Insulin and insulin-like growth factor-I stimulate a common endogenous phosphoprotein substrate (pp185) in intact neuroblastoma cells.

D Leroith,J Shemer,Y Zick,G L Wilson,M Adamo,D Heffez

doi:10.1016/s0021-9258(18)47751-4

Abstract

Mouse neuroblastoma N18 cells contain specific high affinity insulin and insulin-like growth factor-I (IGF-I) receptors. Insulin and IGF-I induce phosphorylation, in intact cells, of their respective receptor beta subunits. The insulin receptor beta subunit is represented by a 95-kDa phosphoprotein that is recognized by a specific antiserum (B10). The IGF-I receptor beta subunit is represented by two phosphoproteins of molecular mass 95 and 105 kDa. The hormone-induced phosphorylation was rapid and dose-dependent occurring on both phosphoserine and phosphotyrosine residues. In addition, both insulin and IGF-I induced phosphorylation of an endogenous protein of molecular mass 185 kDa (pp185). The rapidity and dose dependency of the phosphorylation of pp185 suggested that it may represent a common endogenous substrate for the insulin and IGF-I receptors in these neural-derived cells. Phosphorylation was primarily on phosphoserine and phosphotyrosine residues. pp185 did not absorb to wheat germ agglutinin-agarose and was not stimulated by either epidermal growth factor or platelet-derived growth factor. The finding of pp185 in these neural-related cells as well as in non-neural tissues suggests that it may represent a ubiquitous endogenous substrate for both the insulin and IGF-I receptor kinases.

Highlights

From the Diabetes Branch, National Institute of Diabetes, Digestiveand Kidney Diseases, Nationul Institutes of Health, Bethesda, Maryland 20892 and the $Department of Chemical Immurwbgy, Weizmann Institute of Science, Rehovot,76100 Israel
Mouse neuroblastomaN18 cells contain specific high characterized on primary cultures of neuronal and glial cells affinity insulin and insulin-like growth factor-I (IGF- as well as established neural-derived cell lines [20,21,22,23,25,38]
We demonstrate that insulin and insulin-like growth factor-I (IGF-I) induce phosphorylation of their respective receptor p subunits in intact mouse neuroblastoma cells (N18).they stimulate the phosphorylation of a common endogenous substrate of M, 185,000

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

Vol 262, No 32, Issue of November 15, pp. 15476-15482,1987 Printed in U.S.A. Insulin andInsulin-like Growth Factor-I Stimulate aCommon Endogenous Phosphoprotein Substrate(pp185) in Intact Neuroblastoma Cells*. We demonstrate that insulin and IGF-I induce phosphorylation of their respective receptor p subunits in intact mouse neuroblastoma cells (N18).they stimulate the phosphorylation of a common endogenous substrate of M , 185,000 (pp185). Insulin and insulin-like growth factor-I (IGF-I)' both induce autophosphorylation of their respective receptor @ subunits [1,2,3,4,5,6,7,8] This effect is predominantly on tyrosine residues. Stimulation of 32Pincorporain detergent extracts of cells [9,10,11,12], whereas in intact cells tion into phosphoproteins was achieved using insulin (Elanco Prodexposure to these ligands results in receptor @ subunit phos- ucts Co., Indianapolis, IN) and IGF-I (AMGEN Biologicals, Thouphorylation on both tyrosine and serine residues (7, 8, 1316). The tyrosine-containing phosphoproteins that were stimulated by insulin ( I N S )included 95 kDa (W) and 185 kDa (A);those for IGF-I were 95 and 105kDa (0a)s well as 185kDa (A)(lower panel)

RESULTS

Mr x

ABCDE Mr x

DISCUSSION