Abstract
The interactions between the phosphatidylinositol 3-kinase (PI 3-kinase) and Ras/MAPK kinase pathways have been the subject of considerable interest. In the current studies, we find that epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) lead to rapid phosphorylation of Shc (maximum at 1-2 min), whereas insulin-mediated Shc phosphorylation is relatively delayed (maximum at 5-10 min), suggesting that an intermediary step may be necessary for insulin stimulation of Shc phosphorylation. The Src homology-2 (SH2) domain of Shc is necessary for PDGF- and EGF-mediated Shc phosphorylation, whereas the phosphotyrosine binding (PTB) domain is critical for the actions of insulin. Because the Shc PTB domain can interact with phospholipids, we postulated that PI 3-kinase might be a necessary intermediary step facilitating insulin-stimulated phosphorylation of Shc. In support of this, we found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked insulin-stimulated but not EGF- or PDGF-stimulated Shc phosphorylation. Furthermore, overexpression of a dominant negative PI 3-kinase construct (p85N-SH2) blocked insulin, but not EGF- or PDGF-induced Shc phosphorylation. All three growth factors cause localization of Shc to the plasma membrane, but only the effect of insulin was inhibited by wortmannin, supporting the view that PI 3-kinase-generated phospholipids mediate insulin-stimulated Shc phosphorylation. Consistent with this, expression of a constitutively active PI 3-kinase (p110(C)(AAX)) increased membrane localization of Shc, and this was completely blocked by wortmannin. A mutant Shc with a disrupted PTB domain (Shc S154) did not localize to the membrane in p110(C)(AAX)-expressing cells or after insulin stimulation and was not phosphorylated by insulin. In summary, 1) PI 3-kinase is a necessary early step in insulin-stimulated Shc phosphorylation, whereas the effects of EGF and PDGF on Shc phosphorylation are independent of PI 3-kinase. 2) PI 3-kinase-stimulated generation of membrane phospholipids can localize Shc to the plasma membrane through the Shc PTB domain facilitating phosphorylation by the insulin receptor.
Highlights
Growth factor signaling initiates a variety of biologic responses, many of which are mediated through the PI 3-kinase1 and the Ras/MAP kinase pathway
Activation of the insulin receptor or other growth factor receptors results in the tyrosine phosphorylation of Shc, which interacts with the adapter protein Grb2, which is pre-associated with SOS, a guanine nucleotide exchange factor [4, 5]
Time Course of Insulin, EGF, and PDGF-stimulated Shc Tyrosine Phosphorylation—Growth factor stimulation leads to tyrosine phosphorylation of Shc, with downstream activation of the Ras/MAP kinase pathway (6, 20 –22)
Summary
Vol 277, No 21, Issue of May 24, pp. 18592–18597, 2002 Printed in U.S.A. Phosphatidylinositol 3-Kinase Is Required for Insulin-stimulated Tyrosine Phosphorylation of Shc in 3T3-L1 Adipocytes*. Most of the attention has been focused on potential direct interactions between PI 3-kinase and Ras, but in this study, we have concentrated on an upstream activator of Ras, and we have explored potential interactions between PI 3-kinase signaling and Shc activation These studies have shown that PI 3-kinase activity is necessary for insulin-stimulated tyrosine phosphorylation of Shc, whereas other growth factors, such as PDGF and EGF, can efficiently signal to Shc in the absence of the PI 3-kinase requirement. As such, these experiments demonstrate a novel mechanism of cross-talk between. The PI 3-kinase and Ras/MAP kinase signaling pathways and demonstrate the specificity of this mechanism for the insulin action cascade
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