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J. Biol. Chem., Vol. 282, Issue 49, 35471-35481, December 7, 2007

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Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H⁺-ATPase Leads to Glucose-dependent Activation^*

Silvia Lecchi, Clark J. Nelson, Kenneth E. Allen, Danielle L. Swaney^¶, Katie L. Thompson^¶, Joshua J. Coon^¶||, Michael R. Sussman, and Carolyn W. Slayman¹

From the Departments of Genetics and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06511 and Departments of Biochemistry, ^¶Chemistry, and ^||Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H⁺-ATPase, which is responsible for H⁺-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5–10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal ¹⁴N/¹⁵N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H⁺-ATPase.

Received for publication, July 25, 2007 , and in revised form, October 1, 2007.

^* This work was supported by National Institutes of Health Grant GM 15761 (to C. W. S.), grants from the Department of Energy, National Science Foundation, and the University of Wisconsin College of Agricultural and Life Sciences (to M. R. S.), a National Institutes of Health predoctoral fellowship from Genomic Sciences Training Program HG 002706 (to D. L. S.), grants from University of Wisconsin-Madison, the WiCell Research Institute, Thermo Electron, and the Beckman Foundation, and National Institutes of Health Grant GM 80148 (to J. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental Appendix and Table S1.

¹ To whom correspondence should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Fax: 203-737-1771; E-mail: carolyn.slayman{at}yale.edu.

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