Oh Eunjin, Heise Charles J, English Jessie M, Cobb Melanie H, Thurmond Debbie C
Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
J Biol Chem. 2007 Nov 9;282(45):32613-22. doi: 10.1074/jbc.M706591200. Epub 2007 Sep 11.
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.
可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）介导的颗粒胞吐作用缺陷发生在胰岛β细胞、脂肪细胞和/或骨骼肌细胞中，这与胰岛素抵抗和糖尿病易感性增加相关。丝氨酸/苏氨酸激酶WNK1（无赖氨酸K）最近被认为与胞吐作用有关，并且在这三种细胞类型中均有表达。为了寻找与胞吐作用相关的WNK1底物，我们进行了WNK1双杂交筛选，结果得到了Munc18c。已知Munc18c是靶膜（t-SNARE）蛋白Syntaxin 4参与SNARE核心复合体组装的可及性的关键调节因子，尽管缺乏Munc18c结合因子阻碍了对其精确功能的发现。为了验证WNK1作为一种新的与Munc18c相互作用的伙伴，使用体外结合分析证实了WNK1与Munc18c之间的直接相互作用，并在胰岛β细胞系MIN6的胞质和质膜区室中检测到内源性WNK1-Munc18c复合体。这种结合相互作用是通过Munc18c的N端172个残基和WNK1的激酶结构域残基（残基159 - 491）介导的。这两个最小相互作用结构域中任何一个的表达都会导致葡萄糖刺激的胰岛素分泌受到抑制，这与内源性WNK1-Munc18c复合体在胞吐作用中的功能重要性一致。有趣的是，在激酶活性测定中，Munc18c未能作为WNK1的底物，这表明WNK1在SNARE复合体组装中的作用并非作为激酶。综上所述，这些数据支持了WNK1的新作用以及WNK1-Munc18c复合体调节SNARE复合体组装的新机制。
