Characterization of a Third Metal Binding Site in the Human PP2C-Family Phosphatases, PP2C-alpha and Wip1

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NCI	BIOCHEM-13

Authors

• K. Tanoue
• S.R. Durell
• W.K. Gillette
• L.M. Jenkins
• E. Appella
• S.J. Mazur

Abstract

PP2C serine/threonine protein phosphatases are critical regulators of stress responses in all three kingdoms of life and are distinguished by divalent metal ion-dependent stimulation of phosphatase activity. In humans, PP2C-alpha (PPM1A) functions as a tumor suppressor whereas Wip1 (PPM1D) negatively regulates several tumor suppressors. Although a crystal structure of human PP2C-alpha was shown to contain two bound Mn ions, details of the catalytic mechanism and determinants of substrate specificity remain incompletely understood. Recently, structural studies of several prokaryotic PP2C phosphatases demonstrated the presence of three or four bound metal ions. As most of the coordinating residues for the additional metal ions are highly conserved, these results anticipate additional metal binding sites in human PP2C phosphatases. We have used site-directed mutagenesis, molecular modeling, calorimetry, and phosphatase activity assays to characterize additional metal binding site(s) in PP2C-alpha. Separate mutation of D146 or D243 to alanine abolished phosphatase activity or reduced metal affinity, respectively, suggesting that these are essential residues for additional metal ion binding. Furthermore, the Wip1 D192A mutant (equivalent to PP2C-alpha D146A) also was inactive. These results suggest that additional metal ion binding in human PP2C phosphatases is essential for catalytic activity and open prospects for the development of specific inhibitors.

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