Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors.
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.
Yonezawa
K
K
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Ando
A
A
Kaburagi
Y
Y
Yamamoto-Honda
R
R
Kitamura
T
T
Hara
K
K
Nakafuku
M
M
Okabayashi
Y
Y
Kadowaki
T
T
Kaziro
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D019000:Phosphotyrosine; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D011972:Receptor, Insulin / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)