Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways.
We compared the intracellular insulin-like growth factor-1 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2.Sos complexes, leading to p21ras activation. To compare the functional importance of p21ras for IGF-1-and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21ras. Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21ras antibody completely inhibited both IGF-1-and insulin-induced DNA synthesis, indicating the central role of p21ras in signaling by both hormones. Signal transduction from these receptors to Grb2.Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2.Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2.Sos, to p21ras, the delayed IGF-1 induced mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.
Sasaoka
T
T
First Department of Medicine, Toyama Medical & Pharmaceutical University, Japan. tsasaoka-tym@umin.u-tokyo.ac.jp
Ishiki
M
M
Sawa
T
T
Ishihara
H
H
Takata
Y
Y
Imamura
T
T
Usui
I
I
Olefsky
J M
JM
Kobayashi
M
M
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000906:Antibodies / Q000276:immunology; D005347:Fibroblasts / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism*; D008845:Microinjections; D008934:Mitogens / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000276:immunology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013552:Swine
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D008934:Mitogens; D010750:Phosphoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D007334:Insulin-Like Growth Factor I; D005982:Glutathione Transferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)