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.
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.
Zachary
I
I
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Sinnett-Smith
J
J
Rozengurt
E
E
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*
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