Tyrosine phosphorylation of alpha-tubulin is an early response to NGF and pp60v-src in PC12 cells.
Neuronal differentiation is accompanied by extensive reorganization of the cytoskeleton to initiate the extension of neuritic processes. We have used the rat PC12 pheochromocytoma cell line to examine the role of protein tyrosine kinase activity in the induction of these events. Immunoblotting with phosphotyrosine antibodies revealed that tyrosine phosphorylation of alpha-tubulin in PC12 cells occurred within 10 min of nerve growth factor (NGF) treatment. Tyrosine phosphorylation of alpha-tubulin also occurred on induction of pp60v-src expression in a PC12 cell line (PC12-B9) harboring an inducible v-src gene under transcriptional control of the mouse metallothionine I gene promoter. Two tyrosine phosphorylated proteins in NGF- and pp60v-src induced PC12 cells were identified as alpha-tubulin isoforms by comigration with alpha-tubulin on two-dimensional gel electrophoresis, and by immunoprecipitation with phosphotyrosine antibodies followed by immunoblotting with a monoclonal antibody specific for alpha-tubulin. These results demonstrate that alpha-tubulin is an in vivo tyrosine kinase substrate, which is phosphorylated as an early event in the neuronal differentiation pathway of PC12 cells in response to NGF or pp60v-src. Tyrosine phosphorylation of alpha-tubulin could conceivably alter microtubule dynamics during induction of neurite extension.
Cox
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ME
Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
Maness
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PF
D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000494:pharmacology*; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D019000:Phosphotyrosine; D014443:Tyrosine; D008668:Metallothionein; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)