AJP - Gastrointestinal and Liver Physiology, Vol 267, Issue 2 235-G244, Copyright © 1994 by American Physiological Society

ARTICLES

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

P. Singh, S. Narayan and R. B. Adiga
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555-1043.

Intracellular mechanisms that mediate mitogenic effects of gastrin remain largely unknown. The present studies were designed to examine if protein tyrosine kinases (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 microM gastrin in a double biphasic manner. Cells labeled with H3(32)PO4 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 (Kd) = 1 nM]- and low (Kd = approximately 0.1 microM)-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.

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