Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.     

Afr1p has a role in regulating the localization of Mpk1p at the shmoo tip in Saccharomyces cerevisiae

Afr1p functions to promote adaptation to pheromone-induced growth arrest and morphogenesis. We show here that Afr1p regulates polarized localization of the Mpk1p MAP kinase in shmooing cells. Deletion of AFR1 results in mislocalization of Mpk1p although the scaffold protein Spa2p localizes normally at shmoo tip, and overexpression of Spa2 cannot rescue this defect, indicating Afr1p in required for Spa2p to recruit Mpk1 to the site of polarized growth during mating. Overexpression of SPA2 partially suppresses the morphogenetic defect of afr1Delta cells upon alpha-factor induction, suggesting the two proteins function in the same genetic pathway with Spa2p acts downstream of Afr1p.     

Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.     

Protein-protein interactions in the yeastPKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.     

A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.     

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.     

Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2delta cells, like ssk2delta cells, were defective for actin recovery from osmotic stress. These spa2delta defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2delta and spa2delta yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p-actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.     

Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p)

Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.     

The MpkA MAP kinase module regulates cell wall integrity signaling and pyomelanin formation in Aspergillus fumigatus

Aspergillus fumigatus is the most important air-borne fungal pathogen, causing severe infections in immunocompromised patients. Mitogen-activated protein kinase (MAPK) signaling pathways are involved in the regulation of various cellular responses to environmental changes in eukaryotes. Genome Blast analysis revealed that the central core of the cell wall integrity signaling pathway in A. fumigatus is composed of three protein kinases designated Bck1, Mkk2 and MpkA. This pathway is of particular interest because it represents a possible target for new antifungal drugs. Deletion of these genes resulted in severe sensitivity of the mutants against cell wall-disturbing compounds and drastic alterations of the fungal morphology. Western blot analysis demonstrated that Bck1 and Mkk2 directly activate MpkA during vegetative growth and under cell wall stress conditions further confirming that Bck1, Mkk2 and MpkA form a MAP kinase module. Interestingly, this MAP kinase module affects the formation of pyomelanin derived from tyrosine degradation.     

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.     

Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae

Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.     

NHP6A and NHP6B, which encode HMG1-like proteins, are candidates for downstream components of the yeast SLT2 mitogen-activated protein kinase pathway.

The yeast SLK1 (BCK1) gene encodes a mitogen-activated protein kinase (MAPK) activator protein which functions upstream in a protein kinase cascade that converges on the MAPK Slt2p (Mpk1p). Dominant alleles of SLK1 have been shown to bypass the conditional lethality of a protein kinase C mutation, pkc1-delta, suggesting that Pkc1p may regulate Slk1p function. Slk1p has an important role in morphogenesis and growth control, and deletions of the SLK1 gene are lethal in a spa2-delta mutant background. To search for genes that interact with the SLK1-SLT2 pathway, a synthetic lethal suppression screen was carried out. Genes which in multiple copies suppress the synthetic lethality of slk1-1 spa2-delta were identified, and one, the NHP6A gene, has been extensively characterized. The NHP6A gene and the closely related NHP6B gene were shown previously to encode HMG1-like chromatin-associated proteins. We demonstrate here that these genes are functionally redundant and that multiple copies of either NHP6A or NHP6B suppress slk1-delta and slt2-delta. Strains from which both NHP6 genes were deleted (nhp6-delta mutants) share many phenotypes with pkc1-delta, slk1-delta, and slt2-delta mutants. nhp6-delta cells display a temperature-sensitive growth defect that is rescued by the addition of 1 M sorbitol to the medium, and they are sensitive to starvation. nhp6-delta strains also exhibit a variety of morphological and cytoskeletal defects. At the restrictive temperature for growth, nhp6-delta mutant cells contain elongated buds and enlarged necks. Many cells have patches of chitin staining on their cell surfaces, and chitin deposition is enhanced at the necks of budded cells. nhp6-delta cells display a defect in actin polarity and often accumulate large actin chunks. Genetic and phenotypic analysis indicates that NHP6A and NHP6B function downstream of SLT2. Our results indicate that the Slt2p MAPK pathway in Saccharomyces cerevisiae may mediate its function in cell growth and morphogenesis, at least in part, through high-mobility group proteins.     

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.