A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans

The Hog1 mitogen-activated protein kinase (MAPK) plays a central role in stress responses in the human pathogen Candida albicans. Here, we have investigated the MAPK kinase kinase (MAPKKK)-dependent regulation of the pathway. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, which is regulated by three MAPKKKs (Ssk2, Ssk22, and Ste11), our results demonstrate that Hog1 in C. albicans is regulated by a single MAPKKK Ssk2. Deletion of SSK2 results in comparable stress and morphological phenotypes exhibited by hog1Delta cells, and Ssk2 is required for the stress-induced phosphorylation and nuclear accumulation of Hog1, and for Hog1-dependent gene expression. Furthermore, phenotypes associated with deletion of SSK2 can be circumvented by expression of a phosphomimetic mutant of the MAPKK Pbs2, indicating that Ssk2 regulates Hog1 via activation of Pbs2. In S. cerevisiae, the Hog1 pathway is also regulated by the MAPKKK Ste11. However, we can find no connection between Ste11 and the regulation of Hog1 in C. albicans. Furthermore, expression of a chimeric Pbs2 protein containing the Ste11-dependent regulatory region of S. cerevisiae Pbs2, fails to stimulate Ste11-dependent stress signaling in C. albicans. Collectively, our data show that Ssk2 is the sole MAPKKK to relay stress signals to Hog1 in C. albicans and that the MAPK signaling network in C. albicans has diverged significantly from the corresponding network in S. cerevisiae.     

A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK). Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission. In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK. The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK. We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region. The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain. Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch. Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22. Thus, the docking site contributes to both the efficiency and specificity of signaling. During these analyses, we also found a nuclear export signal and a possible nuclear localization signal in Pbs2.     

A unique fungal two-component system regulates stress responses, drug sensitivity, sexual development, and virulence of Cryptococcus neoformans

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal "two-component" system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component-like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen.     

Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis

The Saccharomyces cerevisiae high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway is required for osmoadaptation and contains two branches that activate a mitogen-activated protein kinase (Hog1) via a mitogen-activated protein kinase kinase (Pbs2). We have characterized the roles of common pathway components (Hog1 and Pbs2) and components in the two upstream branches (Ste11, Sho1, and Ssk1) in response to elevated osmolarity by using whole-genome expression profiling. Several new features of the HOG pathway were revealed. First, Hog1 functions during gene induction and repression, cross talk inhibition, and in governing the regulatory period. Second, the phenotypes of pbs2 and hog1 mutants are identical, indicating that the sole role of Pbs2 is to activate Hog1. Third, the existence of genes whose induction is dependent on Hog1 and Pbs2 but not on Ste11 and Ssk1 suggests that there are additional inputs into Pbs2 under our inducing conditions. Fourth, the two upstream pathway branches are not redundant: the Sln1-Ssk1 branch has a much more prominent role than the Sho1-Ste11 branch for activation of Pbs2 by modest osmolarity. Finally, the general stress response pathway and both branches of the HOG pathway all function at high osmolarity. These studies demonstrate that cells respond to increased osmolarity by using different signal transduction machinery under different conditions.     

Dissection of the HOG pathway activated by hydrogen peroxide in Saccharomyces cerevisiae

Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has long been implicated in transducing the oxidative stress‐induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen‐activated protein kinase (MAPK) Hog1, we reveal that the signal from hydrogen peroxide (H₂O₂) flows through Ssk1, the response regulator of the two‐component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAP3K) Ssk2 but not its paralog Ssk22 or another MAP3K Ste11 of the pathway, culminating in Hog1 phosphorylation via the MAP2K Pbs2. When overexpressed, Ssk2 is also activated in an Ssk1‐independent manner. Unlike in mammals, H₂O₂ does not cause endoplasmic reticulum stress, which can activate Hog1 through the conventional unfolded protein response. Hog1 activated by H₂O₂ is retained in the cytoplasm, but is still able to activate the cAMP‐ or stress‐responsive elements by unknown mechanisms.     

Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological activity

Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity.     

Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress

Genome sequencing analyses revealed that Aspergillus nidulans has orthologous genes to all those of the high-osmolarity glycerol (HOG) response mitogen-activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae. A. nidulans mutant strains lacking sskA, sskB, pbsB, or hogA, encoding proteins orthologous to the yeast Ssk1p response regulator, Ssk2p/Ssk22p MAPKKKs, Pbs2p MAPKK and Hog1p MAPK, respectively, showed growth inhibition under high osmolarity, and HogA MAPK in these mutants was not phosphorylated under osmotic or oxidative stress. Thus, activation of the A. nidulans HOG (AnHOG) pathway depends solely on the two-component signalling system, and MAPKK activation mechanisms in the AnHOG pathway differ from those in the yeast HOG pathway, where Pbs2p is activated by two branches, Sln1p and Sho1p. Expression of pbsB complemented the high-osmolarity sensitivity of yeast pbs2Delta, and the complementation depended on Ssk2p/Ssk22p, but not on Sho1p. Pbs2p requires its Pro-rich motif for binding to the Src-homology3 (SH3) domain of Sho1p, but PbsB lacks a typical Pro-rich motif. However, a PbsB mutant (PbsB(Pro)) with the yeast Pro-rich motif was activated by the Sho1p branch in yeast. In contrast, HogA in sskADelta expressing PbsB(Pro) was not phosphorylated under osmotic stress, suggesting that A. nidulans ShoA, orthologous to yeast Sho1p, is not involved in osmoresponsive activation of the AnHOG pathway. We also found that besides HogA, PbsB can activate another Hog1p MAPK orthologue, MpkC, in A. nidulans, although mpkC is dispensable in osmoadaptation. In this study, we discuss the differences between the AnHOG and the yeast HOG pathways.     

Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical Cryptococcus neoformans serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502 SSK2 alleles were distinguished by two coding sequence changes. Supporting this finding, SSK2 allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the SSK2 gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2Δ, pbs2Δ, and hog1Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the ssk2Δ mutant, similar to what occurred in the pbs2Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in C. neoformans.     

Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway

In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.     

Phosphorylated Ssk1 prevents unphosphorylated Ssk1 from activating the Ssk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway

In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1~P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1~P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.     

Impact of phosphomimetic and non‐phosphorylatable mutations of phospholemman on L‐type calcium channels gating in HEK 293T cells

Background : Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L‐type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods : The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild‐type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results : WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage‐dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM‐induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions : Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects.     

Evidence that the MAPK-docking site in MAPKK Dpbs2p is essential for its function

In eukaryotes, mitogen-activated protein kinase (MAPK) pathways are very important signal transduction modules that regulate various cellular processes. Although eukaryotic cells possess a number of MAP kinase pathways, normally the MAPKKs selectively activate their cognate MAPK. Recent studies suggest that the MAPK-docking site in MAPKK facilitates this specific recognition and activation. However, the role of the docking site under in vivo conditions has not been demonstrated. In yeast external high osmolarity activates HOG (high osmolarity glycerol) MAPK pathway that consists of MAPKKK (Ste11p or Ssk2p/Ssk22p), MAPKK (Pbs2p), and MAPK (Hog1p). Previously, we have isolated a Pbs2p homologue (Dpbs2p) from osmo-tolerant and salt-tolerant yeast Debaryomyces hansenii that complemented pbs2 mutation in Saccharomyces cerevisiae. Here we show, for the first time, the presence of a MAPK-docking domain in Dpbs2p that is essential for its function in vivo. Mutation in this motif completely abolished its binding to Hog1p in vitro.     

Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae

In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.     

Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C.?albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.