WNK1 is a novel regulator of Munc18c-syntaxin 4 complex formation in soluble NSF attachment protein receptor (SNARE)-mediated vesicle exocytosis.
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.
Oh
Eunjin
E
Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
Heise
Charles J
CJ
English
Jessie M
JM
Cobb
Melanie H
MH
Thurmond
Debbie C
DC
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D005089:Exocytosis / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism*; D012995:Solubility
D007328:Insulin; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D017346:Protein Serine-Threonine Kinases