Endocrinology, Vol 129, 301-311, Copyright © 1991 by Endocrine Society

ARTICLES

Insulin and insulin-like growth factors stimulate in vivo receptor autophosphorylation and tyrosine phosphorylation of a 70K substrate in cultured fetal chick neurons

KA Kenner and KA Heidenreich
Department of Medicine, University of California, San Diego, La Jolla 92093.

Insulin and insulin-like growth factors (IGFs) have been shown to regulate neuronal growth and differentiation. To investigate the possible role of tyrosine phosphorylation in the neurotrophic actions of these peptides, we examined the effects of insulin, IGF-I, and IGF- II on tyrosine phosphorylation in cultured fetal chick neurons. Tyrosine phosphorylation was detected by immunoblot analysis using antiphosphotyrosine antibodies. Under basal conditions five major phosphoproteins (170K, 140K, 115K, 103K, and 44K) and a number of minor proteins were detected by three separate antisera. In response to insulin, IGF-I, or IGF-II, an 87K membrane-associated protein (pp87) became phosphorylated on tyrosine in a rapid, dose-dependent manner. The Mr of pp87 was identical to that of the insulin and IGF-I receptor beta-subunits. Comparison of the dose-response curves for pp87 phosphorylation by insulin and IGF-I suggests that each peptide stimulated autophosphorylation of its own receptor beta-subunit. The maximal response obtained with IGF-I was approximately 10-fold higher than that obtained with insulin (3- to 5-fold), consistent with the larger number of IGF-I receptors in these cells. The maximal response obtained with IGF-II was 4- to 7-fold higher than that with insulin, suggesting that it acts through the insulin receptor as well. Tyrosine that IGF-II acts through the insulin receptor as well. Tyrosine phosphorylation of other proteins by the activated receptor kinases was not detected in neurons cultured for 5 days, however neurons cultured for only several h contained a predominant 70K protein (pp70) that was phosphorylated on tyrosine in response to all three hormones. Tyrosine phosphorylation of pp70 was maximal after 2-5 h in culture and was undetectable in neurons cultured longer than 24 h. The time course and dose dependence of pp70 phosphorylation in response to hormone paralleled that of insulin and IGF-I receptor autophosphorylation. These results demonstrate that both insulin and IGF-I receptors are active tyrosine kinases in fetal neurons, and that pp70 represents a potential endogenous substrate for the insulin and IGF-I receptor kinases. The transient phosphorylation of pp70 may be involved in the neurotrophic effects of insulin and insulin-like growth factors.

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