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Molecular and Cellular Biology, May 2005, p. 3831-3841, Vol. 25, No. 9
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.9.3831-3841.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dok-R Mediates Attenuation of Epidermal Growth Factor-Dependent Mitogen-Activated Protein Kinase and Akt Activation through Processive Recruitment of c-Src and Csk

Paul Van Slyke,^1,2 Mariano Loza Coll,^1,2 Zubin Master,^1,2 Harold Kim,² Jorge Filmus,^1,2,3 and Daniel J. Dumont^1,2,3,4*

Division of Molecular and Cellular Biology Research, Sunnybrook and Women's Research Institute, Toronto, Ontario, Canada M4N 3M5,¹ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada,² Toronto-Sunnybrook Regional Cancer Centre, Toronto, Ontario, Canada,³ Heart & Stroke/Richard Lewar Centre of Excellence, Faculty of Medicine, University of Toronto, Ontario, Canada⁴

Received 24 June 2004/ Returned for modification 19 August 2004/ Accepted 23 December 2004

Dok-R has previously been shown to associate with the epidermal growth factor receptor (EGFR) and become tyrosine phosphorylated in response to EGF stimulation. The recruitment of Dok-R to the EGFR, which is mediated through its phosphotyrosine binding (PTB) domain, results in attenuation of mitogen-activated protein kinase (MAPK) activation. Dok-R's ability to attenuate EGF-driven MAPK activation is independent of its ability to recruit rasGAP, a known attenuator of MAPK activity, suggesting an alternate Dok-R-mediated pathway. Herein, we have determined the structural determinants within Dok-R that are required for its ability to attenuate EGF signaling and to associate with c-Src and with the Src family kinase (SFK)-inhibitory kinase, Csk. We demonstrate that Dok-R associates constitutively with c-Src through an SH3-dependent interaction and that this association is essential to Dok-R's ability to attenuate c-Src activity and diminish MAPK and Akt/PKB activity. We further illustrate that EGF-dependent phosphorylation of Dok-R requires SFK activity and, more specifically, that SFK-dependent phosphorylation of tyrosine 402 on Dok-R facilitates the inducible recruitment of Csk. We propose that recruitment of Csk to Dok-R serves to bring Csk to c-Src and down-regulate its activity, resulting in a concomitant attenuation of MAPK and Akt/PKB activity. Furthermore, we demonstrate that Dok-R can abrogate c-Src's ability to protect the breast cancer cell line SKBR3 from anoikis and that an association with c-Src and Csk is required for this activity. Collectively these results demonstrate that Dok-R acts as an EGFR-recruited scaffolding molecule that processively assembles c-Src and Csk to attenuate signaling from the EGFR.

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* Corresponding author. Mailing address: Sunnybrook and Women's Research Institute, 2075 Bayview Avenue, Research Building, S-218, Toronto, Ontario, Canada M4N 3M5. Phone: (416) 480-5748. Fax: (416) 480-5703. E-mail: dan.dumont{at}sw.ca.

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Molecular and Cellular Biology, May 2005, p. 3831-3841, Vol. 25, No. 9
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.9.3831-3841.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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