Protein kinase-ζ interacts with munc18c:: role in GLUT4 trafficking
Protein kinase-ζ interacts with munc18c:: role in GLUT4 trafficking
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DOI：
10.1007/s00125-005-1819-y
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发表时间：
2005-08-01
期刊：
影响因子：
8.2
通讯作者：
Sale, GJ
中科院分区：
文献类型：
--
作者：
Hodgkinson, CP;Mander, A;Sale, GJ
目的/假设：胰岛素刺激的葡萄糖转运需要通过激酶蛋白激酶（PK）C zeta/lambda和PKB的信号级联，导致GLUT 4囊泡移动到质膜。本研究的目的是确定上游胰岛素调节激酶和GLUT 4囊泡运输系统之间的缺失环节。研究方法：酵母双杂交筛选进行，作为诱饵全长小鼠munc 18 c，一种蛋白质已知是GLUT 4囊泡运输机器的一部分。结果：酵母双杂交筛选确定PKC zeta是一种新的与munc 18 c相互作用的蛋白。谷胱甘肽S转移酶（GST）下拉GST标记的munc 18 c结构证实了相互作用，映射的munc 18 c的关键区域结合PKC zeta残基295-338，并表明，PKC zeta的N-末端区域所需的相互作用。内源性munc 18 c在体内不同类型的细胞中与内源性PKC zeta相关。重要的是，胰岛素刺激增加了约三倍的关联。此外，通过缺失munc 18 c的残基295-338或缺失PKC zeta的N-末端区域来破坏PKC zeta与munc 18 c的结合，显著抑制胰岛素刺激葡萄糖摄取或GLUT 4易位的能力。结论/解释：我们已经确定了munc 18 c和PKC zeta之间的生理相互作用是胰岛素调节。这建立了参与胰岛素信号级联的激酶（PKC zeta）与GLUT 4囊泡运输途径（munc 18 c）的已知组分之间的联系。结果表明，PKC zeta调节munc 18 c，并建议胰岛素触发PKC zeta对接munc 18 c的模型，导致增强GLUT 4转运到质膜。
Aims/hypothesis: Insulin-stimulated glucose transport requires a signalling cascade through kinases protein kinase (PK) C zeta/lambda and PKB that leads to movement of GLUT4 vesicles to the plasma membrane. The aim of this study was to identify missing links between the upstream insulin-regulated kinases and the GLUT4 vesicle trafficking system. Methods: A yeast two-hybrid screen was conducted, using as bait full-length mouse munc18c, a protein known to be part of the GLUT4 vesicle trafficking machinery. Results: The yeast two-hybrid screen identified PKC zeta as a novel interactor with munc18c. Glutathione S transferase (GST) pull-downs with GST-tagged munc18c constructs confirmed the interaction, mapped a key region of munc18c that binds PKC zeta to residues 295-338 and showed that the N-terminal region of PKC zeta was required for the interaction. Endogenous munc18c was shown to associate with endogenous PKC zeta in vivo in various cell types. Importantly, insulin stimulation increased the association by approximately three-fold. Moreover, disruption of PKC zeta binding to munc18c by deletion of residues 295-338 of munc18c or deletion of the N-terminal region of PKC zeta markedly inhibited the ability of insulin to stimulate glucose uptake or GLUT4 translocation. Conclusion/interpretation: We have identified a physiological interaction between munc18c and PKC zeta that is insulin-regulated. This establishes a link between a kinase (PKC zeta) involved in the insulin signalling cascade and a known component of the GLUT4 vesicle trafficking pathway (munc18c). The results indicate that PKC zeta regulates munc18c and suggest a model whereby insulin triggers the docking of PKC zeta to munc18c, resulting in enhanced GLUT4 translocation to the plasma membrane.