Characterization of the Net1 inhibitor of the Cdc14 protein phosphatase from budding yeast

Edwin Edgardo Traverso, Purdue University

Abstract

Cdc14 is a protein phosphatase from budding yeast whose activity is required for cells to end mitosis and begin another cycle of cell division. Cdc14 promotes exit from mitosis by dephosphorylating targets that trigger the inactivation of mitotic cyclin dependent kinases, an event that is essential for the completion of mitosis. In a screen for Cdc14 interacting proteins, our laboratory identified the protein Net1. We have shown that Net1 binds directly to Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (Ki = 3 nM). We have demonstrated that the Cdc14 binding site resides within a segment of Ne1 spanning residues 1–341. We propose that Net1 inhibits Cdc14 by occluding its active site because it acts as a competitive inhibitor, binds within the catalytic domain (residues 1–374), binds with reduced affinity to a Cdc14 active site mutant, and is displaced by tungstate, an agent that binds within the catalytic site of protein tyrosine phosphatases. Net1 may recognize other regions within the Cdc14 linear sequence, as indicated by the reduction in affinity of Net1 for a Cdc14 mutant with a substitution in Pro 116. Our studies confirm current models for the regulation of exit from mitosis that postulate that Net1 sequesters Cdc14 within the nucleolus throughout interphase and early mitosis. Our research has helped to demonstrate that Net1 not only restricts Cdc14 to the nucleolus but also inhibits its phosphatase activity. Our work also identifies Net1 as the first protein inhibitor for a member of the protein tyrosine phosphatase family.

Degree

Ph.D.

Advisors

Charbonneau, Purdue University.

Subject Area

Biochemistry

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