title: Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells.
abstract: Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.
authors.affiliation: Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
authors.forename: I
authors.initials: I
authors.lastname: Zachary
authors.forename: J
authors.initials: J
authors.lastname: Sinnett-Smith
authors.forename: E
authors.initials: E
authors.lastname: Rozengurt
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism*; D001839:Bombesin / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002474:Cell-Free System; D016232:Endothelins / Q000494:pharmacology*; D005755:Gastrins / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology; D014638:Vanadates / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology*
chemical_list: D000936:Antigen-Antibody Complex; D016232:Endothelins; D005755:Gastrins; D009479:Neuropeptides; D010455:Peptides; D014667:Vasopressins; C071998:Raytide; D019000:Phosphotyrosine; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D001839:Bombesin