Phosphoinositide 3-kinase binds constitutively to alpha/beta-tubulin and binds to gamma-tubulin in response to insulin.
Recently we reported the localization of phosphoinositide 3-kinase (PI 3-kinase) by immunofluorescence to microtubule bundles and the centrosome (Kapeller, R., Chakrabarti, R., Cantley, L., Fay, F., and Corvera, S. (1993) Mol. Cell. Biol. 13, 6052-6063). In complementary experiments we used the recombinant p85 subunit of PI 3-kinase to identify proteins that associate with phosphoinositide 3-kinase and found that phosphoinositide 3-kinase associates with alpha/beta-tubulin. The association occurs in vivo but was not significantly affected by growth factor stimulation. We localized the region of p85 that interacts with alpha/beta-tubulin to the inter-SH2 domain. These results support the immunofluorescence data and show that p85 directly associates with alpha/beta-tubulin. We then determined whether phosphoinositide 3-kinase associates with gamma-tubulin. We found a dramatic growth factor-dependent association of phosphoinositide 3-kinase with gamma-tubulin. Phosphoinositide 3-kinase associates with gamma-tubulin in response to insulin and, to a lesser extent, in response to platelet-derived growth factor. Neither epidermal growth factor nor nerve growth factor treatment of cells results in association of phosphoinositide 3-kinase and gamma-tubulin. Phosphoinositide 3-kinase is also immunoprecipitated with antibodies to pericentrin in response to insulin, indicating that phosphoinositide 3-kinase is recruited to the centrosome. Neither phosphoinositide 3-kinase activity, nor intact microtubules are necessary for the association. Treatment of cells with 0.5 M NaCl dissociates gamma-tubulin from the centrosome and disrupts the association of phosphoinositide 3-kinase with pericentrin, but not gamma-tubulin. Recombinant p85 binds to gamma-tubulin from both insulin stimulated and quiescent cells. These results suggest that the association of phosphoinositide 3-kinase with gamma-tubulin is direct. These data suggest that phosphoinositide 3-kinase may be involved in regulating microtubule responses to insulin and platelet-derived growth factor.
Kapeller
R
R
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215, USA.
Toker
A
A
Cantley
L C
LC
Carpenter
C L
CL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D018385:Centrosome / Q000378:metabolism; D006224:Cricetinae; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D000941:Antigens; D007328:Insulin; D008869:Microtubule-Associated Proteins; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D014404:Tubulin; C086178:pericentrin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)