The long-term research goal of this project is to understand in molecular terms how intracellular signal transduction is mediated by protein phosphorylation and dephosphorylation. This proposal intends to study the roles of protein phosphatases in MAPK (Mitogen-Activated Protein Kinase) cascades, using budding yeast (Saccharomyces cerevisiae) and cultured human cells. The regulatory mechanism of a MAPK cascade is important to mammalian oncogenesis, because many oncogenes (e.g., Src, Ras, Raf, and Mos) exert their effects through hyper-activation of MAPKS. MAPK cascades are regulated by dephosphorylation as well as by phosphorylation. In spite of intensive and extensive research efforts, however, much remains to be learned about phosphatases that down-regulate MAPK cascades. This project will focus on two specific types of protein phosphatases: protein tyrosine phosphatase (PTPase) and protein serine/threonine phosphatase type 2C (PP2C). The yeast HOG (High Osmolarity Glycerol response) signaling pathway will be studied as a model, because two PTPases (PTP2 and PTP3) and a PP2C (PTC1) have been implicated in the HOG MAPK cascade. Yeast is an ideal model system to study the general principles governing intracellular signal transduction, not only because it is genetically tractable, but also because its signaling pathways have many features in common with human cells.
The specific aims of this proposal are: 1) Study on the regulatory mechanism of the PTP2 and PTP3 tyrosine phosphatase activities by the HOG1 MAP Kinase. The post-translational activation of PTP2/PTP3 will be examined using in vitro assay systems. Whether the PTP2/PTP3 activities are regulated by protein phosphorylation will be tested. Also, the regulatory mechanism of the PTP2/PTP3 activities will be studied using genetic methods. 2) Study on the role of PTC1 and other type 2C protein phosphatases in yeast MAPK signal transduction. The physiological substrate of PTC1 will be defined using genetic epistasis test and an in vitro assay system. The role o the other type 2C protein phosphatases in yeast will be also examined. 3) Study on the role of the mammalian type 2C protein phosphatases in MAPK signal transduction. The regulatory roles of PP2Ca and PP2Cb on mammalian MAPKs will be studied both in vivo and in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM050909-05
Application #
2612928
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1994-05-01
Project End
2002-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
Mita, Hiroaki; Tsutsui, Junichiro; Takekawa, Mutsuhiro et al. (2002) Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding. Mol Cell Biol 22:4544-55
Raitt, D C; Posas, F; Saito, H (2000) Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway. EMBO J 19:4623-31
Takekawa, M; Saito, H (1998) A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95:521-30
Posas, F; Witten, E A; Saito, H (1998) Requirement of STE50 for osmostress-induced activation of the STE11 mitogen-activated protein kinase kinase kinase in the high-osmolarity glycerol response pathway. Mol Cell Biol 18:5788-96
Ferrigno, P; Posas, F; Koepp, D et al. (1998) Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J 17:5606-14
Takekawa, M; Maeda, T; Saito, H (1998) Protein phosphatase 2Calpha inhibits the human stress-responsive p38 and JNK MAPK pathways. EMBO J 17:4744-52
Posas, F; Saito, H (1998) Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator. EMBO J 17:1385-94
Wurgler-Murphy, S M; Maeda, T; Witten, E A et al. (1997) Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. Mol Cell Biol 17:1289-97
Posas, F; Saito, H (1997) Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276:1702-5
Takekawa, M; Posas, F; Saito, H (1997) A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways. EMBO J 16:4973-82

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