Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.
The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration.
Offermanns
S
S
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Bombien
E
E
Schultz
G
G
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D009132:Muscles / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007328:Insulin; D010982:Platelet-Derived Growth Factor; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D000001:Calcimycin; D014443:Tyrosine; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium