The transactivated epidermal growth factor receptor recruits Pyk2 to regulate Src kinase activity.
G protein-coupled receptors such as proteinase-activated receptor 1 induce phosphorylation of mitogen-activated protein kinases through multiple pathways including transactivation of receptor tyrosine kinases. In vascular smooth muscle cells, both matrix-metalloproteinase-dependent extracellular shedding of membrane-bound epidermal growth factor (EGF) receptor ligands and activation of the nonreceptor tyrosine kinases Pyk2 and Src contributed to the thrombin-induced ERK1/2 phosphorylation. Surprisingly, disruption of the HB-EGF-mediated extracellular mode of EGF receptor transactivation also prevented the phosphorylation of the nonreceptor tyrosine kinases Pyk2 and Src, locating these kinases downstream of the transactivated EGF receptor. The ionomycin-induced Pyk2 phosphorylation was partially sensitive to AG1478, heparin, or the matrix-metalloproteinase inhibitor BB2116, and the ionomycin-induced EGF receptor phosphorylation was almost completely blocked by these inhibitors of extracellular transactivation. Coimmunoprecipitation experiments revealed that, upon thrombin stimulation, a signaling complex consisting of Pyk2 and Src assembles at the EGF receptor. Reconstitution of the signaling molecules in HEK293 or vascular smooth muscle cells and subsequent determination of the EGF-induced Src kinase activity applying fluorescent sensor proteins demonstrated that a Ca(2+)-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.
Schauwienold
Dag
D
Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Sastre
Alejandra Pérez
AP
Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany.
Genzel
Nadine
N
Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany; ImaGenes GmbH, 13125 Berlin, Germany.
Schaefer
Michael
M
Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany. Electronic address: michael.schaefer@medizin.uni-leipzig.de.
Reusch
H Peter
HP
Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011799:Quinazolines; D051381:Rats; D013917:Thrombin / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D000925:Anticoagulants; C088670:BB 2116; D004791:Enzyme Inhibitors; D006490:Hemostatics; D006877:Hydroxamic Acids; D007476:Ionophores; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D015759:Ionomycin; D006493:Heparin; C512478:EGFR protein, human; C483803:Egfr protein, rat; D066246:ErbB Receptors; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D045726:Metalloproteases; D013917:Thrombin