Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation.
Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.
Unité Mixte de Recherche 7131, Centre Nationale de la Recherche Scientifique, Hôpital Broussais, 102 rue Didot, 75014 Paris, France.
Régis
R
Bobe
Xing
X
Yin
Marie-Cécile
MC
Roussanne
Olivier
O
Stepien
Evelyne
E
Polidano
Claude
C
Faverdin
Pierre
P
Marche
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D006490:Hemostatics / Q000494:pharmacology*; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D002118:Calcium; D002614:Chelating Agents; D004533:Egtazic Acid; D004791:Enzyme Inhibitors; D066246:ErbB Receptors; D006490:Hemostatics; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011743:Pyrimidines; D019284:Thapsigargin; D013917:Thrombin; D019061:src-Family Kinases; C412373:AG 1879; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester