Characterization of insulin and insulin-like growth factor-I actions in the bovine luteal cell: regulation of receptor tyrosine kinase activity, phosphatidylinositol-3-kinase, and deoxyribonucleic acid synthesis.
The pleiotropic effects of insulin and insulin-like growth factor-I (IGF-I) are mediated via the intrinsic tyrosine kinase activity of their receptors. The potential role of insulin and IGF-I receptor tyrosine kinases in the bovine luteal cell was investigated in terms of autophosphorylation of the receptor and phosphorylation of endogenous and exogenous substrates. Insulin and IGF-I receptors were isolated by wheat germ agglutinin-agarose chromatography and immunoprecipitation with antiphosphotyrosine antibodies (alpha PY20). Insulin and IGF-I treatment of purified receptors or luteal cells in culture resulted in phosphorylation of a protein of 95,000 mol wt. This phosphoprotein was further identified as the autophosphorylated beta-subunit of the insulin/IGF-I receptor by immunoprecipitation with an anti-beta-subunit receptor antibody. The protein tyrosine kinase activity of the receptors was also stimulated after insulin or IGF-I treatment, resulting in a 3- to 4-fold increase in phosphorylation of a synthetic substrate poly(Glu4:Tyr1). Insulin and IGF-I treatment also increased (4.5-fold) phosphatidylinositol-3-kinase (PI-3-kinase) activity in alpha PY20 immunoprecipitates from whole cell lysates, suggesting that this enzyme was regulated by a receptor tyrosine kinase-mediated mechanism. The presence of PI-3-kinase in alpha PY20 immunoprecipitates was further confirmed by the sensitivity of the enzyme to detergents and by immunoblot analysis using a PI-3-kinase antibody. The actions of insulin and IGF-I on tyrosine kinase and PI-3-kinase activity were associated with increased protein and DNA synthesis. Insulin and IGF-I treatment of cultured luteal cells for 18 h increased [3H]thymidine incorporation (6- to 7-fold). The effects of insulin and IGF-I were optimal at 10 micrograms/ml and 50 ng/ml, respectively, and were not additive. The stimulatory effects of insulin and IGF-I on thymidine incorporation were blocked by genistein, a specific inhibitor of protein tyrosine kinase. These findings demonstrate a cascade of insulin and IGF-I receptor tyrosine kinase-mediated pathways in the bovine luteal cell, manifested as increased activity of signal transduction enzymes and increased DNA synthesis.
Chakravorty
A
A
Women's Research Institute, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, Wichita 67214-4716.
Joslyn
M I
MI
Davis
J S
JS
D000818:Animals; D002417:Cattle; D002845:Chromatography; D003338:Corpus Luteum / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D005260:Female; D019833:Genistein; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D017830:Octoxynol; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D004247:DNA; D019833:Genistein; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D007529:Isoflavones; D017830:Octoxynol; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases; D011092:Polyethylene Glycols; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D014909:Wheat Germ Agglutinins; C010615:Nonidet P-40