Type 2C Protein Phosphatases in Fungi · their target, protein phosphatases in general and PP2C en-zymes in particular do not exhibit signiﬁcant site speciﬁcity. This prevents

EUKARYOTIC CELL, Jan. 2011, p. 21–33 Vol. 10, No. 11535-9778/11/$12.00 doi:10.1128/EC.00249-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

MINIREVIEWS

Type 2C Protein Phosphatases in Fungi�†Joaquín Arino,* Antonio Casamayor, and Asier Gonzalez

Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autonoma de Barcelona,Cerdanyola del Valles 08193, Barcelona, Spain

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved ineukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, thecatalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achievedby encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phos-phatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) withdiverse functions can be found. More recently, data on several Candida albicans PP2C proteins becameavailable, suggesting that some of them can be involved in virulence. In this work we review the availableliterature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of theseenzymes in fungi.

Reversible phosphorylation of proteins is a major mecha-nism regulating many biological processes such as metabolism,gene transcription, or cell cycle. It is an extremely commonevent in signal transduction, and it is considered the mainmechanism of posttranslational modification leading, for in-stance, to a change in enzyme activity. The phosphorylationstate of a protein results from the balance of protein kinasesand protein phosphatases activities. Alterations in the phos-phorylation state of proteins are a cause of various diseases,such as cancer, diabetes, rheumatoid arthritis, or hypertension.The importance of this regulatory mechanism is evident whenit is considered that it affects around 30% of the proteome ofSaccharomyces cerevisiae (72) and that the number of genesencoding phosphatases and kinases represents 2 to 4% of allgenes in a typical eukaryotic genome (60).

Most phosphorylation events in eukaryotes involve transferof phosphate to Ser and Thr residues. Removal of this phos-phate is catalyzed by Ser/Thr protein phosphatases. Membersof this large family of enzymes were initially classified, usingenzymological criteria, as type 1 (PP1) and type 2 (PP2) phos-phatases. PP2 enzymes were subsequently divided into threegroups on the basis of the requirements for metal ions: 2A (notrequiring metal ions), 2B (activated by calcium), and 2C (Mg2�

dependent) (10). The molecular cloning of a number ofcDNAs and genes from diverse organisms allowed the estab-lishment of three major subfamilies: PPP (phosphoproteinphosphatases), including type 1, 2A, and 2B phosphatases,among others; PPM (metal-dependent protein phosphatases),including type 2C enzymes (PP2C), which are the subject of

this review; and the catalytically Asp-based subfamily, repre-sented by HAD and FCP/SCP (see reference 89 for a review).

Although PP2C proteins are distantly related in primarysequence to PPP enzymes, their tridimensional structure andcatalytic mechanisms appear to be quite similar. Type 2C phos-phatases are widely represented in bacteria, fungi, plants, in-sects, and mammals (83), suggesting that they are involved inkey cellular processes, such as proliferation, metabolism, andcell survival (11, 48). The understanding of the catalytic pro-cess for PP2C came from the determination of the crystalstructure of the human isoform PP2C� (12), which revealedkey Asp residues required for coordination of Mg2� or Mn2�

ions (mutation of these residues is usually performed to gen-erate catalytically inactive forms of PP2C). In sharp contrastwith PPP enzymes (particularly with PP1 phosphatases), inwhich the catalytic polypeptide interacts with a large numberof very different regulatory subunits that provide functionalspecificity, PP2Cs are normally monomeric enzymes. The myr-iad of functions performed by PP2C enzymes is possibly theresult of the expression of a large number of catalytic isoforms.For instance, at least 14 genes are found in humans, andprobably more than 20 different polypeptides are produced bysplicing mechanisms. A stunning situation is found in themodel plant Arabidopsis thaliana, in which near 80 PP2C pro-teins have been identified or predicted (46, 113). In manycases, the different isoforms have characteristic amino- or car-boxyl-terminal extensions (see below), which could provide thestructural basis for functional specificity, although little aboutthis aspect is known.

Although the reader is directed to recent reviews (48, 55, 83)of the role of PP2C enzymes in these organisms, a brief over-view is provided here for comparative purposes. Human PP2Cshave been implicated in many processes, such as regulation ofmitogen-activated protein kinase (MAPK) cascades (p38 orJun N-terminal protein kinase [JNK]), cell cycle progression,ubiquitination and degradation of proteins, and mechanismsfor cell death and survival (see references 48, 55, and 100 for

* Corresponding author. Mailing address: Departament de Bio-química i Biologia Molecular, Institut de Biotecnologia i Biomedicina,Universitat Autonoma de Barcelona, Cerdanyola del Valles 08193,Barcelona, Spain. Phone: 34-93-5812182. Fax: 34-93-5812011. E-mail:[email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 12 November 2010.

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detailed reviews). PP2Cs can act on several MAPK pathwaysand at different levels within the same pathway. For instance,PP2C� dephosphorylates and inactivates the p38 MAPK bothin vitro and in vivo but also dephosphorylates MKK3/6 (p38MAPKKs) as well as MKK4/7 (JNK MAPKKs) (33, 98). ThePP2Cε isoform negatively regulates both JNK and p38 signal-ing pathways, but in this case by acting on the MAPKKKsASK1 (80) and TAK1 (33). TAK1 (p38 pathway) is also de-phosphorylated and inhibited by the PP2C�-1 isoform (33).

PP2Cs play a role in regulating cell cycle progression sincePP2C� and PP2C�2 dephosphorylate the cyclin-dependent ki-nases Cdk2 and Cdk6 (7). The PP2C�/WIP1 isoform exertsinhibitory functions on several tumor suppressor pathways (in-cluding p53, ATM, p16 [INK4A], and p19 [ARF]), opposes therole of checkpoint kinases Chk1 and Chk2, and adversely af-fects base excision repair. Therefore, WIP1 is catalogued as anoncogene, and it has been proposed as a therapeutic targetagainst cancer and aging (see reference 49 for a recent review).In contrast, PP2C� and PP2C� increase the expression andactivity of p53 (48, 66), and therefore these two phosphataseshave been considered tumor suppressor proteins.

The role of PP2C enzymes in plants has been mainly ex-plored in A. thaliana. PP2Cs have been shown to act as nega-tive regulators in the abscisic acid (ABA) signaling pathway,which participates in the response to different environmentalstresses such as desiccation or high salinity. Examples are ABI1and ABI2, as well as A. thaliana P2C-Hab1 (AtP2C-Hab1) andAtPP2CA (see references 36 and 83 for reviews). It is note-worthy that Arabidopsis AP2C1 (which displays a high degreeof similarity to a PP2C from alfalfa called MP2C) inactivatesthe stress-responsive MAPKs MPK4 and MPK6 and modu-lates innate immunity and jasmonic acid and ethylene levels(84).

It must be stressed that in contrast to many protein kinases,which show a marked specificity for the residues surroundingtheir target, protein phosphatases in general and PP2C en-

zymes in particular do not exhibit significant site specificity.This prevents a simple, sequence-based approach for the de-termination of their protein and amino acid targets. In addi-tion, PP2C-focused research has been hampered by the lack ofspecific inhibitors against these enzymes.

PP2C IN SACCHAROMYCES CEREVISIAE

Even in a relatively simple eukaryotic organism such as theyeast Saccharomyces cerevisiae, there are several PP2C-encod-ing genes. Initially, the family comprised five isoforms calledPtc1 to -5 (for phosphatase two C) (8), although in the follow-ing years two more members were added, Ptc6 (77) and Ptc7(40) (Fig. 1). All these proteins share a conserved PP2C do-main, which can be accompanied by amino-terminal (Ptc5 andPtc7) or carboxyl-terminal (Ptc2 and Ptc3) extensions. As is thecase in higher eukaryotes, yeast PP2Cs were initially involvedin the regulation of cell growth and stress signaling. For in-stance, several PP2C isoforms were initially associated with thenegative regulation of the HOG (high-osmolarity glycerol) sig-naling pathway, inducible after osmotic stress, by dephosphor-ylating and inactivating the Hog1 MAPK. However, our cur-rent knowledge suggests that PP2C functions are much morediverse.

The Ptc1 phosphatase. The Ptc1 isoform is by far the bestcharacterized PP2C in yeasts. Although it shares some of itsfunctions with other members of the family, recent work hasshown that the transcriptional profile of ptc1 mutants growingunder standard conditions is markedly different from that ofptc2, -3, -4, and -5 strains, in a way that could not be anticipatedfrom primary structure comparisons (29). This observation re-inforces the notion that Ptc1 plays specific functional roles thatare not shared by other family members.

(i) The link between Ptc1 and the HOG pathway. PTC1 wasfirst identified in 1993 in a search for mutations that exacerbatethe growth defect of strains lacking the tyrosine phosphatase

FIG. 1. The type 2C protein phosphatase family members in Saccharomyces cerevisiae. A schematic representation of the primary structures ofthe seven PP2C isoforms is shown. The black lines represent each protein, and the blue boxes indicate the catalytic domain of each phosphatase(PP2C domain, SM00332, according to the SMART database [82]). The numbers included within the catalytic domain indicate the amino acidresidues spanning these domains. The number at the C terminus indicates the number of amino acids of the protein. “ORF” refers to the systematicname of the gene. Alternative denominations (aliases) and subcellular localization are indicated (Cyt, cytoplasm; Nuc, nucleus; Mit, mitochondria).Alternative splicing of YHR076W can generate two proteins, Ptc7u (unspliced) and Ptc7s (spliced), with different cellular functions (45). Theorange box denotes the transmembrane domain extending from residue 17 to 39 of the Ptc7u protein. Percentages of similarity and identity ofoverlapping regions of different PP2Cs in comparison to Ptc1 are shown (determined by FASTA using the BLOSUM50 matrix). The dendrogramon the right was generated using the entire protein sequences and the ClustalW algorithm using the default settings (Kyoto UniversityBioinformatic Center).

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Ptp1 or Ptp2 (57). Lack of PTC1 drastically affects growth of aptp2 mutant under normal conditions, which suggested thatboth phosphatases were acting on various substrates with re-dundant functions or on a single substrate phosphorylated atSer/Thr and Tyr residues (as in the case of a MAPK). In thissame study it was shown that the gene product had phos-phatase activity in vitro that required Mn2� or Mg2� ions. Oneyear later it was determined that the overexpression of PTC1 isable to rescue the lethal phenotype of an sln1 mutant, which isdue to hyperactivation of the HOG signaling pathway, and itwas suggested that Ptc1 negatively affects Pbs2, the MAPKkinase upstream of Hog1 (58). In an almost parallel work it wasshown that while the ptc1 mutant is tolerant to the effects ofkiller toxin K1, the pbs2 ptc1 double mutant is hypersensitive(similar to the case for pbs2 cells), thus establishing an epistaticrelationship between PTC1 and PBS2 (39). These authors alsoshowed that in cells lacking PTC1 intracellular glycerol lev-els are higher than in the wild type and that this phenotypeis also suppressed by the deletion of PBS2, suggesting thatthe ptc1 mutation may increase the activity of the HOGpathway (Fig. 2a).

Overall, these results provided genetic evidence placing Ptc1as a negative regulator of the HOG signaling pathway, a notionthat was reinforced by experiments with the fission yeastSchizosaccharomyces pombe, in which the orthologue ptc1�

gene was found to play a role in osmoregulation by counter-acting the effect of the Pbs2 orthologue MAPK kinase Wis1(see below) (90, 91). Later it was shown that the severe growthdefect of a ptc1 ptp2 mutant is indeed due to hyperactivation of

the HOG pathway and that this phenotype disappears whenPBS2 or HOG1 is deleted (38). Four years later it could bedemonstrated that, in vitro, Ptc1 dephosphorylates Hog1 atThr-174. This residue and Tyr-176 are located in the activationloop, and their phosphorylation is indispensable for the acti-vation of the MAPK. It was also shown that the phosphatase,which it is present in the nucleus and the cytoplasm, is neces-sary to regulate the Hog1 phosphorylation state in vivo, both atthe basal level and during adaptation to stress (108).

Large-scale experiments using the two-hybrid method re-vealed that Ptc1 physically interacts with Nbp2, a protein thatcontains an SH3 (Src homology 3) domain (37, 105). Remark-ably, Pbs2, which acts as an scaffold for various elements of theHOG pathway such as Hog1, Sho1, Ste11, and Ssk2/22 (70,101), had been copurified with Nbp2 and Ptc1. Thus, it wasdemonstrated that Ptc1 performs its functions on the HOGpathway through the adaptor protein Nbp2, whose N-terminaldomain is necessary for interaction with the phosphatase,while the SH3 domain is responsible for binding to Pbs2(61). As far as we know, this is the only example of theexistence of a targeting regulatory protein for a PP2C in S.cerevisiae (Fig. 2a).

(ii) Ptc1 and the CWI pathway. In addition to the relativelywell characterized role of Ptc1 in the regulation of the Hog1MAPK pathway, there is evidence linking this phosphatase toanother MAPK-regulated route, the cell wall integrity (CWI)pathway. Schematically, this pathway is composed of severalmembrane sensors that signal through the small GTPase Rho1to the Pkc1 kinase, which is the upstream component of the

FIG. 2. Functional role of PP2C enzymes in yeast MAPK pathway regulation and organelle inheritance. (a) Regulation of MAPK pathways.Different S. cerevisiae and S. pombe pathways are depicted, indicating the triggering stimuli and the transcription factors involved in the response.The three layers defining the MAPK pathways are shaded in gray. Discontinuous lines denote suspected or still poorly characterized interactions.See text for details. (b) The central role of Ptc1 and the different proteins presumed to mediate the phosphatase function on organelle inheritance.Note that only endoplasmic reticulum and mitochondrial inheritances seem to require Slt2 MAPK intervention.

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MAPK cascade that activates the Slt2/Mpk1 MAPK (see ref-erences 21 and 52 for reviews of this pathway). A search formutations that were able to suppress the thermosensitive phe-notype and the high rate of recombination of strains carryingthe hypoactive pkc1-4 allele yielded the KCS2 gene (for Pkc1-4suppressor), which turned out to be allelic with PTC1 (35).Since the lack of PTC1 compensates for reduced Pkc1 kinaseactivity, it could be proposed that Ptc1 might regulate thedephosphorylation of specific targets of Pkc1 or any member ofthe signaling pathway to which Pkc1 belongs (Fig. 2a). It mustbe noted that the ptc1 slt2 double mutant has been described assynthetic lethal (35), suggesting that Ptc1 might have addi-tional substrates that are relevant for cell wall integrity. How-ever, this synthetic phenotype is largely strain dependent. Forinstance, while the lethality of the slt2 ptc1 mutation was re-ported not to be alleviated by the addition of sorbitol to theculture medium (35), in a different genetic background com-monly used in our laboratory (JA100), addition of 1 M sorbitolallows survival (29). Furthermore, in both the S288C and itsderived BY4741 genetic backgrounds, the slt2 ptc1 strain isviable even in the absence of osmotic support (14, 29), al-though it is extremely sensitive to cell wall-damaging agents(29).

The relationship between Ptc1 and the CWI pathway isreinforced by a number of findings. For instance, one of thealiases of the PTC1 gene is CWH47 (for calcofluor white hy-persensitive), revealing that the gene deletion causes hyper-sensitivity to this chitin antagonist (73). Ptc1-deficient cells alsodisplay a phenotype characteristic of some strains with muta-tions of genes involved in cell wall synthesis: they are tolerantto the K1 killer toxin (39, 67). This might be due to a decreasein the amount of �-1,6-glucans, probably because of unusuallyhigh levels of Exg1 exo-�-glucanase activity (39). Furthermore,the ptc1 mutant is sensitive to caspofungin (62), caffeine (27),and alkaline pH (29, 87, 88), circumstances that activate theCWI pathway, and the ptc1 mutation is synthetically lethal withdifferent mutations in genes important for cell wall construc-tion, such as FKS1, GAS1, or SMI1 (51, 102). Furthermore, ithas been shown that cells lacking Ptc1 have larger amounts ofactive Slt2 (14, 29). In addition, mutation of PTC1 results inincreased expression of several genes normally induced by cellwall damage, such as YKL161c, CRH1, or SED1, and thisresponse is dependent on both the Slt2 MAPK module and thedownstream transcription factor Rlm1 (29). All these datareinforce the notion that lack of phosphatase mimics a situa-tion of cell wall damage. Although data collected in recentyears reveal the existence of a interaction between the Slt2 andHOG pathways (4, 24, 25, 32), it must be noted that thehypersensitivity of the ptc1 strain to cell wall-damaging agentsis not attributable to a hyperactivation of Hog1, since a ptc1hog1 double mutant still displays a strong sensitivity to cellwall-damaging agents (29). Similarly, a lack of Nbp2 does notimprove tolerance of the ptc1 strain to calcofluor white orcaffeine (29).

(iii) Ptc1 and cation homeostasis. It has been described thatptc1 cells are highly sensitive to calcium ions (29, 31, 35) andthat this is most probably due to hyperactivation of the phos-phatase calcineurin, because sensitivity is largely abolished bydeletion of the CNB1 gene (29). Similarly, the ptc1 mutant isalso sensitive to amiodarone, an antifungal agent that causes a

dramatic increase in cytoplasmic calcium concentrations (31).In addition, deletion of PTC1 renders cells sensitive to metalssuch as zinc and cesium (29) and, as mentioned above, toalkaline pH. It is worth noting that many of these traits arefound in certain mutants with impaired vacuolar function. Re-markably, the ptc1 mutant displays fragmented vacuoles, mim-icking those of class B vps (vacuolar protein-sorting) mutants(5, 29, 96). Several genome-wide studies have identified Ptc1 asa protein required for maintaining the structure and functionof the vacuole (5, 81, 85), although there is some controversyabout the ability of ptc1 mutants to correctly process car-boxypeptidase Y (CPY). While some reports propose that pro-cessing is not correct (5, 85), other studies have shown normalCPY processing (3, 29). It must be noted that vacuolar mal-function does not always result in deficient CPY processing(85). A further link between Ptc1 and vacuolar function camefrom the finding that overexpression of VPS73, a gene of un-known function involved in vacuolar protein sorting, largelyrescues not only vacuolar fragmentation but also sensitivity tocell wall-damaging agents, high calcium levels, and alkalinepH, as well as other ptc1-specific phenotypes (29). These find-ings prompted the authors to hypothesize that lack of Ptc1would primarily cause vacuolar malfunction, from which otherphenotypes of the mutant would derive.

Deletion of PTC1 confers a lithium (but not sodium)-sensi-tive phenotype that is not shared by other members of thePP2C family (78). A ptc1 mutant is less effective in extrudingLi� and accumulates higher concentrations of this cation, as aconsequence of decreased expression of the Na�-ATPaseENA1 gene. This effect is not attributable to the role of Ptc1 inHog1 regulation, but instead a number of observations suggestthat Ptc1 may regulate the Hal3/Ppz1,2 pathway (13). Forinstance, lack of Hal3 provokes a decrease in ENA1 expressionsimilar to that produced by mutation of PTC1, and the effectsare not additive. Moreover, blocking the mechanism for Ppz-mediated activation of ENA1 (i.e., simultaneous deletion ofthe Trk1,2 potassium transporters and chemical inhibition ofcalcineurin) does not further increase sensitivity of the ptc1strain to lithium (78). How Ptc1 may interact with the Hal3/Ppzsystem is still unknown. It has been recently reported that theprotein kinase Hal5, which is involved in regulation of potas-sium influx, is a multicopy suppressor of the lithium-sensitivephenotype of the ptc1 mutant, but the molecular basis of thiseffect remains to be elucidated (116).

(iv) Ptc1 and the inheritance of cellular organelles. Work inthe past few years has focused on an intriguing role of Ptc1: theinheritance of different organelles, such as mitochondria, vacu-oles, and the endoplasmic reticulum (ER) (Fig. 2b). Cells lack-ing Ptc1 exhibit a marked defect in cortical ER inheritancecaused by a delay in the last step of this process (14), and theptc1 mutation displays strong genetic interactions with muta-tions in SEC3 or AUX1/SWA2, encoding proteins required forcortical ER inheritance (the ptc1 sec3 double mutant shows asevere growth defect, whereas ptc1 aux1 strains are not viable).The absence of the adaptor protein Nbp2 leads to a phenotypesimilar to that of the ptc1 mutation, suggesting that both pro-teins together regulate the inheritance of this organelle. Re-markably, although this phenotype seems to be unrelated tothe HOG pathway (14), both the addition of sorbitol and theinactivation of Slt2 solve the defect in ER inheritance in the



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ptc1 mutant, indicating that activation of the CWI pathway isprobably the cause of this defect in ptc1 cells (14). In thisregard, recent studies have shown that Ptc1 is necessary toinactivate the pool of Slt2 associated with the bud tip thatpromotes the cortical distribution of the ER in daughter cells(53). Moreover, deletion of SPA2, a component of the polari-some that is required for recruitment of Slt2 to the bud tip,suppresses the ER inheritance defect of ptc1 cells (53). Thisdefect is also suppressed by mutation of Mss4, a phosphatidyl-inositol-4-phosphate 5-kinase that plays a predominant role inthe heat shock-induced activation of Slt2. This effect is prob-ably due to the drastically reduced Slt2 phosphorylation levelsobserved in the ptc1 mss4-102 mutant (53).

In wild-type strains, mitochondrial inheritance and budemergence are concomitant processes. However, Roeder andcolleagues found that cells lacking Ptc1 produce buds that areinitially devoid of the mitochondrial compartment (76). Thesesame authors ruled out an involvement of the HOG pathway.However, a recent report shows that the mitochondrial inher-itance defect of ptc1 cells is substantially suppressed in the ptc1slt2 double mutant (53), indicating a role of the Slt2 kinase inthis process. It should be noted that, unlike what happens tothe ER inheritance, in the case of mitochondrial inheritancethe relevant pool of Slt2 is not located at the bud tip and isapparently independent of Mss4 function (Fig. 2b). This dis-tinction makes sense in light of the different stages at whichinheritance of the ER and mitochondria are blocked in ptc1cells. Further links between Ptc1 and mitochondrial functioncome from the observations that in a gem3 background (lack-ing a protein of the Rho family needed to maintain mitochon-drial tubular morphology), deletion of PTC1 causes significantgrowth defects (20). Mitochondrial instability of the ptc1 mu-tant has been proposed as the reason for its sensitivity to theantitumor drug doxorubicin (111).

A recently published study has shown that Ptc1 comple-ments the defects of a vac10-1 mutant, which was identifiedafter a search for strains defective in vacuolar inheritance (43).This strain turned out to contain a mutation that eliminates thecatalytic activity of Ptc1, indicating that Ptc1 phosphatase ac-tivity is necessary to ensure proper vacuolar inheritance. Inlight of the available genetic data, it seems that the mechanismby which Ptc1 controls the movement of the vacuoles from themother to the daughter cell does not involve the HOG pathwayor the CWI pathway (14, 43).

Besides affecting vacuole, mitochondrion, and cortical ERinheritance, the lack of PTC1 alters the inheritance of otherorganelles such as peroxisomes and secretory vesicles (43). Allthese organelles have a common characteristic: they are trans-ported by Myo2 or Myo4, the twin engines of class V myosin.The ability of these proteins to direct multiple organelles todifferent cellular locations is based primarily on the existenceof specific receptors for each organelle. In this regard, it hasbeen proposed (Fig. 2b) that Ptc1 affects the stability of severalof these receptors, such as Vac17, Inp2, and Mmr1, which linkMyo2 to the vacuole, peroxisomes, and mitochondria, respec-tively (43). This would explain a number of phenotypes asso-ciated with the absence of PTC1, placing this phosphatase as akey element in controlling the distribution of cellular or-ganelles (16, 43).

(v) Other phenotypes caused by the mutation of PTC1. Inaddition to the phenotypes described so far, the Ptc1 phos-phatase has been implicated in several processes, although inmost cases the mechanism of action remains unclear. Yearsago, a search for mutants that were unable to correctly processtRNAs allowed the identification of the TPD1 gene (for tRNAprocessing defective), which was later found to be allelic toPTC1, thus implicating the phosphatase in tRNA splicing (75).This same work also revealed other alterations in ptc1 cells; forinstance, they are unable to grow in media containing nonfer-mentable compounds such as lactate, ethanol, or glycerol as acarbon source (this was subsequently confirmed in an indepen-dent study [76]). This defect may be related to the vacuolardefects described above. In addition, ptc1 cells show decreasedefficiency of sporulation and exhibit major defects in cell sep-aration in diploids (especially at 37°C). More recently, it hasbeen shown that haploid ptc1 cells also display cell separationdefects and a random budding pattern at 37°C (29).

The early observations that cells lacking Ptc1 are sensitive torapamycin and caffeine (29, 68, 77, 112) pointed to the possibleexistence of a functional connection between Ptc1 and theTOR pathway. In this regard, it has been recently shown thatPtc1 is required for normal TOR signaling, possibly by regu-lating a step upstream of the type 2A-related Ser/Thr phos-phatase Sit4 in a way independent of the HOG pathway (28).This finding provides the first evidence that a PP2C is linked tothis important and conserved pathway. Remarkably, mutationsin components of the Slt2 MAPK pathway alleviate to a certainextent the rapamycin-sensitive phenotype of ptc1 cells (ourunpublished observations). This result suggests that it would beworth revisiting the link between the TOR and CWI pathways.

Work recently published by Malleshaiah and coworkershighlights the role of Ptc1 in the regulation of the mechanismthat controls the switch-like mating decision (59). It is wellknown that pheromones activate a MAPK cascade that leadsto phosphorylated (active) Fus3, which dissociates from Ste5and phosphorylates downstream targets to mediate mating (fora review, see reference 6). Interestingly, deletion of PTC1substantially prevents shmooing and reduces activation ofFus3, whereas the overexpression of PTC1 has the oppositeeffect. The authors also demonstrated that Ptc1 is responsiblefor the dephosphorylation of four sites on Ste5, a requisite forfull relief and dissociation of the Fus3-Ste5 complex, whichleads to the mating response (Fig. 2a).

Deletion of PTC1 increases sensitivity to a number of agents,such as sorbate, a weak organic acid used to preserve food (64),or oleate (54). Since sensitivity to oleate is typical (though notexclusive) of cells with impaired peroxisomal function, it couldbe explained by the defects in peroxisome inheritance men-tioned above. A ptc1 mutant is also slightly sensitive to linoleicacid hydroperoxide, an oxidizing compound produced by lipidperoxidation that causes a delay in the G1 phase of the cellcycle. It has been speculated that the mechanism by whichPTC1 may be involved in the tolerance to this agent wouldinvolve Mms22, a protein required for resistance to ionizingradiation, although a possible role for the HOG pathway hasnot been discarded (19). Furthermore, the toxicity of oxalateincreases in cells lacking PTC1. In this case, it has been pos-tulated that the HOG pathway could be involved in transduc-tion of signals caused by oxalate stress (9). Interestingly, PTC1



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is a weak multicopy suppressor of the lethal phenotype of a flc1flc2 double mutant. Flc1 and Fcl2 are two flavin carriersneeded to import flavin adenine dinucleotide (FAD) into theendoplasmic reticulum. The reason why PTC1 overexpressionimproves growth in a flc1 flc2 strain could be attributed to thelink between Ptc1 and the CWI route (71). Finally, a genome-wide screen for mutants with affected telomere length revealedthat ptc1 cells display shorter telomeres than the correspondingwild-type strain (1).

The Ptc2, Ptc3, and Ptc4 phosphatases. Ptc2 and Ptc3 aretwo proteins that are 62% identical (77% similar) (8, 57) andwhose origin could be due to a genomic duplication event(110). These two proteins differ structurally from Ptc1 becausethey possess a carboxyl-terminal extension of around 170 res-idues (Fig. 1) that, in the case of Ptc2, is necessary to achievemaximum activity (114). Ptc2 and Ptc3 are unable to fulfill therole of Ptc1 in the HOG pathway (57). However, these twophosphatases are also involved in the regulation of the Hog1MAPK (Fig. 2a), but with a slightly different role than Ptc1,since in this case they serve to limit the maximum of activationof the HOG pathway after stress (114). Unlike Ptc1, Ptc2 andPtc3 do not interact with Nbp2 (61).

Ptc2 also regulates negatively the unfolded-protein responsein the endoplasmic reticulum through dephosphorylation ofthe Ser/Thr protein kinase Ire1 (109), which is necessary toinduce the appropriate transcriptional response. Ptc2 physi-cally interacts with Ire1 through a region, located in the centralpart of the phosphatase, comprising part of the catalytic andcarboxyl-terminal segments (between amino acids 174 and355). Ptc2 is also necessary to preserve cell viability in the faceof agents that compromise the integrity of DNA (63). In thepresence of such agents or under replicative stress, DNAcheckpoints are activated to maintain genomic stability. Spe-cifically, the S-phase checkpoint generates a cellular responsethat halts the cell cycle through a mechanism involving phos-phorylation and activation of the protein kinase Rad53. PTC2overexpression confers sensitivity to genotoxic agents such ashydroxyurea or UV radiation (63) and allows growth of strainscarrying a dominant lethal allele of Rad53. Similarly, ptc2mutants are sensitive to selenite, a compound that inducesDNA damage, probably by inducing double-strand break (86).It has been proposed that both Ptc2 and Ptc3 are necessary forthe inactivation of Rad53 by dephosphorylation after a double-strand break of DNA induced by HO endonuclease (50), al-though there is no evidence for a direct in vitro effect. It hasbeen shown that Rad53 physically interacts with Ptc2 and thatThr-376 (located in the carboxyl terminus outside the phos-phatase catalytic domain) is important for this binding and forfunction in DNA damage recovery (30). That study also dem-onstrated that Ckb1 and Ckb2, the regulatory subunits of ca-sein kinase 2 (CK2), are necessary for the interaction betweenRad53 and Ptc2, and it is suggested that CK2 could act byphosphorylating Thr-376 in Ptc2. However, recent studies haveshown that Ptc2 and Ptc3 are dispensable for Rad53 deactiva-tion after replicative stress or after DNA damage by methyl-ation, and it has been proposed that the inactivation of Rad53would require the presence of various phosphatases in re-sponse to different types of stress (95, 103). A possible role ofPtc2 and Ptc3 in the recently described mechanism of down-

regulation of the damage checkpoint exerted by the polo-likeprotein kinase Cdc5 has been ruled out (106).

In addition, Ptc2 and Ptc3 and have been implicated in theregulation of progression through the cell cycle (8) since theyare capable of dephosphorylating Cdc28 at Thr-169, a residueessential for its activity as a cyclin-dependent kinase (CDK).This function can be extrapolated to higher eukaryotes since,as mentioned above, in humans the PP2C�2 isoform is able todephosphorylate Cdk2 and Cdk6 (7).

The phosphatase activity of Ptc4 was demonstrated byCheng and collaborators through in vitro experiments usingcasein as a substrate (8). Ptc4 is a cytoplasmic protein whoseoverexpression reduces both the level of phosphorylation andthe activity of Hog1 and also suppresses the lethality of a cnb1slt2 double mutant (PTC1, PTC2, and PTC3 are also able to doso) (92). The latter can be explained by the ability of thesephosphatases to inhibit the HOG pathway, since deletion ofHog1 also rescues the lethality of the cnb1 slt2 strain.

The Ptc5, Ptc6, and Ptc7 phosphatases. Little is knownabout the functions of Ptc5. In 1999, Cheng and colleaguesverified in vitro its phosphatase activity (8). Subsequently, itwas reported to have a mitochondrial localization and, to-gether with Ptc6, to dephosphorylate Ser-133 of Pda1, the E1�subunit of the pyruvate dehydrogenase (PDH) complex thatcatalyzes the oxidative decarboxylation of pyruvate to formacetyl coenzyme A (acetyl-CoA) (26, 47). This justifies Ppp1(PDH protein phosphatase 1) as an alias for Ptc5.

It was presumed for many years that the protein encoded byYCR079w was a type 2C phosphatase (94), although the firstattempt to demonstrate the phosphatase activity of the recom-binant protein was unsuccessful (8). It was only recently thatRuan and colleagues could demonstrate that Ycr079w displaysthe typical characteristics of a PP2C, and they renamed theprotein Ptc6 (77). Ptc6 is located both in the intermembranespace and in the mitochondrial matrix (26, 99) and, as men-tioned above for Ptc5, regulates the phosphorylation state ofPda1 (and consequently received the alias of Ppp2) (26). It hasbeen shown that Ptc6 is necessary for survival of stationary-phase cells, and it has recently been found to be involved,probably through the Rtg3 transcription factor, in the mito-chondrial degradation process known as mitophagy. The latterphenotype justifies its alias of Aup1 (autophagy-related proteinphosphatase) (44, 99). It is worth noting that although ptc5 andptc6 phenotypes largely overlap (our unpublished results), alack of Ptc6 (but not of Ptc5) results in sensitivity to rapamycin(28, 77), suggesting distinctive functions for Ptc6.

The biochemical characteristics of Ptc7 were determined in2002 (40). Similarly to Ptc5 and Ptc6, this phosphatase wasinitially located in the mitochondria (74). According to theseauthors, strains lacking Ptc7 do not differ from the wild type ingrowth rate or morphological characteristics. However, over-expression of PTC7 improves growth when ethanol or galac-tose is used as a carbon source in a low-oxygen environment. Itis noteworthy that the expression of PTC7 increases after os-motic stress in a Hog1-dependent manner (79). The existenceof a functional intron in the PTC7 gene has recently beendiscovered (45). As a result, depending on the processing ofthe intron, the gene can produce two mRNAs that encode twodifferent proteins (Fig. 1). Remarkably, they are located indifferent cellular compartments and play different roles. The



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protein translated from the smaller RNA, called Ptc7s, is lo-cated in mitochondria, and its expression depends on thesource of carbon in the growth medium. On the other hand,the translation of the full mRNA leads to the Ptc7u protein,which contains a transmembrane domain, is located in thenuclear envelope, and mediates the effects caused by the toxinlatrunculin A, a compound that affects actin polymerization(45).

PP2Cs IN OTHER FUNGI

Although early work on the fission yeast Schizosaccharomy-ces pombe shed light on the functional role of PP2C in fungi(see below), most information in the last 15 years came fromthe investigation of these enzymes in budding yeast. However,in the last few years, several laboratories have undertaken thestudy of type 2C phosphatases in organisms other than S.cerevisiae. Before initiating a review of the data available in theliterature, we would like to take advantage of the large amountof genomic data currently accessible to provide an overview ofthe existing PP2C enzymes in fungi. A total of 144 sequencesfrom 22 different fungi were selected after a search for simi-larity to the seven PP2C protein sequences from S. cerevisiae atthe NCBI database. The classification of these proteins accord-ing to their primary structure shows that there are five majorgroups of PP2Cs in fungi (Fig. 3a; see Fig. S1 in the supple-mental material). One group is formed by the family of pro-teins related to S. cerevisiae Ptc1, which is present in all ana-lyzed organisms (Fig. 3b). A second group includes the relatedPtc2/Ptc3 and Ptc4 proteins. Interestingly, Ptc4 proteins arepresent in most species of the Saccharomycotina subphylum

but are absent in the rest of the organisms analyzed (Fig. 3b).Another group includes Ptc5-related proteins, which arepresent in all organisms studied. The fourth group contains alarge family of proteins related to S. cerevisiae Ptc6. This in-cludes two subfamilies. One corresponds to the typical Ptc6,which is found in all members of the Saccharomycotina sub-phylum investigated here. Therefore, this distribution almostoverlaps with that of Ptc4, with the exception of Yarrowialipolytica. The second is a branch of Ptc6-related proteins thatis absent only in Saccharomycotina. A relevant member of thisfamily is the Ptc6-related protein of S. pombe named Ptc4 (no.76 in Fig. S1 in the supplemental material) (see below). It isworth noting that almost all analyzed species that contain aPtc4 protein also contain a member of the “classical” Ptc6.Furthermore, all species in which the “classical” Ptc6 is absentdo contain one or more representatives of the Ptc6-relatedfamily. Finally, a large group of proteins with similarity to S.cerevisiae Ptc7 can be found, and these include two families ofproteins: a family of “typical” Ptc7s, present in all organismsanalyzed, and a group of proteins, here named the Ptc7-relatedproteins, that is absent in Saccharomycotina except in thosespecies belonging to the CTG clade (organisms that translatethe CTG codon as serine instead of leucine). This group in-cludes the recently described Ptc8 protein from Candida albi-cans (no. 121) (17). Interestingly, many Ptc7 (but not Ptc7-related) proteins seem to include a transmembrane domain atthe N terminus.

From this analysis it is worthwhile to highlight the followingideas. (i) Yarrowia lipolytica seems to encode two distinct Ptc1proteins (no. 1 and 15 in Fig. S1 in the supplemental material).

FIG. 3. The diversity of PP2Cs in fungi. The NCBI protein database was searched for PP2C-related proteins using the BLAST algorithm andthe seven PP2C sequences from S. cerevisiae. Hits were manually curated, and 144 protein sequences were selected. (a) A ClustalW2 alignmentwas performed, and the results are displayed as a radial tree using the TreeView software. Branches were manually labeled. Ptc6-R, Ptc6-relatedfamily; Ptc7-R, Ptc7-related family. (b) Fungal phylogeny is represented as a tree based on data from reference 15, with some modifications. Thepresence (numbers) or absence (gray boxes) of the indicated PP2C proteins in each organism is represented. The numeric code is described in thelegend to Fig. S1 in the supplemental material, where the accession numbers for each sequence can be found.



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One of them (no. 1) is a protein of 864 amino acids thatpossesses a long (over 400-amino-acid) carboxyl-terminal ex-tension. In this regard, it is worth noting that within the Sac-charomycotina subphylum, Yarrowia lipolytica is an early-di-verging lineage. This may also explain why this species alsolacks Ptc4, which is common to other members of Saccharo-mycotina. (ii) Whereas Ptc7 is present in all organisms ana-lyzed, within the Saccharomycotina, only Yarrowia lipolyticaand the species included in the CTG clade contain Ptc7-relatedproteins. The latter are also missing in Ustilago maydis andSchizosaccharomyces pombe. (iii) Whereas Ptc6 is a family ofproteins found exclusively in species included in the Saccharo-mycotina lineage, Ptc6-related proteins are absent in this lin-eage. (iv) As a conclusion, members of the Ptc1, Ptc2/3, Ptc5,and Ptc7 families are present in all analyzed organisms.

PP2C in Schizosaccharomyces pombe. In 1994, Shiozaki andcoworkers (90) isolated the ptc1� gene (no. 2 in Fig. S1 in thesupplemental material) as a multicopy suppressor of swo1-26,a temperature-sensitive mutation of a gene encoding a closelyrelated homologue to the heat shock protein Hsp90. The ptc1�

gene product is a 40-kDa protein that shares 35% identity withS. cerevisiae Ptc1. Biochemical analyses showed that it displaysMg2�-dependent phosphatase activity using casein as sub-strate. The ptc1 mutant has a normal growth rate and conju-gates and sporulates normally, although it is sensitive to ele-vated temperatures. Furthermore, ptc1� mRNA levelsincrease 5- to 10-fold during heat shock (35°C), suggesting thatPtc1 activity is important for survival under this condition (90).The authors demonstrated that a strain lacking ptc1� hasnearly normal levels of PP2C activity, thus suggesting the pres-ence of multiple PP2C genes in fission yeast. In fact, 1 yearlater the same group (91) isolated and characterized two ad-ditional PP2C genes from S. pombe: ptc2� and ptc3� (no. 25and 24, respectively). Together, Ptc1, Ptc2, and Ptc3 accountfor most of the PP2C activity (approximately 90%) in S. pombecells. The ptc2 and ptc3 mutants are viable and no obviousphenotypes are observed upon deletion of these genes. How-ever, a ptc1 ptc3 double mutant displays aberrant cell morphol-ogy (an increased number of swollen and deformed cells) andtends to lyse at elevated temperatures, defects that are exac-erbated by deletion of ptc2�. Remarkably, these phenotypesare almost completely suppressed by addition of osmotic sta-bilizers such as sorbitol or by inactivation of components of thestress-activated protein kinase (SAPK) pathway, such as Wis1or Spc1/StyI. Wis1 is the MAPK kinase that phosphorylatesThr-171/Tyr-173 of the MAPK Spc1/StyI, whose orthologues inS. cerevisiae are Pbs2 and Hog1, respectively (Fig. 2a). Theseresults reinforced the notion that PP2Cs negatively regulatethe Wis1-Spc1 kinase cascade, possibly via direct dephosphor-ylation of Wis1 and/or Spc1 (91). Furthermore, Gaits andcoworkers reported that the ptc1 ptc3 double mutation shows asynthetic lethal phenotype with pyp1. Pyp1 is a tyrosine phos-phatase that dephosphorylates Tyr-173 of the Spc1 kinase.Interestingly, lethality is rescued by the wis1 mutation (23).Subsequently, it was shown that Ptc1 and Ptc3 (but probablynot Ptc2) dephosphorylate Thr-171 in Spc1 both in vivo and invitro (65), thus reinforcing the role of PP2C activity in atten-uating the heat shock-activated Spc1 kinase. It should benoted, however, that Gaits and coworkers (23) presented sev-eral pieces of evidence indicating that PP2Cs also regulate

events downstream from the MAPK Spc1 and/or additionalMAPK signaling pathways.

Although ptc1 ptc3 or ptc1 ptc2 ptc3 mutants display noapparent significant defect in cell wall composition or structure(91), it has been reported that Ptc1 (but not Ptc2 or Ptc3)controls the activity of the MAPK Pmk1/Spm1 (56). Pmk1 is astructural homologue to Slt2 from S. cerevisiae, and it is in-volved in the maintenance of cell integrity, as well as regulationof morphogenesis, cytokinesis, and ion homeostasis. Pmk1 be-comes phosphorylated and subsequently activated under mul-tiple stresses, including cell wall-damaging compounds, hyper-or hypotonic conditions, glucose deprivation, and oxidativestress (Fig. 2a). Ptc1 copurifies with Pmk1 and dephosphory-lates the active protein kinase both in vivo and in vitro. Anadditional link between type 2C phosphatases and the CWIpathway regulation was established when Takada and cowork-ers reported that Ptc1 and Ptc3 modulate Pmk1 phosphoryla-tion levels induced by the cell wall-damaging agent micafungin(97). These authors also showed that Pmk1 regulates thesePP2Cs at the transcriptional level through Atf1, a transcriptionfactor downstream of the Spc1 MAPK pathway. From the datadescribed so far, it is evident that despite the fact that buddingand fission yeasts largely diverge in evolution, Ptc1 and/orPtc2/3 phosphatases have maintained an important role in theregulation of diverse MAPK signaling pathways in both organ-isms.

As mentioned above, the S. pombe ptc1 ptc2 ptc3 mutant isviable and retains approximately 10% of the PP2C activitymeasured in extracts from wild-type cells (91). In an effort toidentify new PP2C genes, Gaits and Russell performed aBLAST search on the portion of the S. pombe genome se-quenced at that time, using as bait the sequences of Ptc1, Ptc2,and Ptc3 (22). The analysis yielded a gene that was namedptc4�, encoding a protein with an estimated molecular mass of42 kDa that displays the classical Mg2�-dependent phos-phatase activity in vitro. It must be noted that this protein doesnot correspond to the S. cerevisiae Ptc4 isoform, but it belongsto the Ptc6-related branch (no. 76 in Fig. S1 in the supplemen-tal material). Cells lacking ptc4� are viable but grow slowly onminimal medium and undergo premature growth arrest in re-sponse to nitrogen starvation. The phenotype of ptc4 cells wasreminiscent of the growth delay and sterility observed in theautophagy-defective mutants of S. cerevisiae (104). Interest-ingly, when grown in minimal medium, the ptc4 strain displaysa large number of highly fragmented vacuoles, closely relatedto the class B mutants (2). Furthermore, ptc4 cells are unableto undergo vacuole fusion in response to hypotonic stress ornutrient starvation. Since vacuolar function is required for theprocess of autophagy, the vacuolar defect of ptc4 mutantsmight explain why these cells undergo premature growth arrestin response to nitrogen starvation. It is remarkable that Ptc4localizes in vacuolar membranes, which suggests that this phos-phatase regulates vacuole fusion by dephosphorylation of oneor more proteins in the vacuole membrane. Conversely, Ptc4overexpression appears to induce vacuole fusion (22).

PP2C in Candida albicans. Relatively little is known regard-ing PP2C enzymes in the human opportunistic pathogenicyeast Candida albicans, and most available information hasbeen accumulated in the last 2 years. The first evidence of C.albicans PP2Cs dates from the early 2000s, when Jiang and



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coworkers (41) searched for potential homologous sequencesfrom the public Candida genome database using the known S.cerevisiae PP2C genes as queries. According to their sequencesimilarities, they identified six sequences related to buddingyeast PP2C: C. albicans PTC1 (CaPTC1), CaPTC2/3, CaPTC4,CaPTC5, CaPTC6, and CaPTC7. A seventh member, CaPTC8,has recently been added to the list (17).

The first C. albicans PP2C characterized in some detail wasCaPtc7. Jiang and coworkers (41) demonstrated that CaPtc7has the biochemical characteristics of classical PP2C enzymes(i.e., Mn2�- and Mg2�-dependent in vitro phosphatase activityblocked by NaF but not sensitive to okadaic acid). The N-terminal region of CaPtc7 contains a transmembrane domainand a potential mitochondrion-targeting signal that justifies itspresence in this organelle (41, 107). Homologues of CaPtc7can be found in S. cerevisiae (Fig. 1), S. pombe, Caenorhabditiselegans, and A. thaliana, indicating that this phosphatase isconserved in eukaryotes. In addition, the catalytic domain ofCaPtc7 displays an overall 33% sequence identity with theAzr1 protein of Schizosaccharomyces pombe (41) (no. 144 inFig. S1 in the supplemental material). The azr1� gene wasoriginally isolated as a multicopy suppressor of the 5-azacyti-dine sensitivity of G2 checkpoint- and DNA repair-deficient S.pombe strains (69), but the phosphatase activity of Azr1 hasnever been demonstrated. The expression of the CaPTC7 geneis developmentally regulated at both the transcriptional andprotein levels during serum-induced morphogenesis, suggest-ing that CaPtc7 might be an important regulator of morpho-genesis of this organism. However, disruption of CaPTC7 doesnot affect vegetative growth or filamentous development in C.albicans (107). Therefore, the biological function of this phos-phatase still remains unknown.

The CaPTC1 gene (no. 10 in Fig. S1 in the supplementalmaterial) encodes a putative protein phosphatase (to ourknowledge, the activity of the protein has never been deter-mined) which shares 52% identity with the S. cerevisiae Ptc1protein (34). The homozygous null mutant is viable, but, incontrast to what is known for the S. cerevisiae ptc1 strain, it isnot sensitive to calcofluor white, Congo red (indeed, it showsgreater tolerance to Congo red than the wild-type strain),alkaline pH, or high temperature. On the other hand, it showsincreased sensitivity to the echinocandin-derived antifungalscaspofungin and micafungin, which interfere with the synthesisof glucan at the cell wall (34). Interestingly, CaPtc1 may con-tribute to the pathogenicity of C. albicans, since deletion of theCaPTC1 gene significantly reduces the virulence of this organ-ism in both silkworm and mouse models of disseminated can-didiasis (34).

CaPtc4 (no. 57 in Fig. S1 in the supplemental material)shares 31% sequence identity with S. cerevisiae Ptc4 (ScPtc4),and it has only recently been characterized (117). Bioinfor-matic analyses indicate that CaPtc4 has a potential myristoyl-ation site at the N terminus which is conserved in its sequencehomologues of other fungi (i.e., S. cerevisiae, Candida dublini-ensis, Pichia stipitis, and Kluyveromyces lactis). The purifiedPP2C-like catalytic domain of CaPtc4 exhibits a typical PP2Cactivity toward synthetic peptides phosphorylated on threonineresidues (117). Although CaPtc4 lacks a mitochondrion-target-ing sequence, Zhao and coworkers showed that a CaPtc4-green fluorescent protein (GFP) fusion is present mostly in this

organelle. The homozygous Captc4 mutant displays sensitivityto sodium and potassium ions and to antifungal drugs such asfluconazole and ketoconazole (117).

In a recent report, it has been demonstrated that the cata-lytic domain of CaPtc6 (no. 70 in Fig. S1 in the supplementalmaterial) displays phosphatase activity (115). The protein islocated in mitochondria, in accordance with the presence of amitochondrion-targeting sequence at the N terminus. Al-though the Captc6 mutant is not sensitive to rapamycin orcaffeine, the introduction of CaPtc6 into the S. cerevisiaeBY4743 ptc6/ptc6 diploid strain significantly reverses the sen-sitivity to both compounds, suggesting that CaPtc6 could be afunctional homologue of ScPtc6 (115).

A bioinformatic search in the public C. albicans databaseusing the CaPtc7 protein sequence as a query revealed theexistence of a protein that shows 38% sequence identity toCaPtc7. This protein contains an established PP2C domainthat exhibits a low degree of identity (14 to 19%) to othermembers of the C. albicans PP2C family (17). The protein wastherefore designated CaPtc8 (no. 121 in Fig. S1 in the supple-mental material). As indicated by these authors, no evidentsequence homologue of CaPtc8 exists in S. cerevisiae. Accord-ing to our similarity analysis, CaPtc8 could be included in thePtc7-related subfamily (see above). However, CaPTC8-likegenes appear to be evolutionarily conserved, since homologuesin Arabidopsis thaliana, Oryza sativa, or Drosophila melano-gaster can be found (17). The transcription of CaPTC8 is in-duced by several stress conditions, such as high osmolarity,temperature, and serum stimulation, whereas deletion ofCaPTC8 causes cells to be defective in hypha formation, indi-cating that CaPTC8 is required for the yeast-hypha transition(17).

The last member of the C. albicans PP2C family that hasbeen characterized is the product of CaPTC2 (no. 37 in Fig. S1in the supplemental material), whose phosphatase activity hasbeen confirmed (18). CaPtc2 is predominantly cytosolic, al-though it is also associated with the mitochondria, and it has apotential myristoylation site located at the N terminus, sug-gesting a possible anchorage to membranes. The expression ofCaPTC2 is repressed during the early steps of hypha formationand then induced in mature hyphae. Deletion of CaPTC2yields C. albicans cells that are sensitive to azole antifungalssuch as fluconazole and ketoconazole and to the detergentSDS. A Captc2 mutant also displays hypersensitivity to thegenotoxic stress-inducing agents methyl methanesulfonate andhydroxyurea, suggesting that CaPtc2 could share a functionalrole with S. cerevisiae Ptc2 and Ptc3. However, expression ofScPTC2 or ScPTC3 from its own promoter was not able tocomplement the hypersensitivity of the Captc2 mutant to thementioned genotoxic drugs.

The product of the CaPTC5 gene, which contains a pre-dicted transmembrane domain between residues 64 and 86,remains uncharacterized to date.

PP2C in Fusarium graminearum. Fusarium graminearum(also known as Gibberella zeae) is the major causal agent ofFusarium head blast disease on wheat and barley, which causesyield losses and affects the quality of grains. Using ScPtc1 as aquery, Jiang et al. identified a single homologue sequence,encoded by a gene named FgPTC1, in the genome of F. gra-minearum (42). FgPtc1 (no. 16 in Fig. S1 in the supplemental



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material) displays PP2C activity in vitro on a synthetic phos-phopeptide, and deletion of FgPTC1 causes the mycelium of F.graminearum to become sensitive to 0.2 M LiCl but not to 1 MKCl or 1 M NaCl. These results are consistent with the role ofS. cerevisiae PTC1 in lithium toxicity (78). In addition, FgPTC1complemented the function of ScPTC1 in lithium toxicity aswell as tolerance to Congo red, CaCl2, and ZnCl2, albeit notthat to alkaline pH. Therefore, FgPtc1 can be considered afunctional homologue of ScPtc1. Remarkably, deletion ofFgPTC1 attenuates the virulence of F. graminearum on wheat,suggesting that FgPTC1 plays an important role in regulatingthe hyphal growth and virulence of F. graminearum.

PP2C in Aspergillus nidulans. Our knowledge about PP2Cisoforms in Aspergillus nidulans is very scarce. Recently, Sonand Osmani carried out a systematic gene deletion analysis of28 identified phosphatase catalytic subunit-encoding genes (in-cluding six predicted PP2C-encoding genes) and showed thateach single PP2C mutant is viable (93), although no furthercharacterization was carried out. It must be noted that accord-ing to our analysis (see above), this organism does not containtwo Ptc2 isoforms, as proposed by these authors. Instead, thereported Ptc2-Ppm1k (AN2472) could be classified as Ptc6related (no. 83 in Fig. S1 in the supplemental material). Inaddition, our analysis has revealed an additional PP2C mem-ber in A. nidulans (AN0308.2), which can be classified as aPtc7-related protein (no. 114).

CONCLUDING REMARKS AND OUTLOOK

In spite of the growing body of knowledge on PP2C en-zymes, a large number of important questions remain open.From one side, it seems evident that common cellular targetscan be identified for specific PP2C isoforms across differentfungi. For instance, Ptc1 and Ptc2/3 isoforms are regulators ofdiverse MAPK signaling pathways in budding and fissionyeasts, usually with deactivation of these pathways. This roleseems to be conserved across evolution, as has been also doc-umented for animals and plants. Similarly, a role for PP2Cenzymes in cell cycle progression and response to DNA dam-age has been identified in both yeasts and mammals. On theother hand, a given PP2C isoform can exhibit distinctive cel-lular roles. For instance, a lack of Ptc1 in budding yeast resultsin a huge variety of phenotypes compared with those for othermembers of the PP2C family. In this regard, Ptc1 poses anintriguing question: are most phenotypes secondary to the lossof a major primary function (as has been suggested for vacu-olar malfunction), or do they reflect a large variety of differentcellular targets?

A distinctive feature of PP2C catalytic polypeptides is thatthey are usually not associated with regulatory or targetingsubunits. The interaction of Ptc1 with Nbp2 in budding yeastappears to be an exception rather than a rule. However, therecent identification of a family of proteins (RCAR/PYR) inthe plant Arabidopsis thaliana that interact with and regulateseveral PP2C isoforms (36) may reopen this issue, although itmust be noted that this specific family of plant regulatorysubunits does not seem to be present in fungi. An additionalrelevant issue relates to the possible role of specific isoforms(such as Ptc1) in fungal virulence. Examples have been de-scribed here for C. albicans and F. graminearum. However, the

molecular bases for this virulence are still unknown. Exploita-tion of hypothetical differences between fungal and mamma-lian or plant PP2C may help in the development of efficientantifungal drugs.

ACKNOWLEDGMENTS

Work in our laboratory has been supported by grants BFU2008-04188-C03-01 and BFU2009-11593 (MICINN, Spain). J.A. is the re-cipient of an “Ajut de Suport a les Activitats dels Grups de Recerca”(grant 2009SGR-1091) and an “ICREA Academia” award (Generali-tat de Catalunya).

We thank our colleague Amparo Querol for helpful advice.

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Type 2C Protein Phosphatases in Fungi · their target, protein phosphatases in general and PP2C en-zymes in particular do not exhibit signiﬁcant site speciﬁcity. This prevents