The C-terminal domains of human neurofibromin and its budding yeast homologs Ira1 and Ira2 regulate the metaphase to anaphase transition

The human tumor suppressor neurofibromin contains a cysteine and serine-rich domain/Ras-GTPase activating protein domain (CSRD/RasGAP) and a C-terminal domain (CTD). Domain studies of neurofibromin suggest it has other functions in addition to being a RasGAP, but the mechanisms underlying its tumor suppressor activity are not well understood. The budding yeast Saccharomyces cerevisiae is a good model system for studying neurofibromin function because it possesses Ira1 and Ira2, which are homologous to human neurofibromin in both sequence and function. We found that overexpression of CTD or a neurofibromin CTD-homologous domain (CHD) of Ira1/2 in budding yeast delayed degradation of the securin protein Pds1, whereas overexpression of CSRD/RasGAP did not affect Pds1 degradation. We also found that when CTD or CHD was overexpressed, the number of cells in metaphase was higher than in the control. These results demonstrate that CTD and CHD function in the metaphase to anaphase transition. In addition, Δira1Δira2 cells bypassed mitotic arrest in response to spindle damage, indicating that Ira1 and Ira2 may be involved in the spindle assembly checkpoint (SAC). However, Δira1Δira2Δmad2 cells are more sensitive to spindle damage than Δmad2 or Δira1Δira2 cells are, suggesting that Ira1/2 and Mad2 function in different pathways. Overexpression of CTD but not CSRD/RasGAP partially rescued the hypersensitivity of Δira1Δira2Δmad2 cells to microtubule-destabilizing drugs, indicating a role for CTD in the SAC pathway. Taken together, independently of RasGAP activity, the C-terminal domains of neurofibromin, Ira1, and Ira2 regulate the metaphase to anaphase transition in a Mad2-independent fashion.     

Galpha subunit Gpa2 recruits kelch repeat subunits that inhibit receptor-G protein coupling during cAMP-induced dimorphic transitions in Saccharomyces cerevisiae

All eukaryotic cells sense extracellular stimuli and activate intracellular signaling cascades via G protein-coupled receptors (GPCR) and associated heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 and associated Galpha subunit Gpa2 sense extracellular carbon sources (including glucose) to govern filamentous growth. In contrast to conventional Galpha subunits, Gpa2 forms an atypical G protein complex with the kelch repeat Gbeta mimic proteins Gpb1 and Gpb2. Gpb1/2 negatively regulate cAMP signaling by inhibiting Gpa2 and an as yet unidentified target. Here we show that Gpa2 requires lipid modifications of its N-terminus for membrane localization but association with the Gpr1 receptor or Gpb1/2 subunits is dispensable for membrane targeting. Instead, Gpa2 promotes membrane localization of its associated Gbeta mimic subunit Gpb2. We also show that the Gpa2 N-terminus binds both to Gpb2 and to the C-terminal tail of the Gpr1 receptor and that Gpb1/2 binding interferes with Gpr1 receptor coupling to Gpa2. Our studies invoke novel mechanisms involving GPCR-G protein modules that may be conserved in multicellular eukaryotes.     

Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.     

Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiae

Ras proteins are guanine nucleotide-binding proteins that are highly conserved among eukaryotes. They are involved in signal transduction pathways and are tightly regulated by two sets of antagonistic proteins: GTPase-activating proteins (GAPs) inhibit Ras proteins, whereas guanine exchange factors activate them. In this work, we describe Tfs1p, the first physiological inhibitor of a Ras GAP, Ira2p, in Saccharomyces cerevisiae. TFS1 is a multicopy suppressor of the cdc25-1 mutation in yeast and corresponds to the so-called Ic CPY cytoplasmic inhibitor. Moreover, Tfs1p belongs to the phosphatidylethanolamine-binding protein (PEBP) family, one member of which is RKIP, a kinase and serine protease inhibitor and a metastasis inhibitor in prostate cancer. In this work, the results of (i) a two-hybrid screen of a yeast genomic library, (ii) glutathione S-transferase pulldown experiments, (iii) multicopy suppressor tests of cdc25-1 mutants, and (iv) stress resistance tests to evaluate the activation level of Ras demonstrate that Tfs1p interacts with and inhibits Ira2p. We further show that the conserved ligand-binding pocket of Tfs1-the hallmark of the PEBP family-is important for its inhibitory activity.     

Neurofibromin Homologs Ira1 and Ira2 Affect Glycerophosphoinositol Production and Transport in Saccharomyces cerevisiae

Saccharomyces cerevisiae produces extracellular glycerophosphoinositol through phospholipase-mediated turnover of phosphatidylinositol and transports glycerophosphoinositol into the cell upon nutrient limitation. A screening identified the RAS GTPase-activating proteins Ira1 and Ira2 as required for utilization of glycerophosphoinositol as the sole phosphate source, but the RAS/cyclic AMP pathway does not appear to be involved in the growth phenotype. Ira1 and Ira2 affect both the production and transport of glycerophosphoinositol.Membrane phospholipids are continually synthesized and degraded as cells grow and respond to environmental conditions. A major pathway of phosphatidylinositol (PI) turnover in Saccharomyces cerevisiae is its deacylation to produce extracellular glycerophosphoinositol (GroPIns) (3). Plb3, an enzyme with phospholipase B (PLB)/lysophospholipase activity, is thought to be primarily responsible for the production of extracellular GroPIns, with Plb1 playing a lesser role (11, 12, 13). GroPIns is transported into the cell by the Git1 permease (17). GIT1 expression is upregulated by phosphate limitation and inositol limitation. In fact, GroPIns can act as the cell''s sole source of both inositol (17) and phosphate (1).A screening for gene products involved in the process by which GroPIns enters the cellular metabolism identified Ira1 and Ira2, yeast homologs of the mammalian protein neurofibromin. Alterations in NF1, the gene encoding neurofibromin, are associated with the pathogenesis of neurofibromatosis type 1, an autosomal dominant genetic disease (4, 5, 25). Ira1 and Ira2 and neurofibromin function as RAS GTPase-activating proteins (RAS GAPs). S. cerevisiae Ras1 and Ras2 activate adenylate cyclase to modulate cyclic AMP (cAMP) levels. The binding of cAMP to the regulatory subunits of protein kinase A (Bcy1) results in dissociation and activation of the catalytic subunits (Tpk1 to Tpk3). Ira1 and Ira2 inactivate RAS and thereby downregulate the pathway (18, 19). Hydrolysis of cAMP by the phosphodiesterases encoded by PDE1 and PDE2 also downregulate the pathway (7, 20, 23). The RAS/cAMP pathway responds to nutrient signals to modulate fundamental cellular processes, including stress resistance, metabolism, and cell proliferation (7, 20, 21).     

Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1

The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.     

Localization of Ras signaling complex in budding yeast

In Saccharomyces cerevisiae, cAMP/pKA pathway plays a major role in metabolism, stress resistance and proliferation control. cAMP is produced by adenylate cyclase, which is activated both by Gpr1/Gpa2 system and Ras proteins, regulated by Cdc25/Sdc25 guanine exchange factors and Ira GTPase activator proteins. Recently, both Ras2 and Cdc25 RasGEF were reported to localize not only in plasma membrane but also in internal membranes. Here, the subcellular localization of Ras signaling complex proteins was investigated both by fluorescent tagging and by biochemical cell membrane fractionation on sucrose gradients. Although a consistent minor fraction of Ras signaling complex components was found in plasma membrane during exponential growth on glucose, Cdc25 appears to localize mainly on ER membranes, while Ira2 and Cyr1 are also significantly present on mitochondria. Moreover, PKA Tpk1 catalytic subunit overexpression induces Ira2 protein to move from mitochondria to ER membranes. These data confirm the hypothesis that different branches of Ras signaling pathways could involve different subcellular compartments, and that relocalization of Ras signaling complex components is subject to PKA control.     

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.     

Activation state of the Ras2 protein and glucose-induced signaling in Saccharomyces cerevisiae

The activity of adenylate cyclase in the yeast Saccharomyces cerevisiae is controlled by two G-protein systems, the Ras proteins and the Galpha protein Gpa2. Glucose activation of cAMP synthesis is thought to be mediated by Gpa2 and its G-protein-coupled receptor Gpr1. Using a sensitive GTP-loading assay for Ras2 we demonstrate that glucose addition also triggers a fast increase in the GTP loading state of Ras2 concomitant with the glucose-induced increase in cAMP. This increase is severely delayed in a strain lacking Cdc25, the guanine nucleotide exchange factor for Ras proteins. Deletion of the Ras-GAPs IRA2 (alone or with IRA1) or the presence of RAS2Val19 allele causes constitutively high Ras GTP loading that no longer increases upon glucose addition. The glucose-induced increase in Ras2 GTP-loading is not dependent on Gpr1 or Gpa2. Deletion of these proteins causes higher GTP loading indicating that the two G-protein systems might directly or indirectly interact. Because deletion of GPR1 or GPA2 reduces the glucose-induced cAMP increase the observed enhancement of Ras2 GTP loading is not sufficient for full stimulation of cAMP synthesis. Glucose phosphorylation by glucokinase or the hexokinases is required for glucose-induced Ras2 GTP loading. These results indicate that glucose phosphorylation might sustain activation of cAMP synthesis by enhancing Ras2 GTP loading likely through inhibition of the Ira proteins. Strains with reduced feedback inhibition on cAMP synthesis also display elevated basal and induced Ras2 GTP loading consistent with the Ras2 protein acting as a target of the feedback-inhibition mechanism.     

Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae.

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.     

Cyclic AMP-independent regulation of protein kinase A substrate phosphorylation by Kelch repeat proteins

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein alpha-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1Delta gpb2Delta mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1Delta gpb2Delta mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.     

The arginine finger loop of yeast and human GAP is a determinant for the specificity toward Ras GTPase.

In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21. It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase. Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase. The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop. In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase. Analysis of these results and modeling of Ira2p.Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms.     

Molecular basis of the Tfs1/Ira2 interaction: a combined protein engineering and molecular modelling study

Tfs1p and Ylr179cp are yeast proteins belonging to the PEBP family. Tfs1p, but not Ylr179cp, has been shown to interact with and inhibit Ira2p, a GTPase-activating protein of Ras. Tfs1p has been shown to be a specific inhibitor of the CPY protease and the 3D structure of the complex has been resolved. To shed light on the molecular determinants of Tfs1p involved in the Tfs1/Ira2 interaction, the 3D structure of Ylr179cp has been modelled and compared to that of Tfs1p. Tfs1p point mutants and Tfs1 hybrid proteins combining regions of Tfs1p and Ylr179cp were also designed and their function was tested. Results, interpreted from a structural point of view, show that the accessibility of the surface pocket of Tfs1p, its N-terminal region and the specific electrostatic properties of a large surface region containing these two elements, play a crucial role in this interaction.     

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.     

Canonical heterotrimeric G proteins regulating mating and virulence of Cryptococcus neoformans

Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three Galpha, one Gbeta, and two Ggamma subunit proteins. We have found that Galphas Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3alpha, Gbeta subunit Gpb1, and RGS protein Crg1. Crg1 also exhibited in vitro GAP activity toward Gpa2. These findings suggest that Gpa2 regulates mating through a conserved signaling mechanism. Moreover, we found that Ggammas Gpg1 and Gpg2 both regulate pheromone responses and mating. gpg1 mutants were attenuated in mating, and gpg2 mutants were sterile. Finally, although gpa2, gpa3, gpg1, gpg2, and gpg1 gpg2 mutants were fully virulent, gpa2 gpa3 mutants were attenuated for virulence in a murine model. Our study reveals a conserved but distinct signaling mechanism by two Galpha, one Gbeta, and two Ggamma proteins for pheromone responses, mating, and virulence in Cryptococcus neoformans, and it also reiterates that the link between mating and virulence is not due to mating per se but rather to certain mating-pathway components that encode additional functions promoting virulence.     

Multiple regulatory domains on the Byr2 protein kinase.

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.     

Regulation of the Ras‐GTPase activating protein neurofibromin by C‐tail phosphorylation: implications for protein kinase C/Ras/extracellular signal‐regulated kinase 1/2 pathway signaling and neuronal differentiation

PKC, Ras, and ERK1/2 signaling is pivotal to differentiation along the neuronal cell lineage. One crucial protein that may play a central role in this signaling pathway is the Ras GTPase‐activating protein, neurofibromin, a PKC substrate that may exert a positive role in neuronal differentiation. In this report, we studied the dynamics of PKC/Ras/ERK pathway signaling, during differentiation of SH‐SY5Y neuroblastoma cells upon treatment with the PKC agonist, phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Surprisingly, we observed that, among other PKC‐dependent signaling events, TPA induced a rapid and sustained decrease of neurofibromin immunoreactivity which was not due to proteolysis. Instead, we identified a specific phosphorylation event at the C‐tail of neurofibromin. This phosphorylation was acute and correlated perfectly with the signaling dynamics of the Ras/ERK pathway. Moreover, it persisted throughout prolonged treatment and TPA‐induced differentiation of SH‐SY5Y cells, concurrently with sustained activation of ERK1/2. Most importantly, C‐tail phosphorylation of neurofibromin correlated with a shift of neurofibromin localization from the nucleus to the cytosol. We propose that PKC‐dependent, sustained C‐tail phosphorylation is a requirement for prolonged recruitment of neurofibromin from the nucleus to the cytosol in order for a fine regulation of Ras/ERK pathway activity to be achieved during differentiation.     

In Saccharomyces cerevisiae an unbalanced level of tyrosine phosphorylation down-regulates the Ras/PKA pathway

The role of tyrosyl phosphorylation/dephosphorylation in the budding yeast Saccharomyces cerevisiae, whose genome does not encode typical tyrosine kinases, has long remained elusive. Nevertheless, several protein kinases phosphorylating poly(TyrGlu) substrates have been identified. In this work, we use the expression of the low molecular weight tyrosine phosphatase Stp1 from the distantly related yeast Schizosaccharomyces pombe, as a tool to investigate whether an unbalanced level of protein tyrosine phosphorylation affects S. cerevisiae growth and metabolism. We correlate the previously reported down-regulation of the phosphotyrosine level brought about by overexpression of Stp1 with a large number of phenotypes indicative of down-regulation of the Ras pathway. These phenotypes include reduction in both glucose- and acidification-induced GTP loading of the Ras2 protein and cAMP signaling, impaired growth on a non-fermentable carbon source, alteration of cell cycle parameters, delayed recovery from nitrogen starvation, increased heat-shock resistance, attenuated pseudohyphal and invasive growth. Genetic data suggest that Stp1 acts either at, or above, the level of Ras2, possibly on the Ira proteins. Consistently, Stp1 was found to bind to immunoprecipitated Ira2. Since a catalytically inactive mutant form of Stp1 (Stp1(C11S)) effectively binds to Ira2 without producing any effect on yeast physiology, we conclude that down-regulation of the Ras pathway by Stp1 requires its phosphatase activity. In conclusion, our data suggest a possible cross-talk between tyrosine phosphorylation and the Ras pathway in yeast.     

Crosstalk between cAMP-PKA and MAP kinase pathways is a key regulatory design necessary to regulate FLO11 expression

Signal transduction pathways crosstalk with one another and play a central role in regulation of cellular events. Crosstalk brings complexity to the system, and hence, a systematic analysis of these crosstalks helps in relating the signaling network structure to its function. Here, we present a modular steady state approach to quantify the network comprising of cAMP-PKA and MAP kinase pathways involved in the regulation of FLO11, a gene which is required for pseudohyphae growth in Saccharomyces cerevisiae under nitrogen starvation. These two pathways crosstalk by converging on the same target, i.e., FLO11 and through Ras2p, an upstream activator of both cAMP and MAPK pathway. Analysis of crosstalk at the gene level revealed that cAMP-PKA and MAPK pathways are indispensable to FLO11 expression. The dose response was highly sensitive and primarily controlled by cAMP-PKA pathway. We demonstrate that the highly sensitive response in the cAMP-PKA pathway was due to crosstalk and inhibitor ultrsensitivity, key regulatory designs present at the downstream of cAMP-PKA pathway. The analysis of the role of Ras2p in the crosstalk between the cAMP-PKA and MAPK pathways indicated that crosstalk essentially helped in amplification of the Gpa2p signal, another upstream activator of the cAMP-PKA pathway. However, the effect of crosstalk due to Ras2p on FLO11 expression was minimal under normal activation levels of Ras2p. Whereas, the crosstalk itself can bring about FLO11 expression under the hyperactivated Ras2p conditions thereby eliminating the requirement for the other activator Gpa2p. We also observed the presence of system level properties such as amplification, inhibitor ultrasensitvity and bistability, which can be attributed to the regulatory design present in the FLO11 expression system. These system level properties might help the organism to respond to varying nutritional status.