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

Authors

  • Shatarupa De,

    1. Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York
    2. The Graduate Center, The City University of New York, New York
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  • Areti Tsimounis,

    1. Department of Biology, Queens College, The City University of New York, New York
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  • Xiangyu Chen,

    1. Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York
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  • Susan A. Rotenberg

    Corresponding author
    1. Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York
    • Address correspondence to: Susan A. Rotenberg, Ph.D., Department of Chemistry and Biochemistry, Queens College–City University of New York, 65-30 Kissena Boulevard, Flushing, NY 11367. E-mail: Susan.Rotenberg@qc.cuny.edu

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  • Monitoring Editor: George Bloom

  • This work was performed by S.D. in partial fulfillment of the requirements of the Ph.D. Program in Biochemistry at the Graduate Center of The City University of New York.

Abstract

Protein kinase C (PKC) engenders motility through phosphorylation of α-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-α-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-α6-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-α6-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 α6-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-α6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement. © 2014 Wiley Periodicals, Inc.

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