Phosphorylation of α6-tubulin by protein kinase Cα activates motility of human breast cells

Abstract

Engineered overexpression of protein kinase Cα (PKCα) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCα (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that α6-tubulin is phosphorylated in cells expressing traceable PKCα and in vitro by wild type PKCα. Gain-of-function, single site mutations (Ser → Asp) were constructed at each PKC consensus site in α6-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 α6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCα. Expression of a phosphorylation-resistant mutant (S165N-α6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCα or by treatment with PKCα-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous α-tubulin that could be blocked when S165N-α6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCα levels (MDA-MB-231 cells) or lack PKCα and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type α6-tubulin and S165N-α6-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 α-tubulin at Ser165, leading to microtubule elongation and motility. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.