Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation

ABSTRACT 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 Vmax by 1.6-fold and increased Km by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.

[1]  D. Raitt,et al.  Yeast Cdc42 GTPase and Ste20 PAK‐like kinase regulate Sho1‐dependent activation of the Hog1 MAPK pathway , 2000, The EMBO journal.

[2]  R. Sikorski,et al.  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. , 1989, Genetics.

[3]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[4]  Francesc Posas,et al.  Yeast HOG1 MAP Kinase Cascade Is Regulated by a Multistep Phosphorelay Mechanism in the SLN1–YPD1–SSK1 “Two-Component” Osmosensor , 1996, Cell.

[5]  A. Martinez-Arias,et al.  puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. , 1998, Genes & development.

[6]  G. Ammerer,et al.  Kinase activity-dependent nuclear export opposes stress-induced nuclear accumulation and retention of Hog1 mitogen-activated protein kinase in the budding yeast Saccharomyces cerevisiae. , 1999, Molecular biology of the cell.

[7]  Pamela A. Silver,et al.  Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin β homologs NMD5 and XPO1 , 1998, The EMBO journal.

[8]  R. Deschenes,et al.  Vectors for the inducible overexpression of glutathione S‐transferase fusion proteins in yeast , 1993, Yeast.

[9]  J. Broach,et al.  TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation , 1994, Molecular and cellular biology.

[10]  P. Russell,et al.  Protein Phosphatase 2C Acts Independently of Stress-activated Kinase Cascade to Regulate the Stress Response in Fission Yeast* , 1997, The Journal of Biological Chemistry.

[11]  T. Maeda,et al.  Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae , 1993, Molecular and cellular biology.

[12]  Mutsuhiro Takekawa,et al.  Protein phosphatase 2Cα inhibits the human stress‐responsive p38 and JNK MAPK pathways , 1998, The EMBO journal.

[13]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[14]  A. Varshavsky,et al.  The recognition component of the N‐end rule pathway. , 1990, The EMBO journal.

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

[16]  D. Barford.  Molecular mechanisms of the protein serine/threonine phosphatases. , 1996, Trends in biochemical sciences.

[17]  I. Ota,et al.  Differential Regulation of the Cell Wall Integrity Mitogen-Activated Protein Kinase Pathway in Budding Yeast by the Protein Tyrosine Phosphatases Ptp2 and Ptp3 , 1999, Molecular and Cellular Biology.

[18]  T. Maeda,et al.  Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases , 1997, Molecular and cellular biology.

[19]  S. Adam,et al.  The nuclear pore complex , 2001, Genome Biology.

[20]  H. Hirt,et al.  MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Zeitlinger,et al.  Thorax closure in Drosophila: involvement of Fos and the JNK pathway. , 1999, Development.

[22]  D. Mann,et al.  Mammalian protein serine/threonine phosphatase 2C: cDNA cloning and comparative analysis of amino acid sequences. , 1992, Biochimica et biophysica acta.

[23]  M. Mumby,et al.  Protein serine/threonine phosphatases and cell transformation. , 1993, Biochimica et biophysica acta.

[24]  K. H. Wolfe,et al.  Molecular evidence for an ancient duplication of the entire yeast genome , 1997, Nature.

[25]  M. Johnston,et al.  Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. , 1989, Gene.

[26]  I. Ota,et al.  A yeast protein similar to bacterial two-component regulators. , 1993, Science.

[27]  M. Gustin,et al.  MAP Kinase Pathways in the YeastSaccharomyces cerevisiae , 1998, Microbiology and Molecular Biology Reviews.

[28]  C. Hollenberg,et al.  Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p , 1998, Molecular and General Genetics MGG.

[29]  J. Ninomiya-Tsuji,et al.  Regulation of the TAK1 Signaling Pathway by Protein Phosphatase 2C* , 2001, The Journal of Biological Chemistry.

[30]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[31]  M. Cobb,et al.  Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. , 2001, Endocrine reviews.

[32]  K. Shiozaki,et al.  Heat-shock-induced activation of stress MAP kinase is regulated by threonine- and tyrosine-specific phosphatases. , 1999, Genes & development.

[33]  I. Ota,et al.  Two protein tyrosine phosphatases, Ptp2 and Ptp3, modulate the subcellular localization of the Hog1 MAP kinase in yeast. , 2000, Genes & development.

[34]  Francesc Posas,et al.  Requirement of STE50 for Osmostress-Induced Activation of the STE11 Mitogen-Activated Protein Kinase Kinase Kinase in the High-Osmolarity Glycerol Response Pathway , 1998, Molecular and Cellular Biology.

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

[36]  Y. Yanagawa,et al.  Selective suppression of stress‐activated protein kinase pathway by protein phosphatase 2C in mammalian cells , 1998, FEBS letters.

[37]  T. Maeda,et al.  Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. , 1995, Science.

[38]  I. Ota,et al.  Two Protein-tyrosine Phosphatases Inactivate the Osmotic Stress Response Pathway in Yeast by Targeting the Mitogen-activated Protein Kinase, Hog1* , 1997, The Journal of Biological Chemistry.

[39]  Irene Ota,et al.  Ptc1, a Type 2C Ser/Thr Phosphatase, Inactivates the HOG Pathway by Dephosphorylating the Mitogen-Activated Protein Kinase Hog1 , 2001, Molecular and Cellular Biology.

[40]  I. Ota,et al.  Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress , 2002, Eukaryotic Cell.

[41]  Tatsuya Maeda,et al.  A two-component system that regulates an osmosensing MAP kinase cascade in yeast , 1994, Nature.