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,Debbie C Thurmond

doi:10.1074/jbc.m706591200

Abstract

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.

Highlights

An otherwise highly conserved lysine residue present within kinase core domains (1)
We demonstrate that Munc18c is a WNK1-interacting protein and that endogenous Munc18c-WNK1 complexes are important for glucose-stimulated insulin secretion via a syntaxin 4-dependent mechanism
WNK1 has previously been shown to localize to insulin granules and cytosolic fractions in INS-1 beta cells (10), whereas we and others have found Munc18c localized principally to plasma membrane and cytosolic compartments, with only trace amounts in the granule fraction of MIN6 beta cells (14, 27, 28)

Summary

EXPERIMENTAL PROCEDURES

Materials—The rabbit polyclonal anti-WNK1 (Q256) and Munc18c antibodies were generated as described (1, 15). The monoclonal anti-Myc (9E10) antibody and protein G-Plus-agarose were obtained from Santa Cruz Biotechnology, Inc. MIN6 beta cells at 50 – 60% confluence were transfected with 40 ␮g of plasmid DNA/10-cm dish using Transfectin (BioRad) to obtain ϳ50% transfection efficiency. For measurement of human C-peptide release, MIN6 beta cells were transiently co-transfected with each plasmid plus human proinsulin cDNA, using transfectin with 2 ␮g of each DNA per 35-mm dish of cells at 50 – 60% confluence. The interface was collected and diluted to 2 ml with homogenization buffer for centrifugation at 6,000 ϫ g for 10 min, and the resulting pellet was collected as the plasma membrane fraction. Data were evaluated for statistical significance using Student’s t test

RESULTS

Basal Glucose

DISCUSSION