Journal
CYTOSKELETON
Volume 71, Issue 4, Pages 257-272Publisher
WILEY
DOI: 10.1002/cm.21167
Keywords
live cell imaging; partitioning; motility; mutant; Rac1
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Funding
- National Cancer Institute of the National Institutes of Health [CA125632]
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Protein kinase C (PKC) engenders motility through phosphorylation of alpha-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-alpha-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-alpha 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-alpha 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 alpha 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-alpha 6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of alpha-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement. (c) 2014 Wiley Periodicals, Inc.
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