ISSN:
0730-2312
Keywords:
cell density
;
DNA synthesis
;
Mr receptor substrates
;
IRS-1 protein
;
tyrosine phosphorylation
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high Mr cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 × 105 to 3.2 × 106 per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high Mr receptor substrates (“pp185”) was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high Mr proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3′-kinase activity and only a 39% increase in phosphatidylinositol 3′-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP18 and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway. © 1996 Wiley-Liss, Inc.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
