Saccharomyces cerevisiae Ste5 is important for induction and substrate specificity of Fus3 MAP kinase in the pheromone signaling pathway

The pheromone pathway is one of the mitogen activated protein kinase (MAPK) signaling pathways identified in Saccharomyces cerevisiae and is involved in both G1 cell cycle arrest and mating of cells. Fus3 functions at a branching point for G1 cell cycle arrest and mating responses in the signaling cascade, and the Fus3 MAPK uses components of both G1 arrest and mating routes as substrates. The Ste5 is a scaffold protein of the MAPK module and is essential for the activation of Fus3. However, it is not known how Ste5 is involved in the specific activation of Fus3 in G1 arrest and mating. In this study, we characterized several G1 arrest defective Ste5 mutants to better understand the roles of Ste5 in the regulation of Fus3. The level of Fus3 increased by treatment with alpha-factor. However, the alpha-factor effects were not readily apparent in the observation of yeast cells containing G1 arrest defective ste5 mutant. This suggests that Ste5 plays an essential role in Fus3 induction. Fus3 immune kinase assay of G1 arrest defective ste5 transformants revealed that Ste5 is important for substrate specificity of Fus3 for G1 arrest and/or mating.     

Quantitative profiling of dual phosphorylation of Fus3 MAP kinase in Saccharomyces cerevisiae

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracellular stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved TxY motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the TxY motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic TxY motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.     

Phosphorylation and localization of Kss1, a MAP kinase of the Saccharomyces cerevisiae pheromone response pathway.

Kss1 protein kinase, and the homologous Fus3 kinase, are required for pheromone signal transduction in Saccharomyces cerevisiae. In MATa haploids exposed to alpha-factor, Kss1 was rapidly phosphorylated on both Thr183 and Tyr185, and both sites were required for Kss1 function in vivo. De novo protein synthesis was required for sustained pheromone-induced phosphorylation of Kss1. Catalytically inactive Kss1 mutants displayed alpha-factor-induced phosphorylation on both residues, even in kss1 delta cells; hence, autophosphorylation is not obligatory for these modifications. In kss1 delta fus3 delta double mutants, Kss1 phosphorylation was elevated even in the absence of pheromone; thus, cross-phosphorylation by Fus3 is not responsible for Kss1 activation. In contrast, pheromone-induced Kss1 phosphorylation was eliminated in mutants deficient in two other protein kinases, Ste11 and Ste7. A dominant hyperactive allele of STE11 caused a dramatic increase in the phosphorylation of Kss1, even in the absence of pheromone stimulation, but required Ste7 for this effect, suggesting an order of function: Ste11-->Ste7-->Kss1. When overproduced, Kss1 stimulated recovery from pheromone-imposed G1 arrest. Catalytic activity was essential for Kss1 function in signal transmission, but not for its recovery-promoting activity. Kss1 was found almost exclusively in the particulate material and its subcellular fractionation was unaffected by pheromone treatment. Indirect immunofluorescence demonstrated that Kss1 is concentrated in the nucleus and that its distribution is not altered detectably during signaling.     

Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.     

Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.     

Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3(PP)) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3(PP) activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3(PP)-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.     

Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae.

alpha-Factor pheromone inhibits division of yeast a cells. After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division. The sst2 mutation is known to inhibit adaptation. This report examines adaptation in scg1 (also designated gpa1) and STE4Hpl (Hpl indicates haploid lethal) mutants that exhibit constitutive activation of the pheromone response pathway. Recovery of the STE4Hpl mutant was blocked by the sst2-1 mutation, whereas recovery of the scg1-7 mutant was not completely blocked by sst2-1. These results indicate that both SST2-dependent and -independent mechanisms regulate postreceptor events in the pheromone response pathway. Down regulation of receptors in response to alpha-factor was independent of the signal that was generated in the scg1 mutant.     

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.     

The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae

Signal transduction mediated by the single yeast isozyme of protein kinase C (Pkc1p) is essential for the maintenance of cellular integrity in this model eukaryote. The past few years have seen a dramatic increase in our knowledge of the upstream regulatory factors that modulate Pkc1p activity (e.g. Tor2p, Rom1p, Rom2p, Rho1p, Slg1p, Mid2p) and of the downstream targets of the MAP kinase cascade triggered by it (e.g. Rlm1p, SBF complex). The picture that has emerged connects this pathway to a variety of other cellular processes, such as cell cycle progression (Cdc28p, Swi4p), mating (Ste20p), nutrient sensing (Ira1p), calcium homeostasis (calcineurin, Mid2p, Fks2p) and the structural dynamics of the cytoskeleton (Spa1p, Bni1p).     

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.     

Carnitine uptake by AGP2 in yeast Saccharomyces cerevisiae is dependent on Hog1 MAP kinase pathway

The AGP2 gene encodes a plasma membrane carnitine transporter in S. cerevisiae. Here, we report the identification of AGP2 as an osmotic stress response gene. AGP2 was isolated from mTn3 tagged mutants that contained in-frame fusions with lacZ. The expression of AGP2 was down-regulated by osmotic stresses, including NaCl, sorbitol, and KCI. We also found that carnitine uptake was inhibited by NaCl. In the ssk1delta stelldelta double-mutant strain, the expression of AGP2 and the uptake of carnitine were greatly reduced compared to the wild-type strain. Furthermore, carnitine uptake was inhibited by the constitutive expression of PBS2, which encodes a MAPKK that activates Hog1. We concluded, therefore, that the HOG pathway plays an important role in the regulation of carnitine uptake in S. cerevisiae.     

Ca2+ signal is generated only once in the mating pheromone response pathway in Saccharomyces cerevisiae

The mating pheromone, alpha-factor, of the yeast Saccharomyces cerevisiae binds to the heterotrimeric G protein-coupled cell surface receptor of MATa cells and induces cellular responses necessary for mating. In higher eukaryotic cells, many hormones and growth factors rapidly mobilize a second messenger, Ca2+, by means of receptor-G protein signaling. Although striking similarities between the mechanisms of the receptor-G protein signaling in yeast and higher eukaryotes have long been known, it is still uncertain whether the pheromone rapidly mobilizes Ca2+ necessary for early events of the pheromone response. Here we reexamine this problem using sensitive methods for detecting Ca2+ fluxes and mobilization, and find no evidence that there is rapid Ca2+ influx leading to a rapid increase in the cytosolic free Ca2+ concentration. In addition, the yeast PLC1 deletion mutant lacking phosphoinositide-specific phospholipase C, a key enzyme for generating Ca2+ signals in higher eukaryotic cells, responds normally to the pheromone. These findings suggest that the receptor-G protein signaling does not utilize Ca2+ as a second messenger in the early stage of the pheromone response pathway. Since the receptor-G protein signaling does stimulate Ca2+ influx after early events have finished and this stimulation is essential for late events in the pheromone response pathway [Iida et al., (1990) J. Biol. Chem., 265: 13391-13399] Ca2+ may be used only once in the signal transduction pathway in unicellular eukaryotes such as yeast.     

Adenosine 3'',5''-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae

Theophylline, aminophylline, and isobutylmethylxanthine, compounds reported to be inhibitors of adenosine 3',5'-phosphate (cAMP) phosphodiesterase, prevented the alpha-factor-induced cell cycle arrest of Saccharomyces cerevisiae a cells. To determine whether the in vivo effect of these methylxanthines on yeast pheromone response was related to their known biochemical mode of action, two assays for cAMP phosphodiesterase based on affinity of the product of the reaction (5'-AMP) for boronate groups were developed and were used to monitor the activity of the low Km cAMP phosphodiesterase present in yeast extracts. It was found that the relative efficacy of the methylxanthines as inhibitors of this enzyme in vitro was correlated with the degree to which they antagonized alpha-factor action in vivo. These results were consistent with our previous proposal that pheromone action involves a lowering of cAMP level in the target cell.