title: Phosphorylation of α-tubulin by protein kinase C stimulates microtubule dynamics in human breast cells.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Queens College, The City University of New York, New York, New York; The Graduate Center, The City University of New York, New York.
authors.forename: Shatarupa
authors.initials: S
authors.lastname: De
authors.forename: Areti
authors.initials: A
authors.lastname: Tsimounis
authors.forename: Xiangyu
authors.initials: X
authors.lastname: Chen
authors.forename: Susan A
authors.initials: SA
authors.lastname: Rotenberg
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D001940:Breast / Q000166:cytology*; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009362:Neoplasm Metastasis; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064726:Triple Negative Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D014404:Tubulin / Q000378:metabolism*; D014631:Valerates / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: C406230:(1-(hydroxymethyl)-4-(4-methyl-3-(methylethyl)pentylidene)-3-oxo-2-oxolanyl)methyl 4-methyl-3-(methylethyl)pentanoate; C405804:EB1 microtubule binding proteins; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D014631:Valerates; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D011493:Protein Kinase C; D020830:rac1 GTP-Binding Protein; D015107:4-Butyrolactone
keywords: Rac1; live cell imaging; motility; mutant; partitioning