title: Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin.
abstract: 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.
authors.affiliation: Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555-1043.
authors.forename: P
authors.initials: P
authors.lastname: Singh
authors.forename: S
authors.initials: S
authors.lastname: Narayan
authors.forename: R B
authors.initials: RB
authors.lastname: Adiga
mesh_terms: 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
chemical_list: D005755:Gastrins; D011505:Protein-Tyrosine Kinases