Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin.
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.
Singh
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Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555-1043.
Narayan
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S
Adiga
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RB
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005755:Gastrins / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats
D005755:Gastrins; D011505:Protein-Tyrosine Kinases