Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells (original) (raw)

Protein kinase C (PKC) engenders motility through phosphorylation of a-tubulin at Ser-165 in nontransformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-a-tubulin were treated with diacylglycerol (DAG)lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-a6tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-a6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Subcellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudophosphorylation of a6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plusend binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-a6-tubulin promoted Rac1 activation and Rac1-dependent cell motility.