Nbp2 targets the Ptc1-type 2C Ser/Thr phosphatase to the HOG MAPK pathway

The yeast high osmolarity glycerol (HOG) pathway signals via the Pbs2 MEK and the Hog1 MAPK, whose activity requires phosphorylation of Thr and Tyr in the activation loop. The Ptc1-type 2C Ser/Thr phosphatase (PP2C) inactivates Hog1 by dephosphorylating phospho-Thr, while the Ptp2 and Ptp3 protein tyrosine phosphatases dephosphorylate phospho-Tyr. In this work, we show that the SH3 domain-containing protein Nbp2 negatively regulates Hog1 by recruiting Ptc1 to the Pbs2-Hog1 complex. Consistent with this role, NBP2 acted as a negative regulator similar to PTC1 in phenotypic assays. Biochemical analysis showed that Nbp2, like Ptc1, was required to inactivate Hog1 during adaptation. As predicted for an adapter, deletion of NBP2 disrupted Ptc1-Pbs2 complex formation. Furthermore, Nbp2 contained separate binding sites for Ptc1 and Pbs2: the novel N-terminal domain bound Ptc1, while the SH3 domain bound Pbs2. In addition, the Pbs2 scaffold bound the Nbp2 SH3 via a Pro-rich motif distinct from that which binds the SH3 domain of the positive regulator Sho1. Thus, Nbp2 recruits Ptc1 to Pbs2, a scaffold for both negative and positive regulators.     

Ptc1p regulates cortical ER inheritance via Slt2p

Studies in the yeast Saccharomyces cerevisiae have shown that the inheritance of endoplasmic reticulum (ER), mitochondria, and vacuoles involves the capture of a tubular structure at the bud tip. Ptc1p, a serine/threonine phosphatase, has previously been shown to regulate mitochondrial inheritance by an unknown mechanism. Ptc1p regulates the high osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway and has also been implicated in the cell wall integrity (CWI) MAPK pathway. Here we show that the loss of Ptc1p or the Ptc1p binding protein, Nbp2p, causes a prominent delay in the delivery of ER tubules to the periphery of daughter cells and results in a dramatic increase in the level of phosphorylated Slt2p, the MAPK in the CWI pathway. Either loss of Slt2p or inhibition of the CWI pathway by addition of sorbitol, suppresses the ER inheritance defect in the ptc1Delta and nbp2Delta mutants. Our findings indicate that Ptc1p and Nbp2p regulate ER inheritance through the CWI MAPK pathway by modulating the MAPK, Slt2p.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p

The spindle pole body (SPB) in Saccharomyces cerevisiae functions to nucleate and organize spindle microtubules, and it is embedded in the nuclear envelope throughout the yeast life cycle. However, the mechanism of membrane insertion of the SPB has not been elucidated. Ndc1p is an integral membrane protein that localizes to SPBs, and it is required for insertion of the SPB into the nuclear envelope during SPB duplication. To better understand the function of Ndc1p, we performed a dosage suppressor screen using the ndc1-39 temperature-sensitive allele. We identified an essential SPB component, Nbp1p. NBP1 shows genetic interactions with several SPB genes in addition to NDC1, and two-hybrid analysis revealed that Nbp1p binds to Ndc1p. Furthermore, Nbp1p is in the Mps2p-Bbp1p complex in the SPB. Immunoelectron microscopy confirmed that Nbp1p localizes to the SPB, suggesting a function at this location. Consistent with this hypothesis, nbp1-td (a degron allele) cells fail in SPB duplication upon depletion of Nbp1p. Importantly, these cells exhibit a "dead" SPB phenotype, similar to cells mutant in MPS2, NDC1, or BBP1. These results demonstrate that Nbp1p is a SPB component that acts in SPB duplication at the point of SPB insertion into the nuclear envelope.     

PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway,Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.     

Yeast RSC function is required for organization of the cellular cytoskeleton via an alternative PKC1 pathway

RSC is a 15-protein ATP-dependent chromatin-remodeling complex related to Snf-Swi, the prototypical ATP-dependent nucleosome remodeler in budding yeast. Despite insight into the mechanism by which purified RSC remodels nucleosomes, little is known about the chromosomal targets or cellular pathways in which RSC acts. To better understand the cellular function of RSC, a screen was undertaken for gene dosage suppressors of sth1-3ts, a temperature-sensitive mutation in STH1, which encodes the essential ATPase subunit. Slg1p and Mid2p, two type I transmembrane stress sensors of cell wall integrity that function upstream of protein kinase C (Pkc1p), were identified as multicopy suppressors of sth1-3ts cells. Although the sth1-3ts mutant exhibits defects characteristic of PKC1 pathway mutants (caffeine and staurosporine sensitivities and an osmoremedial phenotype), only upstream components and not downstream effectors of the PKC1-MAP kinase pathway can suppress defects conferred by sth1-3ts, suggesting that RSC functions in an alternative PKC1-dependent pathway. Moreover, sth1-3ts cells display defects in actin cytoskeletal rearrangements and are hypersensitive to the microtubule depolymerizing drug, TBZ; both of these defects can be corrected by the high-copy suppressors. Together, these data reveal an important functional connection between the RSC remodeler and PKC1-dependent signaling in regulating the cellular architecture.     

Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the yeast cell integrity pathway

In Saccharomyces cerevisiae, a variety of stresses and aggressions to the cell wall stimulate the activation of the cell wall integrity MAPK pathway, which triggers the expression of a series of genes important for the maintenance of cell wall homeostasis. This MAPK module lies downstream of the Rho1 small GTPase and protein kinase C Pkc1 and consists of MAPKKK Bck1, MAPKKs Mkk1 and Mkk2, and the Slt2 MAPK. In agreement with previous reports suggesting that Mkk1 and Mkk2 were functionally redundant, we show here that both Mkk1 and Mkk2 alone or even chimerical proteins constructed by interchanging their catalytic and regulatory domains are able to efficiently maintain signal transduction through the pathway. Both Mkk1 and Mkk2 are phosphorylated in vivo concomitant to activation of the cell integrity pathway. Interestingly, hyperphosphorylation of the MEKs required not only the upstream components of the pathway, but also a catalytically competent Slt2 MAPK downstream. Active Slt2 purified from yeast extracts was able to phosphorylate Mkk1 and Mkk2 in vitro. We have mapped Ser(50) as a direct phosphorylation target for Slt2 in Mkk2. However, substitution of all (Ser/Thr)-Pro canonical MAPK target sites with alanine did not totally abrogate Slt2-dependent Mkk2 phosphorylation. Mutation or deletion of a conserved MAPK-docking site at the N-terminal extension of Mkk2 precluded its interaction with Slt2 and negatively affected retrophosphorylation. Our data show that the cell wall integrity MAPKKs are targets for their downstream MAPK, suggesting the existence of complex feedback regulatory mechanisms at this level.     

Signalling mucin Msb2 Regulates adaptation to thermal stress in Candida albicans

Temperature is a potent inducer of fungal dimorphism. Multiple signalling pathways control the response to growth at high temperature, but the sensors that regulate these pathways are poorly defined. We show here that the signalling mucin Msb2 is a global regulator of temperature stress in the fungal pathogen Candida albicans. Msb2 was required for survival and hyphae formation at 42°C. The cytoplasmic signalling domain of Msb2 regulated temperature‐dependent activation of the CEK mitogen activated proteins kinase (MAPK) pathway. The extracellular glycosylated domain of Msb2 (100‐900 amino acid residues) had a new and unexpected role in regulating the protein kinase C (PKC) pathway. Msb2 also regulated temperature‐dependent induction of genes encoding regulators and targets of the unfolded protein response (UPR), which is a protein quality control (QC) pathway in the endoplasmic reticulum that controls protein folding/degradation in response to high temperature and other stresses. The heat shock protein and cell wall component Ssa1 was also required for hyphae formation and survival at 42°C and regulated the CEK and PKC pathways.     

Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.     

Lrg1p functions as a putative GTPase-activating protein in the Pkc1p-mediated cell integrity pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ROM2 gene encodes a GDP/GTP exchange factor for the small G-protein Rho1p, a known activator of protein kinase C. In a screen designed to isolate suppressors of a rom2 mutant allele, we identified a mutant defective in the gene coding for the putative GTPase-activating protein Lrg1p. This protein was previously suggested to be involved in sporulation and mating. Here we provide evidence for its role in Pkc1p-mediated signal transduction based on the following results. (1) Deletion of LRG1 suppresses the growth phenotypes associated with mutations in SLG1 (which codes for a putative sensor of cell wall damage). (2) Using two-hybrid assays an interaction between the GAP domain of Lrg1p and Rho1p was demonstrated. (3) The lrg1 mutant shows enhanced activity of the Pkc1p pathway. (4) Overexpression of LRG1 leads to a cell lysis defect that can be suppressed by the addition of osmotic stabilizers. Phenotypic comparison of lrg1 mutants with mutants defective in other GTPase-activating proteins (Sac7p, Bem2p, Bag7p) presumed to act on Rho1p revealed that deletion of SAC7, but not BEM2 or BAG7, suppresses the phenotype of rom2 mutants. Pairwise combination of mutations in all these genes showed that the simultaneous deletion of SAC7 and LRG1 is synthetically lethal. We therefore suggest that Lrg1p acts as a negative regulator of the Pkc1p pathway in conjunction with its known homologue Sac7p.     

Down-regulation of Pkc1-mediated signaling by the deubiquitinating enzyme Ubp3

Regulated ubiquitination and degradation of signaling proteins have emerged as key mechanisms for modulating the strength and duration of signaling pathways. The reversible nature of the ubiquitination process as well as the large number and diversity of the deubiquitinating enzymes raise the possibility that signaling pathways might be modulated by specific deubiquitinating enzyme(s). Here we provide evidence that in the yeast Saccharomyces cerevisiae, the Pkc1-mediated signaling pathway that controls the cell wall integrity is negatively regulated by the deubiquitinating enzyme Ubp3. Disruption of the UBP3 gene leads to an enhanced activation of the cell wall integrity pathway MAPK Slt2 when cells are challenged with a variety of pathway activation agents such as pheromone and Congo red. The ubp3 deletion mutants accumulate high levels of Pkc1, suggesting potential regulation of Pkc1 by Ubp3. Consistent with this, Pkc1 and Ubp3 interact in vivo, and the stability of Pkc1 is markedly increased in the ubp3 deletion mutants. Moreover, disruption of the PKC1 gene, but not the genes that encode components downstream of Pkc1, completely suppresses other phenotypes displayed by the ubp3 deletion mutants such as hyperactivation of the pheromone-responsive MAPK Fus3 (Wang, Y., and Dohlman, H. G. (2002) J. Biol. Chem. 277, 15766-15772). These findings demonstrate that Ubp3 can regulate Pkc1 by facilitating its destruction and provide the initial evidence that Pkc1 plays a positive role in modulating the parallel pheromone-signaling pathway.     

A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth. Loss of PKC1 function results in cell lysis due to an inability to remodel the cell wall properly during growth. The PKC1 gene has been proposed to regulate a bifurcated pathway, on one branch of which function four putative protein kinases that catalyze a linear cascade of protein phosphorylation culminating in the activation of the mitogen-activated protein kinase homolog, Mpk1p. Here we describe two genes whose overexpression suppress both an mpk1 delta mutation and a pkc1 delta mutation. One of these genes is identical to the previously identified PPZ2 gene. The PPZ2 gene is predicted to encode a type 1-related protein phosphatase and is functionally redundant with a closely related gene, designated PPZ1. Deletion of both PPZ1 and PPZ2 resulted in a temperature-dependent cell lysis defect similar to that observed for bck1 delta, mkk1,2 delta, or mpk1 delta mutants. However, ppz1,2 delta mpk1 delta triple mutants displayed a cell lysis defect at all temperatures. The additivity of the ppz1,2 delta defect with the mpk1 delta defect, combined with the results of genetic epistasis experiments, suggested either that the PPZ1- and PPZ2-encoded protein phosphatases function on a branch of the PKC1-mediated pathway different from that defined by the protein kinases or that they play an auxiliary role in the pathway. The other suppressor gene, designated BCK2 (for bypass of C kinase), is predicted to encode a 92-kDa protein that is rich in serine and threonine residues. Genetic interactions between BCK2 and other pathway components suggested that BCK2 functions on a common pathway branch with PPZ1 and PPZ2.     

Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating

Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.     

Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 Signaling Pathway in Response to Thermal Stress

To initiate and establish infection in mammals, the opportunistic fungal pathogen Cryptococcus neoformans must survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase in Saccharomyces cerevisiae. We identified three C. neoformans Rho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion of RHO10 also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion of RHO11 yielded phosphorylation similar to that of wild type. Surprisingly, like a rho10Δ strain, a strain with a deletion of both RHO10 and RHO11 displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway in C. neoformans.