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J. Biol. Chem., Vol. 282, Issue 45, 32613-32622, November 9, 2007

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WNK1 Is a Novel Regulator of Munc18c-Syntaxin 4 Complex Formation in Soluble NSF Attachment Protein Receptor (SNARE)-mediated Vesicle Exocytosis^*

Eunjin Oh, Charles J. Heise, Jessie M. English^¶, Melanie H. Cobb^||, and Debbie C. Thurmond¹

From the Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, the Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239, ^¶Pfizer Research Technology Center, Cambridge, Massachusetts 02139, and the ^||Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159–491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.

Received for publication, August 8, 2007 , and in revised form, September 4, 2007.

^* This work was supported by National Institutes of Health (NIH) Grant DK-067912 and American Diabetes Association Grant 1-03-CD-10) (to D. C. T.), American Heart Association Postdoctoral Fellowship 0720042Z (to E. O.) and NIH grant GM53032 (to M. H. 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.

¹ To whom correspondence and reprint requests should be addressed: 635 Barnhill Dr., MS4053, Dept. of Biochemistry and Molecular Biology, Indianapolis, IN 46202. Tel.: 317-274-1551; Fax: 317-274-4686; E-mail: dthurmon{at}iupui.edu.

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