Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin

P. Singh, S. Narayan, R. B. Adiga

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Intracellular mechanisms that mediate mitogenic effects of gastrin remain largely unknown. The present studies were designed to examine if protein tyrosine kinase (PTKs) mediate growth effects of gastrin on a rat intestinal epithelial cell line (IEC-6 cells). Gastrin (<10 nM) was mitogenic for IEC-6 cells. PTK activity of cell membranes was stimulated in response to 0.01- 10.0 nM and 0.05-10.0 μM gastrin in a double biphasic manner. Cells labeled with H332PO4 were stimulated with gastrin and cellular proteins immunoprecipitated with phosphotyrosine antibodies. Endogenous proteins were phosphorylated in a dose- (100% effective dose = 0.1-1.0 nM) and time- dependent manner; at >10 nM gastrin, the second peak of response was not measured in intact cells. Thus the growth and phosphorylation response of intact cells to gastrin was similar. Both high [dissociation constant (K(d)) = 1 nM] and low (K(d) = ~0.1 μM)-affinity gastrin binding sites are present on IEC-6 cells. The results of the present study suggest, that occupancy of both high- and low-affinity gastrin-binding sites can potentially activate membrane-associated PTKs. However, in intact cells, occupancy of low-affinity sites apparently attenuates kinase activity resulting in reduced protein phosphorylation. Eight protein bands [with relative molecular weight (M(r)) of 32-145 kDa] were tyrosine phosphorylated in intact cells in response to 0.1-1.0 nM gastrin, including two pp60 src-like proteins (with M(r) of 54 and 62 kDa). Thus the growth response pattern of a target cell to gastrin may depend on the stimulation of kinases and other factors (phosphatases?) that phosphorylate and/or dephosphorylate several proteins including c-src-like proteins in a dose-dependent manner.

Original languageEnglish (US)
Pages (from-to)G235-G244
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume267
Issue number2 30-2
DOIs
StatePublished - 1994

Keywords

  • IEC-6 cells
  • gastrin receptor
  • in vitro
  • mitogenic effects
  • tyrosine kinases

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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