Regulation of microtubule nucleation from membranes by complexes of membrane-bound γ-tubulin with Fyn kinase and phosphoinositide 3-kinase
Résumé
Molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is γ-tubulin. Although previous data suggested that tyrosine kinases might serve as regulators of γ-tubulin function, their exact roles remain enigmatic. Here we show that a pool of γ-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells which exhibit elevated expression of Src family kinase Fyn. Microtubule assembly assays demonstrated that membrane-associated γ-tubulin complexes are capable of initiating formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of γ-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated γ-tubulin forms complexes with Fyn kinase and phosphoinositide 3-kinase (PI3K). Furthermore, in vitro kinase assays showed that p85α regulatory subunit of PI3K serves as Fyn kinase substrate. Direct interaction of γ-tubulin with C-terminal Src homology 2 domain of p85α was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85α. The combined data suggest that Fyn kinase and PI3K might take a part in modulation of membrane-associated γ-tubulin activities.
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