Phosphorylation of alpha6-tubulin by protein kinase Calpha activates motility of human breast cells.

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.