be- to the results obtained with a parA mutant strain [177]. tween centromeres of sister chromatids, from separase Both PP2A regulatory subunits of the pathogenic fungus cleavage at the centromeres until meiosis II is reached. As Aspergillus nidulans play important roles in morphogenesis, in mitosis, PP2ARts1 is recruited to the kinetochore in a conidiation, and self-fertilization, being involved in asexual Sgo1-dependent manner, and there the phosphatase coun- and sexual development. In S. pombe both, PP2APar1 and teracts the phosphorylation of Rec8 by the Hrr25 and Cdc7 PP2APab1, inactivate the SIN pathway, which couples mitotic protein kinases, while Rec8 remains phosphorylated exit with cytokinesis. By contrast, in A. nidulans only throughout the rest of the chromosome arms (reviewed by PP2AParA is the negatively regulator of the SIN, counteract- [169]). Separation of dyad chromosomes during meiosis II ing the role of PabA during the septation process [178]. needs the reactivation of separase at the centromers to In C. albicans, septin Sep7 is dephosphorylated by cleave centromeric cohesin. In budding yeast, phosphoryla- Tpd3-bound Pph21. Dephosphorylated Sep7 is important tion of centromeric cohesin is achieved by removing for proper cell separation since tpd3 mutant cells, defec- OPEN ACCESS | www.microbialcell.com 231 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review tive in Sep7 dephosphorylation, are elongated, fail to sepa- binding of the EVH1 domain of the Psy2 regulatory subunit rate cells, have a pseudohyphae-like morphology and are to the polyPro motif of Mth1. defective in hyphal growth. In agreement with these de- On the other hand, Pph3 has been related to the re- scribed phenotypes, tpd3 mutant cells have greatly de- sponse to DNA damage. Initiation of the response to DNA creased their virulence, leading to the proposal of Tpd3 as damage involves the sequential activation of the Mec1 and a target for antifungal drugs. Collectively, these results Rad53 kinases, finally affecting the phosphorylation state sustain a role of PP2A in filamentous fungi pathogeny. of numerous downstream proteins. The role of Pph3 coun- teracting this phosphorylation cascade is multifaceted. For PPH3 instance, Pph3 was recognized as a Rad53 phosphatase, The S. cerevisiae gene PPH3 codes for a type 2A-related forming a complex with Psy2 that binds and dephosphory- phosphatase catalytic subunit, showing 52% and 58% iden- lates activated Rad53 [188], thus allowing resume of cell tity with Pph21 and Pph22, respectively (Figure 4). Alt- cycle progression once the problems have been solved. hough Pph3 is not an essential protein, the gene was found Lack of Pph3-Psy2 caused delayed Rad53 dephosphoryla- required for survival in the absence of PPH21 and PPH22 tion and resumption of DNA synthesis during recovery [106], thus suggesting partially overlapping functions with from DNA damage, due to failure to restart stalled replica- PP2A enzymes. As it occurs with PP2A, Pph3 enzymes are tion forks [188]. These authors reported that the role of present in all fungal species analyzed. However, early en- Pph3/Psy2 seems to be required for cellular responses to zymatic characterization and phenotypic analyses already the DNA-damaging agent methyl methanesulfonate but hinted that Pph3 function(s) differs from that of PP2A or not the DNA replication inhibitor hydroxyurea (HU). Pph3, Sit4 enzymes (see [46] and references therein). In addition, however, is not the only phosphatase participating in and contrary to PP2A enzymes, Pph3 is not methylated by Rad53 dephosphorylation, and a role for Ptc2/3 and Glc7 the Ppm1 methyltransferase [179]. Pph3 has been often phosphatases has been also reported [189–192]. More found associated with two other proteins, Psy2 and Psy4, recent studies suggest that Pph3 mainly acts on pools of which would be acting as regulatory components of a active Rad53 that have diffused from the site where DNA phosphatase complex that has been maintained through lesions occur [193]. evolution and is also found in humans [180, 181]. The hu- Pph3 also impacts on DNA damage checkpoint through man counterparts of Pph3, Psy2, and Psy4 would be PP4c, regulation of the Mec1 kinase, which phosphorylates and R3, and R2, respectively. However, some differences exist, activates Rad53. It has been shown that Mec1-Ddc2 and since while association of the human R3 subunit appears to Pph3-Psy2 physically interact in a DNA damage- depend on preassembly of Pp4c and R2, yeast Pph3 and independent manner and that Mec1-Ddc2 and Pph3 co- Psy2 form a stable complex even in the absence of Psy4. regulate many Mec1-dependent phosphorylation targets in Pph3 is able to interact with the Peptidyl-prolyl cis/trans- response to HU stress, such as Rad53 and Histone 2A (H2A). isomerase Rrd1/Ypa1 that activates phosphotyrosyl phos- In addition, Ser1991 phosphorylation in Mec1 was regulat- phatase activity in PP2A and PP2A-like enzymes [113], and ed in a Pph3-dependent manner [194]. Finally, targets such interaction is relevant for certain cellular functions of downstream Rad53 are also affected by Pph3. H2A is the phosphatase [182–184]. phosphorylated at Ser129 (giving rise to the γH2AX variant) The Pph3 phosphatase has been related to diverse in a Mec1/Tel1 dependent manner in response to either functions. Early work linked Pph3 to a role in the TOR sig- DNA double-strand breaks (DSBs) or stalled replication. It naling pathway that regulates NCR through the GATA-type was shown that Pph3, in conjunction with both Psy2 and transcription factor Gln3, in contrast with the previously Psy4, is required to dephosphorylate γH2AX [195]. This reported involvement of the Tap42-Sit4 complex (see [46] ability was subsequently confirmed for the human PP4 for references). However, further work provided data sug- homolog [196]. gesting a minimal influence of Pph3 on Gln3 regulation Pph3 would participate in the two independent path- compared with that of Sit4 [185]. More recently, it has ways governing the mechanisms for DSB repair: 1) homol- been proposed the requirement of Pph3 activity in ogous recombination, in which it would be redundant with dephosphorylating Maf1, the major repressor of RNA pol- Ptc2/3 [182]; and 2) Non-Homologous End Joining [197]. ymerase III (Pol III) transcription, in response to nutrient Cells deficient in Pph3 activity show coordinate blockage at deprivation (thus counteracting the function of Tor and early stages of both crossover repair and homology- PKA kinases), or to diverse stresses [186]. The role of Pph3 independent pairing of centromeres. Such defect was in the dephosphorylation of Maf1 would involve the scaf- linked to persistent hyperphosphorylation of Zip1, a fila- fold Psy2, as well as Rrd1 and Tip41 (a Tap42-interacting ment protein of the synaptonemal complex and required protein, see above). Pph3 is also connected to the re- for normal levels of meiotic recombination and pairing sponse to glucose starvation, and it has been proposed between homologous chromosomes [198]. Thus, it was that the Pph3-Psy2 complex counteract the major glucose- proposed that Pph3, in association with Psy2, would coun- responsive kinase PKA by dephosphorylating the putative teract Mec1-induced phosphorylation of Zip1 at Ser75, and PKA sites in Mth1, a protein needed for efficient repression promote chromosomal pairing. The participation of Pph3 in of HXT glucose transporters upon glucose deprivation [187]. defining a novel intranuclear quality control compartment Targeting of Mth1 would be accomplished through direct (INQ) that sequesters misfolded, ubiquitylated and sumoy- OPEN ACCESS | www.microbialcell.com 232 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review lated proteins in response to genotoxic stress has been are viable but display a noticeable slow-growth phenotype. recently proposed [199]. The relevant role of Sit4 in cell cycle regulation is highlight- The role of Pph3 in the response to DNA damage, in ed by the observation that, in addition to ssd1, the sit4 particular its relationship with Rad53 dephosphorylation, mutation is synthetically lethal with more than 20 genes, of has been also investigated in the pathogenic yeast C. albi- which almost half are related to the mitotic cell cycle (Fig- cans. It was shown that Pph3 and Psy2 are required for the ure 9). The Sit4 protein is conserved throughout evolution dephosphorylation of Rad53, but not γH2AX, and that dele- in eukaryotes. Indeed, overexpression of human PP6 or tion of the corresponding genes yielded strong filamentous Drosophila PPV reverts the slow-growth defect of a sit4 growth under genotoxic stress [200]. As in mutant, indicating that these proteins are functional S. cerevisiae, Pph3/Psy2 are required for the response to homologs [205, 206]. DNA damage caused by methyl methanesulfonate but not by HU [201]. More recent work has revealed Rad53 Ser Regulation residues 351, 461 and 477 as likely targets for Pph3- Sit4 associates with a number of proteins. Luke and mediated dephosphorylation [202]. Pph3 is also responsi- coworkers [207] reported the cell cycle-dependent interac- ble for dephosphorylation of Rfa2, a subunit of the replica- tion of Sit4 with several proteins, Sap155, Sap185, Sap190 tion protein A complex that is phosphorylated by Mec1 and and Sap4, collectively named as SAPs (Sit4-associated pro- the cyclin-dependent kinase Clb2-Cdc28 in response to the teins) and demonstrated that loss of all four SAP was phe- genotoxic insult [203]. notypically equivalent to the loss of Sit4 in term of delayed G1 to S phase cell cycle progression, slower growth, and The SIT4 (PPH1) phosphatase budding defects. On the basis of amino acid sequence and The S. cerevisiae gene SIT4 (also known as PPH1) encodes a functional behavior, SAPs could be classified into two type 2A-related protein phosphatase of 311 residues (Fig- groups: Sap185 and Sap190 are more similar to each other ure 4) that was initially cloned in a screening for restora- than to Sap155 and Sap4 [207]. Current evidence suggests tion of HIS4 expression in strains lacking Bas1, Bas2 and that the SAP proteins positively regulate Sit4 phosphatase Gcn4. Two years later it was found necessary for progres- activity, and probably also its substrate specificity [207– sion during the G1/S cell cycle transition (see [46] and ref- 210]. SAP proteins could be, up to some extent, conserved erences therein). Sit4 is required for expression of CLN1 through evolution, since it has been shown that three pu- and CLN2 G1-cyclins, as well as of the transcription factor tative human homologs (PP6R proteins) physically interact SWI4, and cells lacking the phosphatase do show defects in with Sit4 and provide some, but not all, Sit4-dependent bud emergence [73, 204]. Deletion of SIT4 in some genetic SAP functions when expressed in budding yeast [211]. Sit4 backgrounds (cells lacking SSD1 or harboring defective can form a different heterotrimeric complex with Tpd3 and ssd1-d alleles) is lethal, whereas in other backgrounds cells Cdc55 (the typical A and B PP2A subunits), giving rise to a FIGURE 9: Genes displaying synthetic lethality with the sit4 mutation. Venn Diagram showing GO categories for genes displaying synthetic lethality with the sit4 muta- tion. The list was collected from SGD and analyzed with the Gene Ontology Slim Map- per tool. Only SSD1, RPB2 and PRE1 interactions are not included. OPEN ACCESS | www.microbialcell.com 233 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review ceramide-activated protein phosphatase [164]. A link be- line intracellular pH than wild type cells. Interestingly, it tween ceramide homeostasis and the UPR through Sit4 has has been very recently shown [226] that rapamycin inhibits been reported [212]. the H+-ATPase Pma1 in a way that depends on Sit4. Since Sit4 also associates, in a SAP-independent fashion, to the sit4 mutant exhibits low Pma1 activity, the authors Tap42 [213], an essential protein that is involved in one of propose a mechanism by which TORC1 activates Sit4 and the branches of TORC1-mediated signaling. When pre- the phosphatase, directly or indirectly, activates Pma1. This ferred nitrogen sources are available, active TORC1 phos- reported effect of Sit4 on Pma1-dependent H+ efflux might phorylates Tap42, thus promoting the interaction between explain the changes in intracellular pH described by Masu- Tap42 and Sit4 (as well as with other type 2A PPases) and da and coworkers. A role for Sit4 has also been proposed in restricting access of the PPases to their cellular targets (see K+ homeostasis [227], likely through regulation of the Nha1 [214] and references therein). Upon starvation of nitrogen, H+/Na+,K+ antiporter. In this case, Sit4-dependent opposite treatment with rapamycin (a TORC1 inhibitor), or in the effects of Sap155 and Sap185 overexpression were ob- presence of certain stresses, the Tap42-PPase complex served, being SAP155 and SAP185 negative and positive dissociates and diverse cellular targets are dephosphory- modulators of K+ efflux, respectively. However, lated, such as Gln3 and Gat1, which translocate to the nu- K+ efflux was not affected by the mutation of SIT4 [227]. cleus and trigger the transcription of genes involved in the Interestingly, NHA1, encoding the yeast H+/Na+,K+ anti- metabolism of non-preferred nitrogen sources [214–216]. porter, was found as a high-copy suppressor of the syn- Tip41 has been described either to collaborate or oppose thetic lethality of the sit4 and hal3/sis2 mutations [85]. to TORC1 signaling through Tap42-Sit4 [111, 217]. It must However, this effect is likely unrelated to the role of Nha1 be noted that a different brand of phosphatase, Ptc1, has in maintaining K+ and pH homeostasis, as deduced from been proposed to act upstream of Sit4 in TORC1 signaling mutagenesis analysis of ScNha1 and heterologous expres- and influence Tip41 stability and hence Tip41-Tip2 associa- sion of the C. albicans antiporter [228, 229]. tion [218]. It has been also shown that Sit4 mediates Sit4 plays a role on lipid metabolism. For instance, mu- TORC1 signaling to maintain nuclear localization and stabil- tants in sit4 and sap190 were catalogued as low-lipid drop- ity of the transcription factor Stp1 and hence promoting let content strains, whereas the content in sap185 cells amino acid uptake [219]. More recently, a role for Tap42- was higher than normal [230]. In addition, it has been Sit4 in controlling site-specific acetylation of histone H3 shown that sit4 deletion mutants have decreased ceramide and H4 N-terminal tails, thus controlling epigenetic traits, levels, display resistance to exogenous ceramides and phy- has been proposed [220]. tosphingosine, and show a shift towards non-hydroxylated Sit4 also interacts physically and is regulated by the forms of long chain bases and sphingolipids, suggesting phosphotyrosyl phosphatase activators (PTPA) Ncs1/Rrd1 that Sit4 could regulate hydroxylase (SUR2) or ceramide and Noh1/Rrd2, also known as Ypa1 and Ypa2 [113, 221, synthase, without involvement of the SAPs regulators [231]. 222], which also regulate other type 2A PPases. It has been A link between the TOR pathway and lipid droplets medi- demonstrated that Rrd1 can be a component of the Tap42- ated by Sit4 and Tap42 and requiring the downstream Sit4 complex, and that rapamycin promotes the release of TORC1-controlled transcriptional activators Gln3, Gat1, the PTPA-Sit4 active complex [223]. Rtg1, and Rtg3 has been reported [232]. The Sit4-Sap190 and/or Sit4-Sap185 complexes are also Functions required for normal Elongator activity [233]. A major func- In addition to its involvement in cell cycle progression and tion of the Elongator complex in yeast (composed of Elp1 TORC1 pathway signaling, mentioned above, Sit4 regulates to Elp6) is the formation of certain modifications at the a broad range of biological processes. For instance, the tRNA anticodon, and it is known that sit4 and elp1–elp6 phosphatase plays a role in the CWI pathway, since dele- mutants display identical tRNA modification defects (see tion of SIT4 enhances both basal and heat-induced phos- [234] for a review). The role of Sit4 seems to antagonize phorylation level of the Slt2 MAP kinase, and the phospha- the phosphorylation of the largest Elongator subunit Elp1 tase appears involved in rapamycin-mediated induction of by the Hrr25 kinase. Recent work has proved that the role Slt2 [209, 224]. It was shown that Sap185 and Sap190 func- of Sit4 on lipid droplet synthesis is independent of its func- tion together with Sit4 to provide an essential role in the tion on Elongator-dependent tRNA modification [235]. absence of Bem2 [207], a GTPase activating protein that Carbohydrate metabolism is also affected by Sit4 activi- down-regulates Rho1. However, the additive effect of the ty. It has been proposed that lack of SIT4 causes rewiring of sit4 and bem2 mutation on Slt2 does not support the no- carbohydrate metabolism, with entry in a futile cycle of tion of Sit4 being a regulator for Bem2 and suggest an in- glycogen synthesis and degradation, down-regulation of dependent role for Sit4 and Bem2 on Slt2 regulation [224]. fermentation, overexpression of genes that are normally A role for Sit4 in monovalent cation tolerance and pH activated by glucose deprivation, and activation of respira- homeostasis was proposed by Masuda and coworkers, on tion [236]. The reduced fermentative capacity of sit4 cells the basis that overexpression of the phosphatase con- has been attributed to a decrease in pyruvate decarbox- ferred lithium tolerance in galactose medium but, in con- ylase activity [237]. In cells actively growing in the presence trast to the mutation of Ppz1, this effect did not affect the of abundant glucose, the Mig1 repressor and Hxk2 are expression of the ENA1 ATPase gene [225]. It was also ob- dephosphorylated and transferred into the nucleus where served that Sit4-overexpressing cells maintain a more alka- this complex exerts a repressor effect on expression of OPEN ACCESS | www.microbialcell.com 234 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review genes required for growth on non-fermentable carbon Initial characterization of ppg1 mutants revealed de- sources. It has been found that in the absence of Sit4, the creased glycogen accumulation that could be attributed to Snf1 kinase is activated and then phosphorylates the Mig1 higher levels of glycogen phosphorylase a, in addition to repressor, which leads to its inactivation [25, 238]. Sit4 also lower amounts of total glycogen synthase activity [247]. influences catabolite repression in a Snf1-independent More recently, it was found that ppg1 mutants were toler- fashion, since lack of the phosphatase promotes the deg- ant to ethanol and heat [250] as well as sensitive to Congo radation of the Mig1 repressor [239] Furthermore, hyper- Red [251], a compound that interferes with normal cell phosphorylation of Hxk2 observed in sit4 mutants prevents wall synthesis. The latter phenotype fits with the finding the formation of the Mig1-Hxk2 complex. Free Mig1 is then that, in C. neoformans, ppg1 mutants are also sensitive to phosphorylated at Ser311 by Snf1 promoting export of the cell wall inhibitors, such as Congo Red or Calcofluor White repressor into the cytosol. This further contributes to inter- (CFW) [252]. It must be noted that although in this paper fere with normal glucose repression [240, 241]. Sit4 is also the Cryptoccocus Ppg1 protein is defined as a "Sit4 homo- involved in the link between Snf1 and protein translation logue", Blastp analysis of the reported GenBank entry since, whereas in histidine starved cells Snf1 promotes the (XP_571206) shows higher identity to S. cerevisiae Ppg1 formation of phospho-eIF2α by activating the Gcn2 kinase, than to Sit4. when cells are shifted from glucose to galactose Snf1 coun- Given its interactions with Tap42 and Tip41, it is con- teracts the likely direct Glc7 and Sit4 phosphatase activity ceivable that Ppg1 might act downstream the TOR pathway. on phospho-eIF2α [242]. Indeed, mutations in PPG1 were found to suppress TORC2 As mentioned, respiration is derepressed in sit4 cells deficiency [253], and it was proposed that the phosphatase grown in glucose medium. Since these mutants are unable might collaborate with the FAR complex in the control of to grow under anaerobic conditions, mitochondrial respira- actin-based cell polarity. As mentioned above, the FAR tion becomes essential for their viability. Mitochondria are complex is composed of Far3 and Far7-11 and components a major source for reactive oxygen species and play key of this complex interact functionally and physically with roles in oxidative stress resistance and chronological PP2A phosphatases [119]. Very recently, Ppg1 has been lifespan. In agreement with the proposed role of Sit4 as a shown to interact with members of the FAR complex with- negative regulator of mitochondrial function, sit4 cells in the context of dephosphorylation of the mitophagy re- show some protection from defects associated with mito- ceptor Atg32, and it has been proposed that Ppg1 and the chondrial DNA damage [243], and increased chronological FAR complex cooperatively counteract casein kinase 2- lifespan [244]. It has been recently found that Hxk2 is hy- mediated phosphorylation of Atg32 to prevent excessive perphosphorylated in sit4-deficient cells by a Snf1- mitophagy [254]. independent mechanism, and that mutation of Ser15 in Studies in C. albicans using the systematic deletion mu- Hxk2 to Ala suppressed diverse phenotypes associated tant library have shown that Ppg1 is important for viru- with the deletion of SIT4, such as transition defects at G1 lence [255]. In addition, Ppg1 was found necessary for fil- phase, derepression of mitochondrial respiration, toler- ament extension, invasion, and virulence in a mouse model ance to H2O2 and lifespan extension [245]. Very recently, of systemic candidiasis. It was also proposed that Ppg1 involvement of Sit4 in the post-translational regulation of controls C. albicans filamentation via the PKA signaling nine mitochondrial proteins has been reported. In the case pathway, and that is also important for downregulation of of the ATP synthase  subunit Atp2, it was proposed that NRG1, encoding a transcriptional repressor required for Sit4-mediated dephosphorylation of Atp2 at T124 and T317 filamentous growth [256]. downregulates Atp2, together with ATP synthase and mi- tochondrial function [246]. PP2B (PP3, CALCINEURIN) AND PP2B-RELATED ENZYMES PPG1 This family comprises two branches: calcineurin (PP3) and The S. cerevisiae PPG1 gene encodes a 368 residues pro- PP5. Both kind of enzymes are widely distributed among tein with some similarity to type 2A phosphatases (Figure eukaryotes, including fungi (Figure 10). 4). However, it features an internal insertion of ten resi- dues (from amino acids 205-215) and a C-terminal exten- Calcineurin sion of 50 residues ending with the highly conserved DYFL Calcineurin is a conserved Ser/Thr protein phosphatase sequence characteristic of type 2A phosphatases [247]. regulated by calcium/calmodulin. In budding yeast cal- This phosphatase is also found in other fungi, but seems cineurin is a heterodimer, made out of one of two redun- absent in human or plants, where the closest sequences dant catalytic subunits (encoded by the CNA1 and CNA2 are that of PP4 or PPX phosphatases. Ppg1 has been found genes), plus a regulatory subunit, encoded by CNB1 [257]. to interact with Tap42 [248] and Tip41 [249], which are The CNA1 gene product has 553 residues, whereas the proteins involved in the TORC1 signaling pathway and also protein encoded by CNA2 is somewhat larger (604 amino able to interact with other type-2A phosphatases. In addi- acids). The phosphatase domain is located at the tion, interactions of Ppg1 with canonical PP2Ac regulatory N-terminal half of the so called calcineurin A (CNA) poly- subunits, such as Cdc55 and Tpd3 [51] or Rrr1/Ypa1 [113], peptide, and is followed by a regulatory subunit binding have been described. helix (BBH), a calmodulin-binding domain (CBD), and a OPEN ACCESS | www.microbialcell.com 235 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review of such motifs, known as PxIxIT sequence (with a [PG]xΦxΦζ consensus) has been identified in several cal- cineurin substrates in S. cerevisiae, such as Crz1, Rcn1, Slm1, Sml2, or Hph1 (see, [262] and references therein). These interactions, which are of relatively low affinity, oc- cur in CNA at a groove distal from the catalytic center and independently of the phosphatase activation state. In addi- tion, a secondary contact can be made through the LxVP motif, also found in some substrates such as Rcn1, which is thought to mediate the orientation of substrates during dephosphorylation, and therefore are only relevant when calcineurin is active. More recently, a combination of phosphoproteomics and bioinformatic approaches has expanded the list of yeast calcineurin candidate substrates, including the kinase Elm1 (acting upstream the Snf1 kinase) or Dig2, involved in pheromone signaling [263] (see below). Calcineurin was recognized long ago as the target for the immunosuppressive drugs cyclosporin A (a cyclic pep- tide) and tacrolimus (FK506, a macrolide). These drugs form complexes with cyclophilin (Cpr1) and the FK506 Binding Protein (Fpr1 or FKBP12, a ubiquitously expressed peptidyl-prolyl isomerase), respectively, and these com- FIGURE 10: Phylogenetic relationship of PP2B and PP5 (Ppt1) plexes are responsible for calcineurin inhibition. The reso- phosphatases from various fungal species. Protein sequences lution of the crystal structures of calcineurin and its com- correspond to organisms described in Figure 2. The analysis plexes with FKBP12-FK506 and cyclophilin-cyclosporin al- was performed as described in Figure 1. lowed the identification of a few common residues in cal- cineurin required for recognition of the complexes [264]. It C-terminal autoinhibitory domain (AID) that blocks the has been documented that the LxVP motif is necessary for catalytic activity [258] (Figure 11). interaction with the immunosuppressant-immunophilin Cnb1 is a relatively small protein (175 residues) that complexes, raising the notion that they inhibit calcineurin contains four EF hand calcium binding domains (EF1-4, by interfering with substrate recognition [265]. Recently, a Figure 11). In vitro analysis of recombinant mammalian mechanism of self-substrate regulation unique to the calcineurin allowed to propose that EF3 and EF4 have a A. fumigatus and C. albicans FKBP12 proteins has been structural role and constitutively bind Ca2+ in the cells, proposed [266]. whereas calcium binding to EF1 and EF2 facilitate respon- RCANs (Regulators of calcineurin) are a family of pro- siveness to changes in intracellular Ca+2 because such in- teins known to modulate calcineurin activity. Although also teraction results in conformational changes in CNB that found in humans, RCANs were first identified in yeast be- trigger partial activation of the phosphatase catalytic sub- cause their ability to interact with and inhibit calcineurin unit. Additional binding of Ca2+/calmodulin to CNA would upon overexpression. Indeed, signaling via calmodulin, cause dissociation of the so called autoinhibitory signal calcineurin, and Crz1 (the transcription factor downstream (AIS) from the LxVP docking pocket and reorientation of calcineurin, see below) induced Rcn1 expression, suggest- the C-terminal AID with respect the catalytic site allowing ing that Rcn1 works as an endogenous feedback inhibitor full enzyme activation [259]. Recent work using yeast cal- of calcineurin [267]. However, there has been some con- cineurin [260] demonstrated that, as previously reported troversy regarding the physiological roles of these regula- for mammalian calcineurin, EF4 is largely dispensable for tors, since in the same work it was shown that loss of RCN1 calcineurin activity in response to intracellular Ca2+ signals, in yeast also gave rise to decreased calcineurin signaling. A whereas EF1, EF2 and EF3 each contribute to calcineurin positive role of Rcn1 (which can be extended to mammali- activation, independently of previously known conforma- an RCANs) was reinforced by the finding that the stimula- tional changes induced upon Ca2+/calmodulin binding. The tory effect of yeast Rcn1 involves its phosphorylation at a regulatory properties of Cnb1 are affected by myristoy- conserved serine residue by Mck1, a member of the GSK-3 lation of the N-terminal Gly by the Nmt1 N- family of protein kinases. This allowed postulating that myristoyltransferase, since mutation of this Gly or expres- Rcn1 might act as activator or inhibitor of calcineurin de- sion of the ts allele nmt1-181 increases calcineurin activity pending of its phosphorylation state [268]. A subsequent [261]. Therefore, Cnb1 myristoylation functions to limit comparative study identified conserved docking motifs calcineurin signaling. that were necessary for inhibition of calcineurin signaling, Calcineurin recognizes its substrates and interacting whereas several additional motifs in RCANs (such as the proteins via conserved short, degenerate docking motifs GSK-3 phosphorylation site) were specifically required for that are distinct from the sites of dephosphorylation. One stimulatory and not for inhibitory effects. The authors sug- OPEN ACCESS | www.microbialcell.com 236 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review FIGURE 11: Schematic depiction of the struc- ture and functional features of the catalyt- ic (Cna1) and regulato- ry (Cnb1) subunits of calcineurin (A), and of Ppt1 (B). BBH, cal- cineurin B binding He- lix; CBD, Calmodulin Binding Domain; AIS, AutoInhibitory Signal; AID, AutoInhibitory Domain. EF1-4, EF hand Ca+2 binding domains. TPR, tetratricopeptide repeats. The number of TRP repeats shown are according to SMART analysis. The number of residues is indicated on the right of each figure. See main text for details. gested that RCANs may function primarily as chaperones also showed that the presence of the C at the 3' position of for calcineurin biosynthesis or recycling [269]. the CDRE was more frequent in promoters showing strong Crz1 recruitment. It has been demonstrated that, in re- Function sponse to external calcium, Crz1 shows pulsatile localiza- In budding yeast calcium is a common second messenger tion dynamics, with stochastic short burst ( 2 min) of nu- for diverse stimuli, such as exposure to mating phero- clear localization [278, 279]. mones, high salt or osmolarity, endoplasmic reticulum The set of genes induced upon calcineurin-mediated stress, and others (see [270, 271] and references therein), activation of Crz1 encode proteins controlling diverse cellu- although the timing of the calcium response varies greatly lar functions. Monovalent cation homeostasis is affected (from seconds in response to alkalinization [272] to nearly by the absence of calcineurin in several ways. In addition to one hour upon stimulation with mating pheromone [273]). a possible direct effect of calcineurin on the Trk potassium Calcineurin is a major cellular target for the calcium signal, transporters switch from low to high affinity transport [58], as exemplified by the observation that nearly 50% of the calcineurin/Crz1 activates expression of HAL5 [280], encod- gene deletions that lead to calcium sensitivity are sup- ing a kinase important for stabilization of Trk1/2 at the pressed by inhibition of calcineurin [274]. plasma membrane. Sodium efflux under cation stress is Activation of calcineurin provokes multiple changes in greatly influenced by Crz1, which plays a major role in the the yeast cells. Many of these changes are promoted by control of ENA1 expression [281–283]. Activation of cal- the calcineurin-mediated dephosphorylation of the zinc- cineurin influences calcium homeostasis and results in finger transcription factor Crz1/Tcn1/Hal8 (from now on, Crz1-mediated increase in the expression of PMC1 and Crz1). Upon binding to its PxIxIT-like motifs, calcineurin PMR1 (encoding Ca2+ ATPases in the vacuole and the Golgi dephosphorylates Crz1 at various sites and this promotes apparatus, respectively). Calcineurin also negatively regu- fast entry of Crz1 into the nucleus, assisted by Nmd5 (see lates the vacuolar Ca2+/H+ exchanger Vcx1 and influences [257] and references therein). Binding of Crz1 to gene Ca2+ influx through the plasma membrane Cch1/Mid1 cal- promoters occurs at somewhat degenerate consensus se- cium channels, likely by controlling the phosphorylation quence, named CDRE (Calcineurin-Dependent Response state of Cch1 (see [273] and references therein). Additional element) that exhibits a constant GCC core. Such CDRE was substrates of calcineurin, independently of its role on Crz1, defined by Yoshimoto and coworkers as [TG(A/C)GCCNC] are Hph1 and the Slm1/Slm2 proteins (see below). Hph1 or [CAGCCTC], depending on the methodology used [275], and Hph2 are homologous proteins with overlapping func- or as A/CGCCNC by means of Protein Binding Microarray tions, required for normal tolerance to saline, alkaline pH, technology [276]. A more recent study using ChIP-Seq and cell wall stress [284]. Calcineurin-mediated technology identified 152 intergenic regions recruiting Crz1 dephosphorylation positively modulates Hph1. More re- upon alkaline stress (the vast majority between 1 and 5 cently it has been described [285] that Hph1 (and Hph2) min upon stress onset), and confirmed the prevalence of act with the Sec63/Sec62 complex and that defects in this the A/CGCCNC motif for Crz1 binding [277]. These authors OPEN ACCESS | www.microbialcell.com 237 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review complex results in destabilization of Vph1, a subunit of the amiodarone, a drug that elicits an immediate influx of Ca2+, vacuolar proton ATPase (V-ATPase). temporarily delayed cell cycle progression at different cell Calcineurin plays a role in the regulation of the biosyn- phases, being the Swe1-mediated delay in G2/M phase the thesis of sphingolipids, and this involves the ancillary one most dependent on calcineurin [300]. These and other TORC2 subunits Slm1 and Slm2 [286], which were initially evidences suggested a role for calcineurin in cell cycle described as direct substrates for calcineurin [287]. It has regulation, usually in response to a variety of stresses, and been proposed that calcineurin negatively regulates the in particular at the level of the morphogenesis checkpoint. sphingolipid pathway at the level of ceramide synthesis, Possibly, the characterization of the role of calcineurin in and this is largely due to the direct dephosphorylation of cell cycle and morphogenesis has been hampered by func- the ceramide synthase subunits Lac1 and Lag1 [288]. Such tional redundancy with other phosphatases, such as PP2A, dephosphorylation would counteract the positive effect of as previously suggested [271]. However, recent work has phosphorylation due to TORC2-Ypk1 signaling. shown that calcineurin is important in the regulation of the Quite often, those conditions that activates the Slt2- forkhead transcription factor Hcm1 [301]. This factor is mediated CWI pathway also triggers influx of calcium and involved in the expression of genes that are transcribed activation of calcineurin (note that Mid1 has been pro- during S-phase and, in this way, it regulates chromosome posed to be a mechanosensitive channel). Both the CWI segregation and is vital for maintaining genome stability. pathway and the calcineurin pathways are important in Hcm1 is activated by multiple phosphorylation at its response to cell wall stress, and when one of them be- C-terminal trans-activation domain by the cyclin- comes non-functional, the other becomes essential [289]. dependent kinase Cdk1, which also phosphorylates More recently, it has been shown that Pkc1-Slt2 and cal- N-terminal residues that promote destabilization [302]. cineurin pathways cooperate to allow cell survival under Upon stress, calcineurin would dephoshorylate the activat- compressive mechanical stress [290]. Knr4, an intrinsically ing residues, while the destabilizing of phosphorylated sites disordered protein, has been proposed to serve as con- would remain. In this way, the function of Hcm1 is abro- necting node between these two signal transmission path- gated, leading to a protective halt in cell cycle progression ways [291]. [303]. Interestingly, parallel evidences point to Slt2- Calcineurin plays a significant role in the adaptation to mediated phosphorylation of the destabilizing N-terminal nutrient availability. In response to glucose addition, yeast sites to decrease Hcm1 amounts in response to cell wall cells raise a calcium signal that occurs through two differ- stress to delay proliferation [304]. ent influx pathways: Mid1/Cch1 and the GIC (for Glucose Calcineurin plays an important role in down- Induced Calcium) system, and that involves IP3 as second modulating the response to mating pheromones as exem- messenger [292, 293]. This signal can stimulate calcineurin plified by the early finding that calcineurin mutants die and results in the up-regulation of the expression of di- upon prolonged exposure to the pheromone (Figure 12). verse genes encoding carbohydrate transporters and me- During mating, the pheromone-activated receptor recruits tabolizing enzymes. Such activation is sufficient to allow the small GTPase Cdc24 to the membrane that, in its acti- growth under glucose limitation even in the absence of the vated GTP-bound form, initiates a kinase cascade that Snf1 kinase [294]. Likewise, in response to excess amino leads to the activation of two partially overlapping MAPK, acids, dephosphorylation of the α-arrestin trafficking adap- namely Fus3 and Kss1. Activation of these kinases (mainly tor, Aly1/ Art6 by calcineurin activates endocytosis of the Fus3) leads to phosphorylation of the repressor proteins dicarboxylic amino acid permease Dip5 (but not that of the Dig1 and Dig2, thus allowing the function of the Ste12 Gap1 permease), suggesting that the action of calcineurin transcription factor and the consequent specific changes in on a given α-arrestin can affect the trafficking of specific gene expression pattern, which are accompanied by mor- cargo proteins [295]. More recently, it has been shown phological changes (shmoo formation) and cycle arrest (see that calcineurin negatively regulates Aly1-mediated traf- [305] for a review). Indeed, exposure to mating factor ficking to the plasma membrane of the heterogously ex- stimulates the Mid1/Cch1 high-affinity Ca2+ channel, induc- pressed mammalian potassium channel Kir2.1 [296]. ing an increase in cytosolic Ca2+ that leads to calcineurin It has been described that, during amino acid starvation, activation (Figure 12). The regulatory effect of calcineurin TORC2 promotes autophagy via its downstream target the on the mating response occurs at different levels. From protein kinase Ypk1, which inhibits calcineurin. In turn, one side, calcineurin reduces the levels of pheromone re- calcineurin inhibits the activation of Gcn2, the eIF2α kinase ceptor at the plasma membrane by dephosphorylating the and, consequently, the translational de-repression of the arrestin Rod1/Art4 thus promoting endocytosis [306] while, transcription factor Gcn4 [297]. These events are required in parallel, dephosphorylation of the Ste12 repressor Dig2 for both initiation of the general amino acid control (GAAC) inhibits the pheromone-induced transcriptional response response and autophagy during amino acid starvation. [263]. More recently, a third level of regulation has been More recently it has been proposed that Mid1, inde- proposed involving Rga2 [307], one of the Cdc42 GAPs pendently of Cch1, is required to maintain calcineurin ac- (GTPase-activating protein). Rga2 is phosphorylated by tive and prevent autophagy [298]. Fus3 on inhibitory Ser and Thr sites, thus avoiding deactiva- Early work showed that overexpression of calcineurin tion of Cdc42 upon pheromone signaling. Calcineurin causes severe morphologic changes [299]. It was also would dephosphorylate Rag2, and reactivated Rag2 would found that treatment of cells with moderate doses of promote transition of Cdc42 to its (inactive) GDP-bound OPEN ACCESS | www.microbialcell.com 238 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review form. In this way, calcineurin action would counteract physiological characteristics, in most cases the fungal pro- Fus3-mediated downstream signaling in response to pher- cesses affected by lack of calcineurin are the same: growth omone stimulus (Figure 12). rate, morphological changes (often required for virulence, such as filamentation), and the ability to respond to stress Calcineurin as virulence determinant in pathogenic fungi conditions. It is worth noting that calcineurin has been The study of the calcineurin signaling pathway has received found to be involved in cell wall maintenance in most fun- considerable attention in the last years since this enzyme gal pathogens, and that this same process (specifically cell seems to be a relevant virulence factor in major pathogenic wall -glucan synthesis) is the target of the lipopeptide fungi, such as C. albicans, A. fumigatus or C. neoformans. drugs echinocandins. In contrast, calcineurin is necessary Thus, calcineurin has been proposed as an important drug for survival at 37°C in some pathogens, as in C. neoformans, target for antifungal treatments [308, 309]. The im- but not in other, such as C. albicans (see [310] and refer- portance of calcineurin in virulence processes is quite ences therein). widespread and has been also identified in fungal plant Homologs of the Crz1 transcription factor can be found pathogens. Despite their very diverse morphological and FIGURE 12: Roles of calcineurin during pheromone response. Upon stimulation with pheromone (α-factor in the model) entry of calci- um promotes activation of calcineurin. To attenuate pheromone signaling, calcineurin acts at least at three levels: a) dephosphorylates Rod1 to promote endocytosis of the pheromone receptor; b) dephosphorylates Rga2, leading to decrease in Cdc42-GTP levels and at- tenuation of signaling through the Fus3 MAP kinase module; c) dephosphorylation and activation of the Ste12 inhibitor Dig2, to de- crease pheromone-regulated specific gene transcription. Other targets downstream Fus3 are not included for simplicity. OPEN ACCESS | www.microbialcell.com 239 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review in pathogenic fungi (CrzA in A. fumigatus). Mutation of clients. Subsequent work by mass spectrometry revealed CRZ1 does not fully reproduce the phenotypes caused by that Hsp90 is phosphorylated at multiple sites and that elimination of calcineurin, suggesting that other down- Ppt1 directly dephosphorylates two of these sites, S485 stream targets relevant for virulence exist. Thus, calcineu- and S604 [321]. It is worth noting that interaction of PP5 rin is necessary for growth in the presence of serum in and Hsp90 has been also observed in the plant Arabidopsis C. glabrata and C. albicans, but Crz1 is not [311, 312]. The thaliana [322]. identification of a Crz1 homolog in C. neoformans has been Investigation on modern sake yeast strains, which are somewhat controversial, but a recent work provided highly sensitive to environmental stresses, revealed that strong evidence that loss of Crz1 results in intermediate the transcription factor Hsf1, relevant in the activation of phenotypes compared to wild type and calcineurin mutant multiple genes in response to various stresses, was consti- strains. This same work identified a subset of calcineurin- tutively hyperphosphorylated even in the absence of stress. regulated genes that, in response to a 24 to 37°C shift, Hsf1 hyperphosphorylation was caused by a spontaneous were not dependent on the presence of Crz1, as well as a deletion of the PPT1 locus [323]. Indeed, deletion of PPT1 smaller number of genes dependent on Crz1 but not on in a laboratory strain led to constitutive hyperphosphoryla- calcineurin [313]. A recent study demonstrated that in tion of Hsf1, and introduction of functional PPT1 derived Cryptococcus deneoformans the initiation of the yeast- from laboratory yeast recovered the HSE-mediated stress hyphal morphological transition is independent of Crz1, response of sake yeast. These findings suggested that Ppt1 whereas the sporulation process is dependent on the tran- is a positive regulator of Hsf1 [323]. Subsequently, it was scription factor [314]. found that Ppt1 directly interacts with Hsf1 via its TPR do- main and that the phosphatase counteracts the CK2- PPT1 (PP5) dependent phosphorylation of the transcription factor on In S. cerevisiae, the Ppt1 phosphatase is 513-residue long S608, which is an ethanol stress-specific repression mech- protein with a distinct C-terminal half displaying the typical anism of Hsf1 [324]. It must be noted that a functional re- features of the PPP family and an N-terminal extension lationship between Hsf1 and Hsp90 has been proposed, (200 residues) that includes several TPR (tetratricopep- although there is some controversy about its nature [325]. tides repeats; four according the original paper, three upon Quantitative phosphoproteomic analysis using SILAC al- our own analysis). The TPR repeats are structural motifs lowed specific comparison of wild type yeast cells and consisting of a degenerate 34 amino acid sequence often those carrying the PPT1 deletion [326]. These authors occurring in clusters and involved in protein-protein inter- found 33 phosphorylation sites significantly up-regulated in actions. Ppt1 is well conserved across evolution and is the mutant, corresponding to 28 different proteins (includ- found not only fungi, but also in plants and animals, includ- ing Hsf1, Cdc37 and Hsp90), but no dephosphorylation ing humans, named as PP5 [315]. events. In addition to proteins related to heat stress, the A number of roles for PP5 in mammals were identified authors identified biosynthetic enzymes as particularly relatively soon (see [316] and references therein). In con- prominent among Ppt1-regulated phosphoproteins, sug- trast, our knowledge on yeast Ppt1 was scarce for quite a gesting still uncharacterized roles for Ppt1 in metabolic few years. Ppt1 was present in both the nucleus and cyto- regulation. plasm and was found to be expressed during logarithmic growth [317]. The same authors showed that although, as PP2C in PP5, Ppt1 activity was stimulated by lipids, the mamma- Type PP2C phosphatases are represented by the family of lian and yeast enzymes differed in several characteristics, Ptc proteins, included in the PPM group of phosphatases such as the effect caused by removal of the TPR domain on since their activity depends on metal ions. In S. cerevisiae, Ppt1 activity. The lack of inhibitory effect upon removal of this family is encoded by 7 different genes (Figure 13), the TPR moiety was also observed during the characteriza- ranging from PTC1 to PTC7. Not all members are repre- tion of the Ppt1 homolog from Aspergillus oryzae [318]. sented in all fungi and, in some species, more than one A major advance in understanding Ppt1 function was gene may exist for a specific Ptc [327]. In contrast to other the finding that Hsp90 was specifically dephosphorylated PPP’s the catalytic subunits of the PP2C family are not usu- by purified Ppt1 [319]. Hsp90 is a conserved ATP- ally associated with regulatory subunits. Evidence has been dependent molecular chaperone involved in the conforma- reported that deletion of all seven Ptc-encoding genes is tional maturation of client proteins implicated in various not lethal [328]. and essential processes in eukaryotes. It was also shown that Ppt1 binds to Hsp90 through the phosphatase TPR Ptc1 phosphatase domain and that lack of Ppt1 leads to hyperphosphoryla- Fungi Ptc1 constitute a well-defined group of proteins that tion of Hsp90 in vivo and to a client-specific apparent de- are involved in many cellular functions often not shared by crease in the efficiency of the Hsp90 chaperone system other members of the PPM family [327, 329]. In S. cere- [319]. Remarkably, Ppt1 also dephosphorylates the Hsp90 visiae, cells lacking Ptc1 show a large number of functional co-chaperone Cdc37 at Ser13 specifically when Cdc37 is defects, including sensitivity to high pH, LiCl, CaCl2, ZnCl2, associated with Hsp90 [320], and this dephosphorylation CFW, caffeine, rapamycin, and inability to grow on ethanol affects the activation of the Hsp90-Cdc37 protein kinase as the only carbon source. These mutants also show de- OPEN ACCESS | www.microbialcell.com 240 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review FIGURE 13: Phylogenetic relation- ships among PP2C phosphatases from various fungal species. Pro- tein sequences correspond to or- ganisms described in Figure 2. The analysis was performed as de- scribed in Figure 1. fects in budding pattern, vacuolar morphology or inher- the regulation of the CWI pathway is a major cellular role itance of cellular organelles (see [327] and references for Ptc1 [331]. There is some evidence that Ptc6 might con- therein). Ptc1 also plays a role in the TOR pathway and in tribute, although to a much lesser extent, to the regulation cation homeostasis, among other functions. of the CWI pathway (see below). In addition to mediate the interaction between Ptc1 and the HOG pathway, Nbp2 also Ptc1 and MAP kinase pathways functions as a negative regulator of the CWI pathway by A role of Ptc1 in regulating MAP kinase pathways was es- facilitating the interaction of Ptc1 with Bck1. This role of tablished long time ago. Thus, the regulation of the high- Nbp2 in Ptc1 signaling might be extended to other MAPK osmolarity glycerol (HOG) pathway is achieved by pathways and seems to be conserved across the Ascomy- dephosphorylation of Hog1 MAP kinase, and this is accom- cete species. plished by binding of the phosphatase to the N-terminal Ptc1 is also involved in the MAPK pathway activated by domain of the adaptor protein Nbp2, which in turn inter- pheromone, regulating the mechanism that controls the acts through a SH3 domain with the scaffold protein Pbs2 switch-like mating decision [332]. Ptc1 and the MAPK Fus3 [330]. compete for the control of the phosphorylation state of A number of independent evidences placed Ptc1 in Ste5, the scaffold protein of this MAP kinase module. close relationship with the CWI pathway, including the Dephosphorylation of Ste5 is a requisite for full relief and observation that mutation of PTC1 results in increased dissociation of the Fus3-Ste5 complex, which leads to the expression of genes induced by cell wall damage in a way mating response. Lack of Ptc1, thus, prevents shmooing that was dependent on the Slt2 MAPK module [329]. and reduces activation of Fus3. Therefore, lack of Ptc1 mimicked the activation of the CWI pathway. Recently, it has been shown that Ptc1 specifically Ptc1 and cation homeostasis dephosphorylates and inactivates Mkk1, one of the two Diverse evidences indicate that, among Ptc enzymes, Ptc1 MAP kinase kinases upstream Slt2 [331]. Remarkably, is the major responsible for Li+ tolerance, as only ptc1 sin- many phenotypic defects described for the ptc1 mutant gle mutants display increased sensitivity to this cation. It can be attributed to the abnormal activation of the Slt2 has been determined that Ptc1 is necessary for the proper pathway, as a result of the incapacity to properly extrusion of Li+ since ptc1 mutant cells accumulate higher dephosphorylate and down-regulate Mkk1, indicating that concentrations of this cation due to a decreased expres- OPEN ACCESS | www.microbialcell.com 241 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review sion of the Na+-ATPase ENA1 gene. Evidences suggest a since the close human orthologs Ppm1E, and probably possible role of Ptc1 in the regulation of Ppz1/2-Hal3 (see PpmF, are implicated in the dephosphoryation of human PPZ section above) that could be exerted through the Hal5 AMPK [336]. protein kinase [333, 334]. The inheritance of several cellular organelles is a pro- A systematic analysis performed with all possible com- cess in which Ptc1, together with the adaptor protein Nbp2, bination of ptc disruptions showed that only Ptc1 and Ptc2 is involved and it has been extensively reviewed [327]. and/or Ptc4 had a redundant role in the tolerance at high More recently, Ptc1 was found to promote the association concentrations of Na+ ions in the medium [328]. A strain of myosin-V, encoded by MYO2 and MYO4, with its orga- containing ptc2 ptc3 ptc5 ptc7 simultaneous deletions tol- nelle-specific adaptor proteins such as Mmr1, Vac17 and erates 1.5 M NaCl similarly to wild type cells, but the same Inp2, being important for the proper inheritance of mito- strain was unable to grow in the presence of 0.4 M LiCl chondria, vacuole and peroxisomes, respectively [337, 338]. (and grew poorly in YPD medium supplemented with ade- The phosphorylated form of Mmr1 has been found in- nine). The same report showed that the Li+-sensitive phe- creased in cells lacking Ptc1 [338]. In the same cells, the notype displayed by the ptc1 mutant cells is suppressed by steady-state level of Mmr1, Vac17 and Inp2 was reduced additional deletion of PTC7 [328], although no explanation [337]. was proposed for this effect. The Ptc2, Ptc3, and Ptc4 phosphatases Ptc1 as regulator of the cell cycle at the G2/M transition The analysis of the primary structure of these proteins de- Several recent evidences support a functional positive role notes that they are closely related, sharing one only branch of Ptc1 in the G2/M transition. Under stress conditions where two main groups of proteins can be differentiated: activating the CWI pathway ptc1 mutant cells display hy- Ptc2/3 and the Ptc4 (Figures 1 and 13). The number of perphosphorylated Cdc28 kinase and reduced Clb2- genes coding for proteins in any of these groups varies in associated Cdc28 activity, and they accumulate with dupli- different fungi species, ranging from none (in the case of cated DNA, pointing out to a delay in the G2/M transition Tuber melanosporum) to three. S. pombe, Schizosaccharo- [331, 335]. Consistently, overexpression of MIH1, coding myces japonicus, C. glabrata and S. cerevisiae are the only for the Swe1 tyrosine phosphatase that promotes progres- species analyzed containing two genes encoding the close- sion through cell cycle, suppressed sensitivity of ptc1 mu- ly related Ptc2/Ptc3. On the other side, Ptc4-related pro- tant cells to cell wall stressors, whereas deletion of MIH1 teins are specific of fungi included in the family of Saccha- increased the sensitivity of ptc1 mutant cells to CFW [335]. romycetaceae, being absent in the rest of the Saccharomy- Also, overexpression of PPH22 or ZDS1, important actors in cetales. Mih1 regulation, are able to suppress certain defects of the Although Ptc2/Ptc3, as Ptc1, are involved in the regula- ptc1 mutant [335]. These cell cycle-related alterations also tion of the HOG MAP kinase pathway, they do not interact are attenuated by mutation of the MKK1 gene, encoding a with Nbp2 and, since they cannot replace Ptc1 (see [327] MAP kinase kinase upstream Slt2, leading to the proposal and references therein), they are probably involved in dif- that their primary origin is the hyperactivation of the Slt2 ferent cellular roles. As in the case of Ptc1, Ptc2 and Ptc4 pathway described above. have recently been identified as phosphatases that physi- cally interact to and regulate Slt2, the MAPK of the CWI Other functions of Ptc1 pathway [50]. Several evidences pointed out that ptc1 mutant cells were It has been suggested that Ptc2/3 act as negative regu- hypersensitive to rapamycin, indicating a possible func- lator of the UPR by interacting to the ER membrane local- tional connection between Ptc1 and the TOR pathway (see ized Ire1, a protein with Ser/Thr protein kinase and RNase [327] and references therein). In fact, lack of Ptc1 impairs activities that triggers the UPR. Ptc2/3 attenuate the signal- TOR-mediated signaling on Gln3 and Msn2/4 transcription ing by decreasing the phosphorylation level of Ire1 and its factors, leading to a general attenuation of the transcrip- activation which, in turn, decreases the levels of the HAC1 tional changes triggered by rapamycin. These defects are, cytosolic splicing [29, 339]. in part, caused by the low levels and impaired This subfamily of PP2C were identified as phosphatases dephosphorylation of Tip41 observed in ptc1 mutant cells. required for the recovery from the DNA DSB since, in the Hyperphosphorylated Tip41 cannot bind to nor inhibit absence of Ptc2 and Ptc3, Rad53 remains hyperphosphory- Tap42, which is required for normal signaling through the lated even after a DSB is repaired. In the suggested model, pathway involving inhibition of Sit4 [218]. It has been Ptc2 is phosphorylated by CKII and thereby dephosphory- shown [185] that, in contrast to Gln3, rapamycin-elicited lates Rad53 [189]. In S. cerevisiae, cells deleted in PTC2, Ure2 dephosphorylation occurred independently of Ptc1 PTC3 and PPH3 display defects in repairing a HO endonu- (as well as of Sit4 and Pph21/22, Siw14 and Ppz1). clease-induced DSB. This phenotype is not observed in ptc2 Ptc1 has been described as one of the phosphatases, ptc3 double mutant cells [182], suggesting that Ptc2/3 are together with Glc7-Reg1 and Sit4, directly or indirectly par- dispensable for both, Rad53 deactivation and checkpoint ticipating in the glucose-dependent dephosphorylation of recovery after replication stress and for the dephosphory- the activation loop (Thr210) of Snf1, the yeast AMPK [26] lation of Rad53 that has been activated in S phase in re- when growing on medium containing high glucose concen- sponse to DNA methylation. trations. This seems to be a conserved function of the PP2C, The Ptc5, Ptc6, and Ptc7 phosphatases OPEN ACCESS | www.microbialcell.com 242 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review By using classical genetics and biochemistry techniques form is determined by the carbon source, being Ptc7u more Ptc5 and Ptc6 were identified as mitochondrial proteins prominent when cells were grown in fermentable media acting on the pyruvate dehydrogenase complex (PDC), [347]. The transmembrane domain and the mitochondrial specifically on the E1 subunit Pda1 [see [327] for targeting signal are largely conserved between Saccharo- references]. The activity of the PDC is important for mycetaceae PTC7 orthologs and are mutually exclusive. regulating the entry of pyruvate into the TCA cycle and, Interestingly, there is at least one example (the budding consequently, the overall maintenance of cellular glucose yeast Tetrapisispora blattae) of the existence of two indi- homeostasis. More recently, using comparative vidual genes, one encoding a predicted nuclear envelope- mitochondrial phosphoproteomics and analyses of associated Ptc7, while the other encodes a predicted mito- protein–protein interactions by affinity enrichment–mass chondrial phosphatase [348]. spectrometry, Ptc6 (also known as Aup1) has been It has been reported that the mitochondrial version of identified as the primary PDC phosphatase in S. cerevisiae Ptc7 activates coenzyme Q (CoQ, also known as ubiqui- and the only able to dephosphorylate Pda1 in vitro [340]. none) biosynthesis by dephosphorylation of the demeth- According to this study, Ptc5 and Ptc7 have other distinct, oxy-Q6 hydroxylase Coq7, a key regulatory point in the non-overlapping functions, such as dephosphorylation of yeast CoQ biosynthetic pathway [349]. In this way, Ptc7 the glycerol-3-phosphate dehydrogenase Gpd1. promotes respiratory metabolism. The impact of the ab- Ptc6 has been found necessary for survival of stationary sence of Ptc7 in the phosphorylation status of mitochon- phase cells and it has recently been found involved in mi- drial proteins was recently analyzed [350]. Nearly 20 mito- tophagy, the autophagic degradation of mitochondria, chondrial proteins increased their phosphorylation levels, which is an important housekeeping process in eukaryotic including the citrate synthase Cit1. It was found that hy- cells [341]. It was subsequently established that in Ptc6- perphosphorylation of Cit1 at S362 disrupted the enzyme deficient cells the retrograde signaling pathway (RTG) was function and that this residue was dephosphorylated in defective, and the phosphorylation pattern of Rtg3, the vitro by Ptc7. The authors suggested that, by regulating transcription factor mediating RTG response, was altered. citric acid production, Ptc7 might impact not only in the This allowed proposing that the role of Ptc6 in mitophagy functioning of the TCA cycle, but also might influence de could be explained through regulation of Rtg3-dependent novo fatty acid synthesis. Remarkably, they found that lack transcription [342]. of Ptc7 did not alter CoQ levels. It must be noted, however, Cells lacking Ptc6 (but not the ptc5 mutant) are sensi- that the methodologies and growth conditions used in tive to rapamycin and caffeine [218], and this phenotype is both apparently contradictory studies largely differ. A very independent of the alteration in the PDC activity [343]. In recent report [351] has shown that the lack of Snf2, the fact, the transcriptional profile of the ptc6 mutants was catalytic subunit of the SWI/SNF chromatin remodeling more similar to that of the ptc1 strain than to other Ptc- complex, alters the relative levels of Ptc7s and Ptc7u deficient cells, including ptc5, albeit the sensitivity to ra- isoforms, promoting predominance of the spliced Ptc7s pamycin is not rescued by overexpression of PTC1. It was isoform. The increase in PTC7 splicing was attributed to also found that this mutant suffers a considerable attenua- down-regulation of the very abundant transcripts of the tion in its transcriptional response to rapamycin, being genes encoding ribosomal proteins, leading to an increase particularly affected the subset of repressed genes encod- in the available pool of spliceosomes. The authors found ing ribosomal proteins or involved in rRNA processing [343]. that exclusive expression of Ptc7s increased CoQ synthesis, The sensitivity of S. cerevisiae ptc6 mutants to rapamycin whereas exclusive expression of Ptc7u had the opposite and caffeine is complemented by low-copy expression of effect. This would explain the observed increase in both the C. albicans CaPTC6 gene, although deletion of CaPTC6 the rate of synthesis and steady-state levels of CoQ in Snf2- does not alter C. albicans sensitivity to these compounds deficient cells and might contribute to justify the contradic- [344]. Lack of Ptc6 renders cells tolerant to cell wall damag- tory results on CoQ production described above. Lack of ing agents, such as Congo Red or CFW [343], although it Ptc7 has been also related to the shortening of the chrono- has been hypothesized that this effect might be strain- logical life span (CLS) and prevention of mitophagy, where- specific [345]. as overexpression of the protein had the opposite effects Ptc7 (YHR076w) was biochemically identified as a pro- [352]. Since CLS shortening could not be rescue by exoge- tein phosphatase more than 15 years ago and a mitochon- nous yeast CoQ supplementation, this was additional evi- drial localization was initially reported [346]. It was subse- dence pointing out that Ptc7 could function beyond the quently found that the gene contains a functional intron, regulation of CoQ biosynthesis. It is worth noting that a very near to the N-terminus, that lacks a consensus branch- very recent report [353] has shown that PPTC7, the human point sequence [347]. This intron could be spliced out or orthologue of yeast Ptc7, rescues CoQ deficiency in yeast retained, producing in the first case a slightly shorter ver- and that phosphorylated recombinant human COQ7 is an sion (Ptc7s) that is targeted to the mitochondria and, in the in vitro substrate for PPTC7. In addition, modulation of second case, a version (Ptc7u) that was localized to the PPTC7 levels did result in changes in CoQ content in human nuclear envelope. Targeting to the nuclear envelope has cells, suggesting that the regulatory role of Ptc7 enzymes been attributed to a predicted trans-membrane helix en- could have a very broad distribution. Collectively, all these coded by the retained intron. Both versions retain the findings point to Ptc7 being an important regulatory phos- PP2C catalytic domain, and the prevalence of a given iso- phatase in mitochondrial metabolism. OPEN ACCESS | www.microbialcell.com 243 Microbial Cell | MAY 2019 | Vol. 6 No. 5 J. Ariño et al. (2019) Fungal Ser/Thr phosphatases: a review PP2c’s in other fungi strates require the substrates be previously phosphory- Most of the current knowledge about PP2C proteins in lated and, as far as we know, the conditions and/or kinases other fungi has previously been reviewed [327], so we will required for the phosphorylation of all possible substrates focus here in the most recent findings. are still unknown. For these reasons, it seems plausible Fungal Ptc1 proteins seem to be required for patho- that the next steps for the identification of (direct or indi- genesis in both, animal and plant pathogenic fungi (see rect) substrates will be based on detecting the overall [327] for references). BcPtc1 or BcPtc3 proteins of the As- changes in the phosphorylation state of proteins when comycota fungus Botrytis cinerea, a plant pathogen, are cells are under specific cell growth conditions, either in the required for growth in the presence of osmotic and oxida- absence of one (or several redundant) specific phospha- tive stressors, and cell wall degrading enzymes. Lack of tases, or overexpressing a given enzyme. these proteins also showed decreased virulence on differ- ent host plant tissues [354]. In addition to Ptc1, Ptc3 has ACKNOWLEDGMENTS been found to be involved in virulence in the cereal crops Work in the author’s laboratory has been supported by grants pathogen Fusarium graminearum [355]. BFU2014-54591-C2-1-P (Ministerio de Economía y Competi- In fission yeast, SpPtc4 plays a major role in inactivating tividad, Spain) and BFU2017-82574-P (Ministerio de Ciencia, the mitochondrial pool of the stress-activated MAP kinase Innovación y Universidades, Spain). DV has been recipient of a Sty1, most similar to ScHog1, specifically upon oxidative UAB-PIF fellowship. We thank Dr. Ethel Queralt (IDIBELL, Bar- but not under other types of stress. SpPtc4 is strongly as- celona), for critical review and suggestions. sociated with mitochondrial membranes and is targeted The authors wish to state that, due to constriction in the there by an N-terminal mitochondrial targeting sequence number of allowed references, many relevant publications (MTS), which is cleaved upon import. Cleavage of the could not be explicitly cited. SpPtc4 MTS is greatly reduced specifically upon oxidative stress, resulting in the full-length form of the phosphatase SUPPLEMENTAL MATERIAL that displays a stronger interaction with Sty1. Consistently, All supplemental data for this article are available online at the mitochondrial pool of Sty1 is hyperactivated in the www.microbialcell.com. absence of SpPtc4 [356]. CONFLICT OF INTEREST FINAL REMARKS The authors declare no conflict of interest. One of the most important challenges in this field is to identify and characterize the physiological substrates of COPYRIGHT phosphatases. This objective presents several difficulties. © 2019 Ariño et al. This is an open-access article released As in the case of protein kinases, a given protein may be under the terms of the Creative Commons Attribution (CC the substrate of a phosphatase only for a short period (i.e., BY) license, which allows the unrestricted use, distribution, when cells are in a definite physiological state or under a and reproduction in any medium, provided the original specific stress). An additional difficulty is that some phos- author and source are acknowledged. phatases may have different substrates depending on which regulatory subunit they bind to. Similarly, some en- zymes could likely exhibit a high degree of functional re- Please cite this article as: Joaquín Ariño, Diego Velázquez and dundancy (such as Pph21/Pph22, Ppz1/Ppz2, or Antonio Casamayor (2019). Ser/Thr protein phosphatases in fungi: structure, regulation and function. Microbial Cell 6(5): 217-256. Ptc2/Ptc3/Ptc4). Unlike protein kinases, massive tech- doi: 10.15698/mic2019.05.677 niques for in vitro determination of phosphatase sub- REFERENCES 1. Offley SR, and Schmidt MC (2019). Protein phosphatases of 10.1146/annurev.bi.58.070189.002321 Saccharomyces cerevisiae. Curr Genet 65(1): 41-55. doi: 10.1007/s00294-018-0884-y 6. Gerasimaitė R, and Mayer A (2017). Ppn2, a novel Zn 2+ -dependent polyphosphatase in the acidocalcisome-like yeast vacuole. J Cell Sci 2. Lee J, and Levin DE (2018). Intracellular mechanism by which 130(9): 1625–1636. doi: 10.1242/jcs.201061 arsenite activates the yeast stress MAPK Hog1. 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