Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC-12 pheochromocytoma cells.
Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including two proteins, Shc and Grb2, that are thought to lie upstream from the ras protooncogene in a signaling cascade. The phosphorylation and molecular associations of these proteins were evaluated in PC-12 pheochromocytoma cells treated with nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Both NGF and EGF stimulated the tyrosine phosphorylation of Shc proteins and their subsequent association with the receptors. In contrast, insulin had no effect on Shc phosphorylation, despite the expression of functional insulin receptors in these cells at levels comparable to those observed for NGF and EGF. NGF and EGF also induced the association of Shc proteins with a Grb2 fusion protein or endogenous Grb2, whereas insulin had no effect. All of the tyrosine-phosphorylated Shc proteins associated with the Grb2 fusion protein, although only about half of the endogenous Shc was phosphorylated in response to NGF or EGF. However, all three hormones induced the association of several additional tyrosine phosphorylated proteins with Grb2, some of which also coprecipitated with antiserum against the 85-kDa subunit of phosphatidylinositol-3 kinase. Moreover, these growth factors stimulated the association of phosphatidylinositol-3 kinase activity with the Grb2 fusion protein, although this activity was not detected in anti-Shc immunoprecipitates. These results provide further evidence for the divergence of signaling pathways in insulin action, and suggest that Grb2 forms separate complexes with tyrosine-phosphorylated proteins in PC-12 cells.
Ohmichi
M
M
Department of Physiology, University of Michigan School of Medicine, Ann Arbor 48109.
Matuoka
K
K
Takenawa
T
T
Saltiel
A R
AR
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein; D007328:Insulin / Q000494:pharmacology*; D046911:Macromolecular Substances; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; D007328:Insulin; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D010750:Phosphoproteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin