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

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.

WNK1 is required for mitosis and abscission

“…Depletion of WNK1 showed multipolar spindles, disorganized chromosomes, failed abscission, multinucleated cells, and reduced cell survival. WNK1 binds a number of other proteins, including protein kinases, through its noncatalytic domains (30)(31)(32). Thus, WNK1 may exert its mitotic function through its enzymatic activity, protein-protein associations, or likely both.…”

The Tyrosine Phosphorylation of Munc18c Induces a Switch in Binding Specificity from Syntaxin 4 to Doc2β

“…Plasmids-The generation of pcDNA3.1-FLAG-Munc18c, pET28a(ϩ)-His-Munc18c, and pGEX-Munc18c plasmids has been previously described (14,15,17). The TAP-Munc18c DNA constructs were generated by subcloning a PCR-generated Munc18c fragment into the XhoI and ClaI sites of the pNTAP and pCTAP shuttle vectors (Stratagene, La Jolla CA) for adenoviral particle production by Viraquest (North Liberty, IA).…”

“…The vesicle-associated SNARE protein (VAMP2) is functional in both glucose uptake and insulin granule exocytosis processes and complexes with the binary cognate receptor complex at the target membrane composed of SNAP23 and syntaxin 4 proteins (t-SNAREs) to form a SNARE heterotrimeric complex (3-11). This SNARE core complex is further regulated by the Munc18c isoform of the Sec1/Munc18 secretory protein family through its ability to directly bind to syntaxin 4 at the plasma membrane (8,(12)(13)(14)(15)(16)(17)(18)(19)(20). Although genetic models of Munc18c over-expression (12) and under-expression (13, 21) have both unequivocally substantiated Munc18c as a key and physiologically relevant regulator of glucose homeostasis by its impact upon syntaxin 4-mediated exocytosis, its precise role as a negative or positive factor in the molecular mechanism has remained unresolved.…”

Cab45b, a Munc18b-interacting Partner, Regulates Exocytosis in Pancreatic β-Cells

“…The total membrane ⌬C m in IgG-treated control cells was 91 Ϯ 15 fF/pF (Fig. 3, B and C) in response to a train of 10 depolarizations (pulse [1][2][3][4][5][6][7][8][9][10] ). Under the same conditions, cells treated with anti-Cab45b demonstrated markedly weaker capacitance responses (36 Ϯ 9 fF/pF, p Ͻ 0.01 versus control) (Fig.…”