Functions of protein tyrosine phosphatases in <italic>Saccharomyces cerevisiae</italic> signal transduction.

Abstract

The mitogen activated protein kinases (MAPKs) play essential roles in many signal transduction processes and coordinated regulation of MAPK activity is critical for proper cellular function. This thesis work is aimed to elucidate the critical regulatory mechanisms for MAPKs using <italic> S. cerevisiae</italic> as a model system. Biochemical and genetic analyses revealed that Fus3 MAPK in mating pheromone response is coordinately regulated by two phosphatases. Constitutively expressed PtP3 regulates basal tyrosine phosphorylation and activity of Fus3 whereas a pheromone-inducible dual-specificity phosphatase, Msg5, contributes to Fus3 inactivation following mating response. In the absence of Ptp3, a homologous phosphatase Ptp2 can partially substitute for Ptp3 function. Therefore, tyrosine phosphatase and dual-specificity phosphatase play differentiated roles in the regulation of MAPK. Multiple MAPKs have been found in <italic>S. cerevisiae</italic>, including Fus3 in the mating response and Hog 1 in the osmotic-stress response. Fus3 activity is negatively regulated by Ptp3 and Msg5 whereas Hog 1 is mainly dephosphorylated by Ptp2 even though Ptp2 and Ptp3 share extensive sequence similarity. The N-terminal non-catalytic domain of Ptp3 was found to directly interact with Fus3 <italic>via</italic> a CH2 (Cdc25 homology) domain conserved between yeast and mammals, and to be responsible for the <italic>in vivo</italic> substrate selectivity of the phosphatase. Interaction between Ptp3 and Fus3 is required for dephosphorylation and inactivation of Fus3 under physiological conditions. Mutations in either Ptp3 or Fus3 that abolish this interaction cause a dysregulation of the Fus3 MAPK. Therefore, the selectivity of MAP kinase inactivation <italic>in vivo </italic> by phosphatases is determined by specific protein-protein interactions outside of the catalytic domain. In addition to regulating MAPKs, Ptp2/Ptp3 were found to be required for yeast meiotic differentiation. Diploid <italic>ptp2Delta</italic>/-<italic> ptp3Delta</italic>/-cells were blocked at the early stage of the meiosis and tyrosine phosphorylation of several proteins was elevated in the deletion cells, suggesting Ptp2/Ptp3 regulate meiosis by controlling tyrosine phosphorylation. In parallel, a member of glycogen synthase kinase 3 (GSK3), Rim11, was found to be phosphorylated on tyrosine and the phosphorylation is required for its activity and function in meiosis. Therefore, regulated tyrosine phosphorylation through the actions of Ptp2/Ptp3 and Rim11 GSK3 plays important roles in yeast meiotic: differentiation.