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J. Biol. Chem., Vol. 279, Issue 7, 5975-5983, February 13, 2004

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Mechanism of Csk-mediated Down-regulation of Src Family Tyrosine Kinases in Epidermal Growth Factor Signaling^*

Hidetada Matsuoka, Shigeyuki Nada, and Masato Okada

From the Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

The Src family tyrosine kinases (SFKs) play pivotal roles as molecular switches that link a variety of extracellular cues to intracellular signaling pathway. The function of SFK is regulated by phosphorylation at the C-terminal regulatory site mediated by Csk. Recently a novel SFK target Cbp (or PAG) was identified as a membrane-anchored scaffold protein for Csk. To establish the mechanism of Csk/Cbp-mediated regulation of SFK in vivo, we observed dynamic changes in the interaction of Csk with Cbp by utilizing fusion proteins with modified green fluorescent proteins: cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Upon SFK activation induced by epidermal growth factor stimulation, fluorescent resonance energy transfer (FRET) response was detected transiently at membrane ruffles in COS1 cells co-expressing CFP-Csk and Cbp-YFP and in cells expressing a single-molecule FRET indicator consisting of CskSH2 and Cbp. Suppression of SFK by PP2 or use of a mutant Cbp that lacks the Csk binding site abolished the FRET response, although a dominant-negative form of Csk enhanced and sustained the FRET response, demonstrating that the FRET response is dependent upon the SFK activity. These observations show that Csk/Cbp-mediated down-regulation of SFK takes place at membrane ruffles in an early stage of epidermal growth factor signaling and suggest that the Csk/Cbp-based FRET indicators are useful for monitoring the status of SFK in living cells.

Received for publication, October 14, 2003

^* This work was supported by a Grant-in aid for Scientific Research of Priority Areas, Cancer, from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains video images for Figs. 1A, 4A, 5B, and 7B.

To whom correspondence should be addressed: Dept. of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-8297; Fax: 81-6-6879-8298; E-mail: okadam{at}biken.osaka-u.ac.jp.

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