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Phosphorylation of α‐tubulin by protein kinase C stimulates microtubule dynamics in human breast cells
Author(s) -
De Shatarupa,
Tsimounis Areti,
Chen Xiangyu,
Rotenberg Susan A.
Publication year - 2014
Publication title -
cytoskeleton
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.95
H-Index - 86
eISSN - 1949-3592
pISSN - 1949-3584
DOI - 10.1002/cm.21167
Subject(s) - biology , phosphorylation , tubulin , protein kinase c , microbiology and biotechnology , activator (genetics) , microtubule , motility , kinase , diacylglycerol kinase , biochemistry , receptor
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.