SER/THR AND TYR REVERSIBLE PHOSPHORYLATION

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Linked publications & trials

Abstract

DESCRIPTION: Protein phosphorylation is a highly regulated process utilized by cells to communicate events occurring between the cell surface and the nucleus. This cellular network is controlled by the interplay of protein kinases and protein phosphatases. A major class of protein phosphatases, the dual-specificity phosphatases, play an important role in cell cycle control, mitogenesis, and differentiation. This proposal will develop a better understanding of the catalytic mechanism and biological function for the dual-specificity phosphatases. Experiments are outlined to determine the structure of the dual-specific phosphatase, VHR. In addition, the X-ray structure of VHR complexed with phosphopeptide will also be determined. The catalytic mechanism of the dual-specificity phosphatases will be probed with the use of site-directed mutagenesis, detailed kinetic analyses, nuclear magnetic resonance spectroscopy, and kinetic isotope effects. The applicant will also study the catalytic properties of the dual-specificity phosphatase p80cdc25. Because the physiological substrate for p80cdc25 is the cyclin dependent kinase p34cdc2, this investigation offers the opportunity of directly understanding the molecular details of this biologically important reaction which is required for passage of the cell through mitosis. These phosphatases have an essential active-site cysteine residue involved in the formation of a phosphoenzyme intermediate during catalysis. The investigators have identified a partial cDNA clone which appears to encode a glycine substitution for the active-site cysteine. This molecule would be predicted to bind phosphoprotein(s) but would be catalytically inactive. It is likely to play a role in regulating the phosphorylation status of proteins within the cell. Experiments are described to fully characterize the cDNA clone(s) and encoded protein(s). Finally, the applicant has identified a dual-specificity phosphatase in yeast which plays a key role in cell cycle progression. He will attempt to understand the function of this novel phosphatase using biochemistry and yeast genetics.