Insulin and interleukin-4 induce desensitization to the mitogenic effects of insulin-like growth factor-I. Pivotal role for insulin receptor substrate-2.
Insulin-induced desensitization to insulin-like growth factor-I (IGF-I) stimulated mitogenesis in bovine fibroblasts involves steps distal to IGF-I binding to its tyrosine kinase receptor. When quiescent cultures of bovine fibroblasts were stimulated with 10 nM IGF-I and total cell lysates immunoblotted with anti-phosphotyrosine antibody, we observed a band at approximately 97 kDa, representing the beta-subunit of the IGF-I receptor, and a predominant tyrosyl-phosphorylated species migrating as a broad band between 170 and 190 kDa. The majority of proteins in this latter band were immunoprecipitated by antibodies against insulin receptor substrate (IRS)-2 and not by antibodies against IRS-1. Pretreatment of bovine fibroblasts with 10 nM insulin for 48 h blocked subsequent IGF-I-stimulated DNA synthesis and the IGF-I-induced increase in tyrosyl-phosphorylated IRS-2. Insulin pretreatment did not alter IRS-1 or IRS-2 expression by these cells, as assessed by metabolic labeling and direct immunoblotting with IRS antibodies. The interleukin-4 (IL-4) cytokine receptor also has IRS-2 as its major substrate for tyrosine phosphorylation. Although 10 nM IL-4 was as effective as 10 nM IGF-I in stimulating IRS-2 phosphorylation, 10 nM IL-4 did not have comparable mitogenic power in these cells. Nonetheless, pretreatment of bovine fibroblasts with IL-4 inhibited IGF-I-stimulated DNA synthesis by 50-60%, concomitant with a decrease in IGF-I-induced IRS-2 phosphorylation. Insulin-induced desensitization could be prevented if a specific inhibitor of phosphatidylinositol 3-kinase (LY294002), but not an inhibitor of mitogen-activated protein kinase (PD98059), was present during the preincubation period. LY294002 also prevented the shift in IRS-2 molecular mass in response to prolonged incubation of cells with insulin. These data indicate that, in a nontransformed cell system, IRS-2 plays a key role in cellular desensitization to IGF-I-stimulated mitogenesis most likely through a feedback mechanism in the phosphatidylinositol 3-kinase pathway. Furthermore, they suggest that signaling through IRS-2 may provide an important point of integration for hormone, growth factor, and cytokine receptor systems that regulate critical cellular growth responses.
Haddad
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Endocrine Research Unit, Mayo Clinic and Mayo Foundation, 5-164 W. Joseph, Rochester, Minnesota 55905, USA.
Conover
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CA
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008934:Mitogens / Q000494:pharmacology*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D017526:Receptor, IGF Type 1 / Q000378:metabolism
D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008934:Mitogens; D010750:Phosphoproteins; D015847:Interleukin-4; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases