Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor.
The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D008214:Lymphocytes / Q000201:enzymology; D046911:Macromolecular Substances; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D046911:Macromolecular Substances; D010768:Phosphoserine; D005576:Colforsin; D019000:Phosphotyrosine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014443:Tyrosine; D000242:Cyclic AMP; D011494:Protein Kinases; D011972:Receptor, Insulin