Phosphorylation of ?-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells.

ABSTRACT: Protein kinase C (PKC) engenders motility through phosphorylation of ?-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-?-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-?6-tubulin 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-?6-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 ?6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plus-end binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-?6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of ?-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement.

SUBMITTER: De S 

PROVIDER: S-EPMC4113324 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature