Abeyweera Thushara P, Chen Xiangyu, Rotenberg Susan A
Department of Chemistry and Biochemistry of Queens College of the City University of New York, Flushing, NY 11367-1597, USA.
J Biol Chem. 2009 Jun 26;284(26):17648-56. doi: 10.1074/jbc.M902005200. Epub 2009 Apr 29.
Engineered overexpression of protein kinase Calpha (PKCalpha) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCalpha (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that alpha6-tubulin is phosphorylated in cells expressing traceable PKCalpha and in vitro by wild type PKCalpha. Gain-of-function, single site mutations (Ser-->Asp) were constructed at each PKC consensus site in alpha6-tubulin (Ser158, Ser165, Ser241, and Thr337) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser165 as the only site in alpha6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCalpha. Expression of a phosphorylation-resistant mutant (S165N-alpha6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCalpha or by treatment with PKCalpha-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous alpha-tubulin that could be blocked when S165N-alpha6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCalpha levels (MDA-MB-231 cells) or lack PKCalpha and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type alpha6-tubulin and S165N-alpha6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser165 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 Ser165, leading to microtubule elongation and motility.
先前的研究表明，蛋白激酶Cα（PKCα）的工程化过表达赋予了非运动性的MCF-10A人乳腺细胞侵袭性运动能力。PKCα的一种可追踪突变体（Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364 - 2370）显示，α6-微管蛋白在表达可追踪PKCα的细胞中以及在体外被野生型PKCα磷酸化。在α6-微管蛋白的每个PKC保守位点（Ser158、Ser165、Ser241和Thr337）构建功能获得性单点突变（Ser→Asp）以模拟磷酸化。在MCF-10A细胞中表达每个构建体后，运动性分析确定Ser165是α6-微管蛋白中唯一其假磷酸化能重现PKCα引发的运动行为的位点。磷酸化抗性突变体（S165N-α6-微管蛋白）的表达导致由PKCα表达或用PKCα选择性激活剂二酰基甘油内酯处理所刺激的MCF-10A细胞运动性受到抑制。用二酰基甘油内酯处理的MCF-10A细胞显示内源性α-微管蛋白强烈磷酸化，当表达S165N-α6-微管蛋白时这种磷酸化可被阻断。S165N突变体还抑制表达高内源性PKCα水平的固有运动性人乳腺肿瘤细胞（MDA-MB-231细胞）或缺乏PKCα和其他传统同工型的细胞（MDA-MB-468细胞）。对在MDA-MB-468细胞中表达的Myc标签野生型α6-微管蛋白和S165N-α6-微管蛋白的比较表明，Ser165也是内源性活性非传统PKC同工型的主要磷酸化位点。PKC刺激的MCF-10A细胞运动性对诺考达唑敏感，从而表明微管延长参与了该机制。这些发现支持了一个模型，即PKC在Ser165处磷酸化α-微管蛋白，导致微管延长和运动。
