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1
,¶
* Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland;
Biotechnology Centre, University of Oslo, Oslo, Norway;
Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland;
Geneart, Regensburg, Germany; and
¶ Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany
Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3
. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3 to Eps8L1 and Nck SH3 domains. CD3 bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3 in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3 to interact with either SH3 or SH2 domain-containing proteins.
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 This work was supported by grants from the Academy of Finland, Medical Research Council of Tampere University Hospital, Medical Research Council of Helsinki University Hospital, and Sigrid Juselius Foundation (to K.S.), and from the Functional Genomics Program (FUGE), The Research Council of Norway, Norwegian Cancer Society, and Novo Nordic Foundation Committee (to K.T.).
2 Address correspondence and reprint requests to Dr. Kalle Saksela, Department of Virology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, FIN-0014 Helsinki, Finland. E-mail address: kalle.saksela{at}helsinki.fi
3 Abbreviations used in this paper: SH, Src homology domain; TBP, TATA-box binding protein; HA, hemagglutinin.
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