Mitogen-Activated Protein Kinases with Distinct Requirements for Ste5 Scaffolding Influence Signaling Specificity in Saccharomyces cerevisiae

ABSTRACT Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding.

[1]  B. Errede,et al.  MAP kinase-related FUS3 from S. cerevisiae is activated by STE7 in vitro , 1993, Nature.

[2]  Wendell A Lim,et al.  Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway. , 2004, Molecular cell.

[3]  F. Posas,et al.  Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. , 1997, Science.

[4]  G. Fink,et al.  Combinatorial Control Required for the Specificity of Yeast MAPK Signaling , 1997, Science.

[5]  G. Fink,et al.  Saccharomyces cerevisiae S288C has a mutation in FLO8, a gene required for filamentous growth. , 1996, Genetics.

[6]  C. Hollenberg,et al.  Mutations in the SAM domain of STE50 differentially influence the MAPK-mediated pathways for mating, filamentous growth and osmotolerance in Saccharomyces cerevisiae , 2001, Molecular Genetics and Genomics.

[7]  G. Fink,et al.  Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. , 1994, Genes & development.

[8]  E. Elion,et al.  Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae , 1994, Cell.

[9]  S. Fields,et al.  Analyzing protein-protein interactions using two-hybrid system. , 1995, Methods in enzymology.

[10]  M. Gustin,et al.  Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants. , 1999, Genetics.

[11]  E. Elion,et al.  Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes , 2004, The EMBO journal.

[12]  G. Boguslawski.  PBS2, a yeast gene encoding a putative protein kinase, interacts with the RAS2 pathway and affects osmotic sensitivity of Saccharomyces cerevisiae. , 1992, Journal of general microbiology.

[13]  E. Elion,et al.  The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism , 1996, Molecular and cellular biology.

[14]  T. Hughes,et al.  Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins , 2001, Current Biology.

[15]  B. Cairns,et al.  Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase. , 1992, Genes & development.

[16]  T. Pawson,et al.  Protein-protein interactions define specificity in signal transduction. , 2000, Genes & development.

[17]  B. Cairns,et al.  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 , 1996, Molecular and cellular biology.

[18]  H. Lodish.  Molecular Cell Biology , 1986 .

[19]  L. Bardwell,et al.  Inhibitory and activating functions for MAPK Kss1 in the S. cerevisiae filamentous- growth signalling pathway , 1997, Nature.

[20]  L. Bardwell.  A walk-through of the yeast mating pheromone response pathway , 2004, Peptides.

[21]  L. Flatauer,et al.  A Conserved Docking Site in MEKs Mediates High-affinity Binding to MAP Kinases and Cooperates with a Scaffold Protein to Enhance Signal Transmission* , 2001, The Journal of Biological Chemistry.

[22]  I. Herskowitz,et al.  Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[23]  L. Hartwell.  Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone , 1980, The Journal of cell biology.

[24]  M. Tyers,et al.  MAPK specificity in the yeast pheromone response independent of transcriptional activation , 2001, Current Biology.

[25]  D. Drubin,et al.  Spontaneous receptor-independent heterotrimeric G-protein signalling in an RGS mutant , 2003, Nature Cell Biology.

[26]  J. Thorner,et al.  Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions. , 1997, Genetics.

[27]  W. Sabbagh,et al.  Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. , 2001, Molecular cell.

[28]  H. Ruis,et al.  The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene. , 1994, The EMBO journal.

[29]  Lee Bardwell,et al.  A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1 , 2004, The Journal of cell biology.

[30]  E. Elion,et al.  The Ste5p scaffold. , 2001, Journal of cell science.

[31]  Harvey F. Lodish,et al.  MOLECULAR.CELL.BIOLOGY 5TH.ED , 2003 .

[32]  E. Elion,et al.  Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components. , 2003, Molecular biology of the cell.

[33]  L. Bardwell,et al.  Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  G. Fink,et al.  The riddle of MAP kinase signaling specificity. , 1998, Trends in genetics : TIG.

[35]  Gerald R. Fink,et al.  MAP Kinases with Distinct Inhibitory Functions Impart Signaling Specificity during Yeast Differentiation , 1997, Cell.

[36]  G F Sprague,et al.  Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae , 1991, Molecular and cellular biology.

[37]  J. Thorner,et al.  Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling. , 1997, Science.

[38]  G. Fink,et al.  Elements of the yeast pheromone response pathway required for filamentous growth of diploids. , 1993, Science.

[39]  B. Errede,et al.  Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. , 1992, Genes & development.

[40]  I. Herskowitz,et al.  The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. , 1998, Genes & development.

[41]  J. Thorner,et al.  Mutational analysis suggests that activation of the yeast pheromone response mitogen-activated protein kinase pathway involves conformational changes in the Ste5 scaffold protein. , 2000, Molecular biology of the cell.

[42]  P J Cullen,et al.  Glucose depletion causes haploid invasive growth in yeast. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Davis,et al.  Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. , 1998, Trends in biochemical sciences.

[44]  Lee Bardwell,et al.  A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. , 2004, Genes & development.

[45]  M. Peter,et al.  Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo , 2000, Current Biology.

[46]  T. Hughes,et al.  Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. , 2000, Science.

[47]  Henrik G. Dohlman,et al.  Persistent Activation by Constitutive Ste7 Promotes Kss1-Mediated Invasive Growth but Fails To Support Fus3-Dependent Mating in Yeast , 2004, Molecular and Cellular Biology.

[48]  P. Pryciak,et al.  Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway. , 1998, Genes & development.

[49]  J. Ferrell,et al.  Enforced proximity in the function of a famous scaffold. , 2003, Molecular cell.

[50]  Wendell A. Lim,et al.  Rewiring MAP Kinase Pathways Using Alternative Scaffold Assembly Mechanisms , 2003, Science.