Control of insulin granule dynamics by AMPK dependent KLC1 phosphorylation
Control of insulin granule dynamics by AMPK dependent KLC1 phosphorylation
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DOI：
10.4161/isl.1.3.9608
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发表时间：
2009-11-01
期刊：
影响因子：
2.2
通讯作者：
Rutter, Guy A.
中科院分区：
文献类型：
--
作者：
McDonald, Angela;Fogarty, Sarah;Rutter, Guy A.
胰岛素颗粒沿着微管的运动由驱动蛋白-1/Kif5 B驱动，对于葡萄糖刺激的胰腺β细胞胰岛素分泌是必不可少的。5 'AMP活化蛋白激酶（AMPK）是异源三聚体丝氨酸/苏氨酸激酶，其在低葡萄糖浓度下在β细胞中被活化，但随着葡萄糖水平增加而被抑制。AMPK激活阻断葡萄糖刺激的分泌颗粒向细胞表面的募集和胰岛素分泌，表明马达蛋白可能是这种激酶的靶点。虽然驱动蛋白-1/Kif5B和驱动蛋白轻链-1（KLC 1）都含有共有AMPK磷酸化位点，但在小鼠KLC 1和纯化的重组GST-KLC 1中，只有对应于Ser 520的肽在体外被纯化的AMPK磷酸化。为了验证该位点的磷酸化可能调节驱动蛋白1介导的颗粒运动的假设，我们开发了一种新方法，使用Nokigawa旋转圆盘共聚焦显微镜在三维空间中研究细胞内颗粒的动力学。这种细胞范围的方法显示，在对照MIN 6细胞中，响应于升高的葡萄糖浓度（30对3 mM），较长的偏移（>10 μ m）的数量显著增加。然而，在过表达野生型KLC 1、KLC 1的磷酸化模拟物（S517/520 D）或非磷酸化突变体（S517/520 A）的细胞中观察到类似的变化。此外，使用抗磷酸化Ser 520特异性抗体，没有获得AMPK活化后KLC 1在Ser 520的磷酸化状态发生变化的证据。因此，KLC 1在Ser 517/520的磷酸化状态的变化不太可能影响运动功能。总之，我们描述了一种新的三维细胞范围的方法，用于分析活β细胞中的分泌颗粒动力学。
The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic beta-cells. 5'AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three dimensions using Nokigawa spinning disc confocal microscopy. This cell-wide approach revealed that the number of longer excursions (>10 mu m) increased significantly in response to elevated glucose concentration (30 vs. 3 mM) in control MIN6 cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1. Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function. In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living beta-cells.