Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1

The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae. Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway. Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Delta strain due to hyperactivation of the HOG pathway. In this work, we show that PTC2 also suppresses sln1Delta lethality. Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs. Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2. In the wild type, Hog1 kinase activity increased transiently, approximately 12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to approximately 3-fold. In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2. Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation. In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip. Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm. Thus, one function of Ptc1 is to inactivate Hog1.     

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.     

Regulation of the osmoregulatory HOG MAPK cascade in yeast

The budding yeast Saccharomyces cerevisiae has at least five signal pathways containing a MAP kinase (MAPK) cascade. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity environment. This mini-review surveys recent developments in regulation of the HOG pathway with specific emphasis on the roles of protein phosphatases and protein subcellular localization. The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3. Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations. The subcellular localizations of the osmosensors (Sln1 and Sho1), kinases (Pbs2, Hog1), and phosphatases in the HOG pathway are intricately regulated to achieve their specific functions.     

Two adjacent docking sites in the yeast Hog1 mitogen-activated protein (MAP) kinase differentially interact with the Pbs2 MAP kinase kinase and the Ptp2 protein tyrosine phosphatase

Functional interactions between a mitogen-activated protein kinase (MAPK) and its regulators require specific docking interactions. Here, we investigated the mechanism by which the yeast osmoregulatory Hog1 MAPK specifically interacts with its activator, the MAPK kinase Pbs2, and its major inactivator, the protein phosphatase Ptp2. We found, in the N-terminal noncatalytic region of Pbs2, a specific Hog1-binding domain, termed HBD-1. We also defined two adjacent Pbs2-binding sites in Hog1, namely, the common docking (CD) domain and Pbs2-binding domain 2 (PBD-2). The PBD-2 docking site appears to be sterically blocked in the intact Hog1 molecule, but its affinity to Pbs2 is apparent in shorter fragments of Hog1. Both the CD and the PBD-2 docking sites are required for the optimal activation of Hog1 by Pbs2, and in the absence of both sites, Hog1 cannot be activated by Pbs2. These data suggest that the initial interaction of Pbs2 with the CD site might induce a conformational change in Hog1 so that the PBD-2 site becomes accessible. The CD and PBD-2 docking sites are also involved in the specific interaction between Hog1 and Ptp2 and govern the dynamic dephosphorylation of activated Hog1. Thus, the CD and the PBD-2 docking sites play critical roles in both the activation and inactivation of Hog1.     

Yeast Nap1-binding protein Nbp2p is required for mitotic growth at high temperatures and for cell wall integrity

Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Delta mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.     

Role of Ptc2 type 2C Ser/Thr phosphatase in yeast high-osmolarity glycerol pathway inactivation

Three type 2C Ser/Thr phosphatases (PTCs) are negative regulators of the yeast Saccharomyces cerevisiae high-osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway. Ptc2 and Ptc3 are 75% identical to each other and differ from Ptc1 in having a noncatalytic domain. Previously, we showed that Ptc1 inactivates the pathway by dephosphorylating the Hog1 MAPK; Ptc1 maintains low basal Hog1 activity and dephosphorylates Hog1 during adaptation. Here, we examined the function of Ptc2 and Ptc3. First, deletion of PTC2 and/or PTC3 together with PTP2, encoding the protein tyrosine phosphatase that inactivates Hog1, produced a strong growth defect at 37°C that was dependent on HOG1, providing further evidence that PTC2 and PTC3 are negative regulators. Second, overexpression of PTC2 inhibited Hog1 activation but did not affect Hog1-Tyr phosphorylation, suggesting that Ptc2 inactivates the pathway by dephosphorylating the Hog1 activation loop phosphothreonine (pThr) residue. Indeed, in vitro studies confirmed that Ptc2 was specific for Hog1-pThr. Third, deletion of both PTC2 and PTC3 led to greater Hog1 activation upon osmotic stress than was observed in wild-type strains, although no obvious change in Hog1 inactivation during adaptation was seen. These results indicate that Ptc2 and Ptc3 differ from Ptc1 in that they limit maximal Hog1 activity. The function of the Ptc2 noncatalytic domain was also examined. Deletion of this domain decreased V_max by 1.6-fold and increased K_m by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.     

Phosphoproteomic analyses reveal novel cross‐modulation mechanisms between two signaling pathways in yeast

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen‐activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time‐resolved phosphorylation site dynamics after pathway co‐stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone‐induced down‐regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.     

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1⁺ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1.     

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.     

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.     

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 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.     

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.     

MAPKKK-independent regulation of the Hog1 stress-activated protein kinase in Candida albicans

The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2(AA)). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2(DD)) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2(DD) cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2(DD) cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2(AA) and Pbs2(DD) cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2(AA) cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.     

Rck1 up-regulates Hog1 activity by down-regulating Slt2 activity in Saccharomyces cerevisiae

We previously reported that the over-expression of KDX1 up-regulates RCK1 gene expression. To further understand the function of Rck1, microarray analysis was performed using a RCK1 over-expressing strain. Based on microarray and Northern blot analyses, we determined that the expression of KDX1 was down-regulated when RCK1 was over-expressed. Furthermore, we determined that phosphorylated forms of Slt2 and Mkk2 were down-regulated by the over-expression of RCK1. Ptp2, a phosphatase that is regulated by the Slt2 MAP kinase pathway, was down-regulated by the over-expression of RCK1. Ptp2 is a negative regulator of Hog1; thus, the phosphorylated form of Hog1 was up-regulated by RCK1 over-expression. A point mutation of lysine 152 to arginine resulted in a failure to up-regulate Hog1 and the subsequent down-regulation of CTT1, which is a Hog1 pathway target gene. Furthermore, using microarray and Northern blot analyses, we determined that genes that are regulated by Msn2/Msn4 were up-regulated by Rck1 and that this was the result of Hog1 activation by RCK1 over-expression. Together, our results suggest that Rck1 inhibits Slt2 MAP kinase pathway activity and then Ptp2, which subsequently activates Hog1.     

Osmostress Induces Autophosphorylation of Hog1 via a C-Terminal Regulatory Region That Is Conserved in p38��

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.     

The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage

In photosynthetic eukaryotes assembly components of iron-sulfur (Fe-S) cofactors have been studied in plastids and mitochondria, but how cytosolic and nuclear Fe-S cluster proteins are assembled is not known. We have characterized a plant P loop NTPase with sequence similarity to Nbp35 of yeast and mammals, a protein of the cytosolic Cfd1-Nbp35 complex mediating Fe-S cluster assembly. Genome analysis revealed that NBP35 is conserved in the green lineage but that CFD1 is absent. Moreover, plant and algal NBP35 proteins lack the characteristic CXXC motif in the C terminus, thought to be required for Fe-S cluster binding. Nevertheless, chemical reconstitution and spectroscopy showed that Arabidopsis (At) NBP35 bound a [4Fe-4S] cluster in the C terminus as well as a stable [4Fe-4S] cluster in the N terminus. Holo-AtNBP35 was able to transfer an Fe-S cluster to an apoprotein in vitro. When expressed in yeast, AtNBP35 bound 55Fe dependent on the cysteine desulfurase Nfs1 and was able to partially rescue the growth of a cfd1 mutant but not of an nbp35 mutant. The AtNBP35 gene is constitutively expressed in planta, and its disruption was associated with an arrest of embryo development. These results show that despite considerable divergence from the yeast Cfd1-Nbp35 Fe-S scaffold complex, AtNBP35 has retained similar Fe-S cluster binding and transfer properties and performs an essential function.     

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.