Antiphosphotyrosine immunoprecipitation of an insulin-stimulated receptor phosphatase activity from FRTL5 cells.
Phosphotyrosine-containing proteins (PYPs) from FRTL5 epithelial cells were immunoprecipitated with Sepharose-linked phosphotyrosine antibody and phosphorylated in vitro with insulin and insulin-like growth factor-I (IGF-I) receptor kinases. Insulin preactivation of the kinases resulted in increased phosphorylation of a single 175,000 mol wt PYP (p175) beside insulin and IGF-I receptors. Phosphorylation of both p175 and receptors exhibited almost identical time courses and insulin dose responses, suggesting that p175 may serve as a substrate for the insulin and IGF-I receptor kinases. Preincubation of autophosphorylated receptors with PYPs derived from insulin-stimulated cells decreased 32P-labeled receptor precipitation by antiphosphotyrosine-Sepharose by 50-70% compared to that obtained upon preincubation with PYPs from unstimulated cells. This effect was not the result of PYP-receptor competition, was observed on both in vivo and in vitro phosphorylated receptors, and was almost completely blocked by 100 microM sodium vanadate, suggesting PYP copurification of a phosphotyrosine phosphatase. No phosphatase was recovered in lysates from insulin-unstimulated cells. Maximum activity was detected after 2 min of insulin stimulation, decreasing by more than 50% after 30 min. Antiphosphotyrosine recovery of p175 from cell lysates exhibited almost identical insulin dependency. Complete recovery of both p175 and phosphatase activity was obtained after receptor immunodepletion of the extracts. Thus, a receptor phosphatase activity from insulin-stimulated FRTL5 cells is retained on an antiphosphotyrosine affinity matrix and correlates with phosphorylation of the p175 substrate for the insulin and IGF-I receptor kinases.
Formisano
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P
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, II Facoltà di Medicina, Università degli studi di Napoli, Italy.
Condorelli
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G
Condorelli
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Beguinot
F
F
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D014443:Tyrosine / Q000037:antagonists & inhibitors* / Q000378:metabolism
D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin; D010744:Phosphoric Monoester Hydrolases