Growth hormone stimulates tyrosine phosphorylation of insulin receptor substrate-1
AuthorsSouza, Sandra C.
Frick, G. Peter
Yip, Rupert Guk-Chor
Lobo, Raysildo B.
Goodman, H. Maurice
Document TypeJournal Article
KeywordsAdipocytes; Adipose Tissue; Animals; Blotting, Western; Cells, Cultured; Epididymis; Growth Hormone; Insulin; Kidney; Kinetics; Male; Phosphoproteins; Phosphorylation; Phosphotyrosine; Rats; Rats, Inbred Strains; Triglycerides; Tyrosine
Medicine and Health Sciences
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AbstractGrowth hormone (GH) produces insulin-like effects in rat adipocytes that have been deprived of GH for at least 3 h. The effect of a saturating concentration of GH is qualitatively and quantitatively similar to that produced by 2-4 ng/ml insulin but differs from that of insulin in the respect that adipocytes become refractory to prolonged or repeated stimulation with GH. Since activation of tyrosine kinase is an early event in the action of both hormones, we investigated the possibility that GH stimulation of tyrosine phosphorylation of some protein in the insulin transduction cascade might result in the similar effect of the two hormones. Adipocytes were preincubated for 3 h in the absence of hormones and then reincubated without or with 500 ng/ml GH or 4-400 ng/ml insulin for 10 min. The cells were lysed with an equal volume of buffer containing 1% SDS and preheated to 100 degrees C. Proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes, and tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antiserum coupled to horseradish peroxidase and reagents to produce chemiluminescence. The faint band seen at 185 kDa in control lanes was increased by GH treatment in five independent experiments. Insulin produced a similar effect at a concentration of 4 ng/ml, and phosphorylation increased in a dose-related manner in cells treated with higher concentrations of insulin. A prominent approximately 95-kDa band that is probably not the beta subunit of the insulin receptor was also seen in GH-treated cells. The beta subunit of the insulin receptor has similar electrophoretic mobility to the 95-kDa protein, but was not phosphorylated to an extent that allowed detection when insulin was added at concentrations below 400 ng/ml. Phosphorylation of the 185- and 95-kDa bands was evident within 1 min after addition of GH, persisted for at least 30 min, and was equally prominent in sensitive and refractory cells. Antiserum to IRS-1 immunoprecipitated the tyrosine-phosphorylated 185-kDa protein. The data suggest that IRS-1 is a substrate for a GH-activated tyrosine kinase, possibly JAK-2, which may account for the insulin-like effects of GH. The data further suggest that refractoriness to insulin-like stimulation by GH may result from an additional GH-dependent action that is distinct from phosphorylation of IRS-1.
J Biol Chem. 1994 Dec 2;269(48):30085-8.