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J. Biol. Chem., Vol. 282, Issue 37, 27086-27099, September 14, 2007

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Protein Kinase C Phosphorylation Disrupts Na⁺/H⁺ Exchanger Regulatory Factor 1 Autoinhibition and Promotes Cystic Fibrosis Transmembrane Conductance Regulator Macromolecular Assembly^*

Jianquan Li, Poulikos I. Poulikakos¹, Zhongping Dai, Joseph R. Testa, David J. E. Callaway^¶, and Zimei Bu²

From the Basic Science Division and Population Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 and the ^¶New York University School of Medicine, New York, New York 10016-6402

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Received for publication, March 8, 2007 , and in revised form, June 27, 2007.

^* This work was supported by NCI Grant CA06927 from the National Institutes of Health, American Cancer Society Grant IRG-92-027-09, the W.W. Smith Charitable Trust and National Institutes of Health Grant R01 HL086496 (to Z. B.), and NCI Grant CA114047 from the National Institutes of Health (to J. R. T.). 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 Equations and Tables A.1–A.3.

¹ Present address: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.

² To whom correspondence should be addressed: Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111. Tel.: 215-728-7051; Fax: 215-728-3574; E-mail: Zimei.bu{at}fccc.edu.

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