Phosphorylation of α6-Tubulin by Protein Kinase Cα Activates Motility of Human Breast Cells

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.