Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase.
We investigated the involvement of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the initiation of signal transduction by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. PtdIns 3-kinase catalyzes the formation of phosphoinositides with phosphate in the D-3 position of the inositol ring and previously has been found to associate with other activated protein tyrosine kinases, including growth factor receptor tyrosine kinases. Anti-phosphotyrosine immunoprecipitates had PtdIns 3-kinase activity that reached a maximum (9 times the basal activity) after a 5-min exposure of PC12 cells to NGF (100 ng/ml). Since NGF activates the tyrosine kinase activity of gp140trk, the protein product of the trk proto-oncogene, we also examined the association of PtdIns 3-kinase with gp140trk. Anti-gp140trk immunoprecipitates from NGF-stimulated PC12 cells had increased PtdIns 3-kinase activity compared to that of unstimulated cells, and larger increases were detected in cells overexpressing gp140trk, indicating that PtdIns 3-kinase associates with gp140trk. NGF produced large increases in [32P]phosphatidylinositol 3,4-bisphosphate and [32P]phosphatidylinositol 3,4,5-trisphosphate in PC12 cells labeled with [32P]orthophosphate, indicating an increase in PtdIns 3-kinase activity in intact cells. Using an anti-85-kDa PtdIns 3-kinase subunit antibody, we found that NGF promoted the tyrosine phosphorylation of an 85-kDa protein and two proteins close to 110 kDa. These studies demonstrate that NGF activates PtdIns 3-kinase and promotes its association with gp140trk and also show that NGF promotes the tyrosine phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Thus, PtdIns 3-kinase activation appears to be involved in differentiation as well as mitogenic responses.
Soltoff
S P
SP
Department of Physiology, Tufts University, Boston, Massachusetts 02111.
Rabin
S L
SL
Cantley
L C
LC
Kaplan
D R
DR
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D007700:Kinetics; D051379:Mice; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D010710:Phosphates / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011519:Proto-Oncogenes; D020917:Receptor, trkA; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D009414:Nerve Growth Factors; D010710:Phosphates; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D020917:Receptor, trkA