help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Formisano, P.
Right arrow Articles by Beguinot, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Formisano, P.
Right arrow Articles by Beguinot, F.

Endocrinology, Vol 128, 2949-2957, Copyright © 1991 by Endocrine Society

ARTICLES

Antiphosphotyrosine immunoprecipitation of an insulin-stimulated receptor phosphatase activity from FRTL5 cells

P Formisano, G Condorelli, G Condorelli and F Beguinot
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, II Facolta di Medicina, Universita degli studi di Napoli, Italy.

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.


This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
S. P. Mirza and M. Olivier
Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry
Physiol Genomics, October 8, 2008; 33(1): 3 - 11.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals
Copyright © 1991 by The Endocrine Society