Phosphorylation of 6-Tubulin by Protein Kinase C Activates Motility of Human Breast Cells*

Engineered overexpression of protein kinase C (PKC ) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKC (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364–2370) revealed that 6-tubulin is phosphorylated in cells expressing traceable PKC and in vitro by wild type PKC . Gain-of-function, single site mutations (Ser3 Asp) were constructed at each PKC consensus site in 6-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 6-tubulin whose pseudophosphorylation reproduced themotile behavior engendered by PKC . Expression of a phosphorylation-resistant mutant (S165N6-tubulin) resulted in suppression of MCF10A cell motility stimulated either by expression of PKC or by treatment with PKC -selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous -tubulin that could be blocked when S165N6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKC levels (MDAMB-231 cells) or lack PKC and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type 6-tubulin and S165N6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser165 is also amajor site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazolesensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates -tubulin at Ser165, leading to microtubule elongation and motility.