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J. Biol. Chem., Vol. 282, Issue 46, 33879-33887, November 16, 2007

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Phosphorylation of PDZ1 Domain Attenuates NHERF-1 Binding to Cellular Targets^*

James W. Voltz¹, Matthew Brush, Suzanne Sikes, Deborah Steplock, Edward J. Weinman, and Shirish Shenolikar²

From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and the Department of Medicine, University of Maryland School of Medicine, Department of Veterans Affairs, Baltimore, Maryland 21201

NHERF-1 (Na⁺-H⁺ exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including ₂-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.

Received for publication, April 26, 2007 , and in revised form, August 31, 2007.

^* This work was supported by National Institutes of Health Grant DK55881 (to S. S. and E. J. W.). 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 supplemental Figs. S1 and S2.

¹ Present address: NIEHS, Laboratory for Epithelial Biology, Research Triangle Park, NC 27709.

² To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, LSRC C315, Research Dr., Durham, NC 27710. Tel.: 919-613-8460; Fax: 919-681-9567; E-mail: Sheno001{at}mc.duke.edu.

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