The 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 results suggested that tyrosine kinases might serve as regulators of γ-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of γ-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59Fyn). Microtubule-assembly assays demonstrated that membrane-associated γ-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the γ-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated γ-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85α (regulatory p85α subunit of PI3K) serves as a Fyn substrate. Direct interaction of γ-tubulin with the 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 results suggest that Fyn and PI3K might take part in the modulation of membrane-associated γ-tubulin activities.

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