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.
Women's Research Institute, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, Wichita 67214-4716.
A
A
Chakravorty
M I
MI
Joslyn
J S
JS
Davis
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