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
A. Chakravorty et al., 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, Endocrinology, 133(3), 1993, pp. 1331-1340
The pleiotropic effects of insulin and insulin-like growth factor-I (I
GF-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 autopho
sphorylation of the receptor and phosphorylation of endogenous and exo
genous substrates. Insulin and IGF-I receptors were isolated by wheat
germ agglutinin-agarose chromatography and immunoprecipitation with an
tiphosphotyrosine antibodies (alphaPY20). Insulin and IGF-I treatment
of purified receptors or luteal cells in culture resulted in phosphory
lation 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 an
tibody. The protein tyrosine kinase activity of the receptors was also
stimulated after insulin or IGF-I treatment, resulting in a 3- to 4-f
old increase in phosphorylation of a synthetic substrate poly(Glu4:Tyr
1). Insulin and IGF-I treatment also increased (4.5-fold) phosphatidyl
inositol-3-kinase (PI-3-kinase) activity in alphaPY20 immunoprecipitat
es 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 s
ensitivity of the enzyme to detergents and by immunoblot analysis usin
g a PI-3-kinase antibody. The actions of insulin and IGF-I on tyrosine
kinase and PI-3-kinase activity were associated with increased protei
n and DNA synthesis. Insulin and IGF-I treatment of cultured luteal ce
lls for 18 h increased [H-3]thymidine incorporation (6- to 7-fold). Th
e 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 insul
in and IGF-I on thymidine incorporation were blocked by genistein, a s
pecific inhibitor of protein tyrosine kinase. These findings demonstra
te a cascade of insulin and IGF-I receptor tyrosine kinase-mediated pa
thways in the bovine luteal cell, manifested as increased activity of
signal transduction enzymes and increased DNA synthesis.