Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells.
Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells.
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DOI：
10.1002/cm.21167
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发表时间：
2014-04
期刊：
影响因子：
2.9
通讯作者：
Rotenberg, Susan A.
中科院分区：
文献类型：
--
作者：
De, Shatarupa;Tsimounis, Areti;Chen, Xiangyu;Rotenberg, Susan A.
蛋白激酶C（PKC）通过在未转化的MCF-10A细胞中α-微管蛋白Ser-165的磷酸化产生运动性。活细胞成像探索PKC介导的磷酸化对微管（MT）动力学的影响。将GFP-α-tubulin荧光标记的MT用二酰基甘油（DAG）-内酯（一种膜渗透性PKC激活剂）处理，或与假磷酸化的S165 D-α6-tubulin突变体共转染。每种条件都通过刺激生长阶段的速率和持续时间以及降低灾难的频率来增加MT的动态性。在固有PKC活性高的MDA-MB-231转移性乳腺细胞中，这些MT生长参数也很高，但可通过表达磷酸化抗性S165 N-α6-微管蛋白或用泛PKC抑制剂（双吲哚马来酰亚胺）处理来抑制。MCF-10A细胞的亚细胞分级分离和免疫荧光显示，与对照组相比，α6-微管蛋白的磷酸化（通过DAG-内酯）或假磷酸化增加了其向MT的分配，并产生更长、更稳定的MT。在正末端结合蛋白GFP-EB 1表达后，DAG-内酯加速了新生MT的形成并增加了新生MT的数量。S165 D-α6-微管蛋白的表达促进了Rac 1的激活和Rac 1依赖的细胞运动。这些发现引起了人们对PKC介导的α-微管蛋白磷酸化的关注，认为这是一种控制MT动力学的新机制，从而导致细胞运动。
Protein kinase C (PKC) engenders motility through phosphorylation of α-tubulin at Ser-165 in non-transformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-α-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or co-transfected with a pseudo-phosphorylated S165D-α6-tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-α6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Sub-cellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudo-phosphorylation of α6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plus-end binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-α6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement.
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影响因子：
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作者：
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