Phosphorylation of α6-Tubulin by Protein Kinase Cα Activates Motility of Human Breast Cells
Phosphorylation of α6-Tubulin by Protein Kinase Cα Activates Motility of Human Breast Cells
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DOI：
10.1074/jbc.m902005200
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发表时间：
2009-06-26
影响因子：
4.8
通讯作者：
Rotenberg, Susan A.
中科院分区：
文献类型：
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作者：
Abeyweera, Thushara P.;Chen, Xiangyu;Rotenberg, Susan A.
蛋白激酶Cα(PKCα)的工程过表达以前被证明赋予不能运动的MCF-10A人乳腺细胞具有侵袭性运动。一个可追踪的PKCα突变体(Abyweera，T.P.，and Rotenberg，S.A.(2007)BioChemical 46,2364-2370)揭示了α6-微管蛋白在表达可追踪的PKCα的细胞中被磷酸化，并在体外被野生型PKCα所磷酸化。在α-6-微管蛋白的每个PKC共有位点(Ser(158)、Ser(165)、Ser(241)和Thr(337))构建功能获得单点突变(Ser-&Gt；Asp)以模拟磷酸化。在MCF-10A细胞中表达每个结构后，运动性分析发现Ser(165)是α6-微管蛋白中唯一一个伪磷酸化重现PKCα所产生的运动行为的位点。表达抗磷酸化突变体(S165N-α6-微管蛋白)可抑制由PKCα表达或PKCα选择性激活剂二酰甘油-内酯处理所刺激的MCF-10A细胞的运动。二酰甘油-内酯处理的MCF-10A细胞表现出强烈的内源性α-微管蛋白的磷酸化，当S165N-α6-微管蛋白表达时，这种磷酸化可以被阻断。S165N突变体还抑制了内源性PKCα高水平表达或缺乏PKCα和其他传统亚型的人乳腺肿瘤细胞(MDA-MB-468细胞)。比较Myc标记的野生型α6-微管蛋白和S165N-α6-微管蛋白在MDA-MB-468细胞中的表达，发现Ser(165)也是内源性活性的非常规PKC亚型的主要磷酸化位点。PKC刺激的MCF-10A细胞的运动是诺康唑敏感的，从而提示微管延长在其机制中。这些发现支持一个模型，在该模型中，PKC在Ser(165)处磷酸化α-微管蛋白，导致微管延长和运动。
Engineered overexpression of protein kinase C alpha (PKC alpha) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKC alpha (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that alpha 6-tubulin is phosphorylated in cells expressing traceable PKC alpha and in vitro by wild type PKC alpha. Gain-of-function, single site mutations (Ser -> Asp) were constructed at each PKC consensus site in alpha 6-tubulin (Ser(158), Ser(165), Ser(241), and Thr(337)) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser(165) as the only site in alpha 6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKC alpha. Expression of a phosphorylation-resistant mutant (S165N-alpha 6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKC alpha or by treatment with PKC alpha-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous alpha-tubulin that could be blocked when S165N-alpha 6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKC alpha levels (MDA-MB-231 cells) or lack PKC alpha and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type alpha 6-tubulin and S165N-alpha 6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser(165) is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates alpha-tubulin at Ser(165), leading to microtubule elongation and motility.