Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells.
Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.
Kukharskyy
Vitaliy
V
Institute of Molecular Genetics, Department of Biology of Cytoskeleton, Academy of Sciences of the Czech Republic, Prague 4.
Sulimenko
Vadym
V
Macůrek
Libor
L
Sulimenko
Tetyana
T
Dráberová
Eduarda
E
Dráber
Pavel
P
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology*; D054278:Embryonal Carcinoma Stem Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D046911:Macromolecular Substances; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000502:physiology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000906:Antibodies; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D011518:Proto-Oncogene Proteins; D014404:Tubulin; D050257:Tubulin Modulators; D019000:Phosphotyrosine; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases