Regulation of microtubule nucleation from membranes by complexes of membrane-bound γ-tubulin with Fyn kinase and phosphoinositide 3-kinase

被引:44
作者
Macurek, Libor [1 ]
Draberova, Eduarda [1 ]
Richterova, Vera [1 ]
Sulimenko, Vadym [1 ]
Sulimenko, Tetyana [1 ]
Draberova, Lubica [2 ]
Markova, Vladimira [1 ]
Draber, Pavel [1 ]
机构
[1] Acad Sci Czech Republic, Inst Mol Genet, Dept Biol Cytoskeleton, CR-14220 Prague 4, Czech Republic
[2] Acad Sci Czech Republic, Inst Mol Genet, Dept Signal Transduct, CR-14220 Prague 4, Czech Republic
关键词
detergent-resistant membrane (DRM); Fyn (protein tyrosine kinase p59(Fyn)); phosphoinositide 3-kinase (PI3K); PI3K regulatory subunit (p85 alpha); Src homology 2 (SH2) domain; gamma-tubulin;
D O I
10.1042/BJ20080909
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present Study, we show that a pool of gamma-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 p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-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 gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85 alpha (regulatory p85 alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85 alpha wits determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85 alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.
引用
收藏
页码:421 / 430
页数:10
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