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

被引:42
作者
CHAKRAVORTY, A
JOSLYN, MI
DAVIS, JS
机构
[1] UNIV KANSAS,SCH MED,WOMENS RES INST,DEPT OBSTET & GYNECOL,2903 E CENT,WICHITA,KS 67214
[2] DEPT VET AFFAIRS MED CTR,WICHITA,KS 67214
关键词
D O I
10.1210/en.133.3.1331
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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 (alphaPY20). 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 alphaPY20 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 alphaPY20 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 [H-3]thymidine incorporation (6- to 7-fold). The effects of insulin and IGF-I were optimal at 10 mug/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.
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页码:1331 / 1340
页数:10
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