Regulation of microtubule nucleation from membranes by complexes of membrane-bound gamma-tubulin with Fyn kinase and phosphoinositide 3-kinase.
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 p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.
Macurek
Libor
L
Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
Dráberová
Eduarda
E
Richterová
Vera
V
Sulimenko
Vadym
V
Sulimenko
Tetyana
T
Dráberová
Lubica
L
Marková
Vladimíra
V
Dráber
Pavel
P
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D019869:Phosphatidylinositol 3-Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn