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Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors
K Yonezawa1, A Ando, Y Kaburagi
1Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Abstract:
We have examined the involvement of insulin receptor (IR) substrate-1 (IRS-1) and/or Shc in the upstream of Ras activation in insulin signaling using Chinese hamster ovary (CHO) cell lines overexpressing wild-type (CHO-IR) cells) or mutant insulin receptors. In CHO-IR cells, insulin rapidly phosphorylated IRS-1 and Shc at tyrosine residues and stimulated the formation of the active GTP-bound Ras (Ras.GTP). In contrast, a CHO cell line overexpressing the kinase-negative mutant insulin receptor substituting Arg1018 for Lys1018 was unable to tyrosine-phosphorylate IRS-1 and Shc and failed to activate Ras in response to insulin. A CHO cell line overexpressing the mutant insulin receptor, substituting Ala960 for Tyr960 and which was known to exhibit impaired tyrosine phosphorylation of IRS-1 and biological effects evoked by insulin, showed severely impaired insulin-dependent tyrosine phosphorylation of Shc and moderately impaired activation of Ras. Another cell line overexpressing the mutant insulin receptor, lacking 82 amino acids of the C terminus of beta-subunit and which was recently reported to retain normal insulin-dependent tyrosine phosphorylation of IRS-1, showed slightly impaired Ras activation at 10(-7) M insulin with severely reduced tyrosine phosphorylation of Shc protein. Furthermore, insulin did not induce the association of tyrosine-phosphorylated IRS-1 and Shc in CHO-IR cells. These results suggest that Shc and IRS-1 lie in the separate signaling pathways and that the tyrosine phosphorylation of IRS-1 with or without some low level of Shc phosphorylation may be enough to stimulate the submaximal accumulation of Ras.GTP complex and may need synergistically the higher level of tyrosine phosphorylation of Shc to induce the full activation of Ras in insulin signaling.
Insights
Insulin receptor substrate-1 (IRS-1) and Shc proteins act in separate pathways to activate Ras in insulin signaling. IRS-1 phosphorylation alone partially activates Ras, while Shc phosphorylation is needed for full Ras activation.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Endocrinology
Background:
- Insulin receptor (IR) signaling is crucial for glucose metabolism and cellular growth.
- Ras activation is a key downstream event in insulin signaling, but its upstream regulators are not fully elucidated.
- Insulin receptor substrate-1 (IRS-1) and Shc are known signaling adaptors in various growth factor pathways.
Purpose of the Study:
- To investigate the distinct roles of IRS-1 and Shc in the upstream activation of Ras by insulin.
- To determine whether IRS-1 and Shc function in parallel or converging pathways in insulin signaling.
- To characterize the impact of specific insulin receptor mutations on IRS-1 and Shc phosphorylation and Ras activation.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cell lines overexpressing wild-type or mutant insulin receptors.
- Assessed insulin-induced tyrosine phosphorylation of IRS-1 and Shc using immunoprecipitation and Western blotting.
- Measured the activation of Ras by quantifying the levels of GTP-bound Ras (Ras.GTP).
Main Results:
- Insulin treatment of CHO-IR cells induced rapid tyrosine phosphorylation of IRS-1 and Shc, leading to Ras activation.
- Kinase-negative IR mutants abolished IRS-1, Shc phosphorylation, and Ras activation.
- Mutant IRs with impaired IRS-1 or Shc phosphorylation exhibited differential effects on Ras activation, suggesting distinct pathway contributions.
- IRS-1 and Shc did not associate upon insulin stimulation, supporting their operation in separate pathways.
Conclusions:
- IRS-1 and Shc function in parallel signaling pathways upstream of Ras activation in insulin signaling.
- Tyrosine phosphorylation of IRS-1, with or without low-level Shc phosphorylation, is sufficient for submaximal Ras activation.
- Full Ras activation requires synergistic signaling, involving a higher level of Shc tyrosine phosphorylation alongside IRS-1 phosphorylation.