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J. Biol. Chem., Vol. 283, Issue 41, 27748-27756, October 10, 2008

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The Transactivated Epidermal Growth Factor Receptor Recruits Pyk2 to Regulate Src Kinase Activity^*

Dag Schauwienold, Alejandra Pérez Sastre, Nadine Genzel^¶, Michael Schaefer¹, and H. Peter Reusch

From the Abteilung Klinische Pharmakologie, Ruhr-Universität Bochum, 44780 Bochum, Germany, Neurowissenschaftliches Forschungszentrum, Molecular Pharmacology and Cell Biology, Charité-Universitätsmedizin Berlin, 14195 Berlin, Germany, and ^¶ImaGenes GmbH, 13125 Berlin, Germany

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 Ca²⁺-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.

Received for publication, February 21, 2008 , and in revised form, July 23, 2008.

^* This work was supported by Forschergruppe 341 and Graduiertenkolleg 276 and 865 of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Present address: Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Härtelstrasse 16-18, 04107 Leipzig, Germany. Tel.: 49-341-9724600; Fax: 49-341-9724609; E-mail: michael.schaefer{at}medizin.uni-leipzig.de.

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