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Ε Phosphorylation of a Common Site in CD3 Domain Binding Via Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3ε This information is current as Tapio Kesti, Anja Ruppelt, Jing-Huan Wang, Michael Liss, of October 2, 2021. Ralf Wagner, Kjetil Taskén and Kalle Saksela J Immunol 2007; 179:878-885; ; doi: 10.4049/jimmunol.179.2.878 http://www.jimmunol.org/content/179/2/878 Downloaded from References This article cites 34 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/179/2/878.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Reciprocal Regulation of SH3 and SH2 Domain Binding via Tyrosine Phosphorylation of a Common Site in CD3␧1 Tapio Kesti,* Anja Ruppelt,† Jing-Huan Wang,‡ Michael Liss,§ Ralf Wagner,§¶ Kjetil Taske´n,† and Kalle Saksela2*‡ 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 Downloaded from 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 Journal of Immunology, 2007, 179: 878–885. http://www.jimmunol.org/ cells recognize foreign peptides presented by MHC mol- sponses result in the activation of multiple signaling pathways, ecules of APCs. The TCR consists of a ␣␤ TCR het- changes in actin cytoskeleton, and induction of numerous genes. T erodimer (or ␥␦ TCR in ␥␦ T cells) responsible for ligand Although phosphorylation of ITAMs has been regarded as the binding and is associated with the signal-transducing CD3 com- earliest signaling event following TCR triggering, it has been plex composed of heterodimeric CD3␥-CD3␧ and CD3␦-CD3␧ shown that the TCR-CD3 complex undergoes a conformational and homodimer CD3␨ (see Ref. 1). The cytoplasmic tails of CD3 change that occurs even earlier and independently of phosphory- chains include tyrosine- and leucine-containing motifs called lation, which exposes a proline-rich region in the cytoplasmic tail ␥ ␦ ␧ ␧ ITAM (YxxL/I(x6–8)YxxL/I) (2). CD3 , CD3 , and CD3 have of CD3 (5–7). This alteration enables binding of Nck via its first by guest on October 2, 2021 one ITAM each, whereas CD3␨ contains three ITAM. These are SH3 domain (in the following referred to as Nck(I/III)-SH3) (5). phosphorylated upon TCR ligation by the Src family protein ty- This conformational change has been proposed to be essential for rosine kinase Lck (3), generating docking sites for Src homology T cell activation (5), and shown to correlate spatially and tempo- (SH)3 2 domain-containing proteins. Zap70 is the principal CD3 rally with negative selection of T cells (8), but the functional im- ITAM-binding SH2 protein, but other interacting proteins have portance of the proline-rich residues in CD3␧ has been recently also been reported (2). Recruitment of Zap70 leads to its phos- challenged (9). phorylation and activation, further tyrosine phosphorylation, and The SH3 domain is the most common modular protein binding recruitment of other kinases and adapter proteins (4). These re- domain in nature. It binds proline-rich sequences, which often con- tain the consensus sequence proline-x-x-proline (for reviews, see Refs. 10, 11). SH3 domains are typically found in proteins in- *Department of Virology, Haartman Institute, University of Helsinki and Helsinki volved in signal transduction, membrane trafficking, and cytoskel- University Central Hospital, Helsinki, Finland; †Biotechnology Centre, University of Oslo, Oslo, Norway; ‡Institute of Medical Technology, University of Tampere and etal organization. We have recently generated an essentially com- Tampere University Hospital, Tampere, Finland; §Geneart, Regensburg, Germany; plete collection (n ϭ 296) of human SH3 domains in the form of ¶ and Institute of Medical Microbiology and Hygiene, University of Regensburg, Re- a phage display library, and used this system for identification of gensburg, Germany preferred SH3 partners for different ligand proteins (12). Because Received for publication April 17, 2007. Accepted for publication May 1, 2007. studies by Gil et al. (5) suggested an important role for SH3 bind- 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 ing by CD3 in TCR function, but did not exclude the possibility with 18 U.S.C. Section 1734 solely to indicate this fact. that SH3 proteins other than Nck(I/III) could be involved, we de- 1 This work was supported by grants from the Academy of Finland, Medical Research cided to carry out a comprehensive and unbiased characterization Council of Tampere University Hospital, Medical Research Council of Helsinki Uni- of SH3 binding preferences of CD3␧. versity 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 Materials and Methods Virology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, FIN-0014 Reagents and cell culture Helsinki, Finland. E-mail address: kalle.saksela@helsinki.fi ␧ 3 Abbreviations used in this paper: SH, Src homology domain; TBP, TATA-box The expression constructs for CD3 and Eps8L1 (epidermal growth binding protein; HA, hemagglutinin. factor receptor pathway substrate 8-like 1) were generated from cDNA clones purchased from Deutsches Resourcenzentrum fu¨r Genomfors- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 chung (CD3␧ (IRATp970D0474D) and Eps8L1 (IMAGp998E1911431Q www.jimmunol.org The Journal of Immunology 879 and IRALp962L1134Q); RZPD). These cDNA clones and the GST con- structs were cloned into pEBB (13), a mammalian expression vector with a strong EF-1 promoter. Plasmid pEBB-Nck-Myc was obtained from B. J. Mayer (University of Connecticut, Farmington, CT) and pLckY505F sfrom T. Mustelin (Burnham Institute, La Jolla, CA). The CD8/CD3␧ chimera consists of the ectodomain and transmembrane domain of human CD8␣ with two tandem copies of the Myc epitope inserted after the signal peptide FIGURE 1. Amino acid sequence of the cytoplasmic tail of human cleavage site, fused to the cytoplasmic tail of human CD3␧. In the Myc- CD3␧. These residues were expressed as a GST-fusion protein as indicated, ␧ ␣ TM-CD3 chimera, the ectodomain of CD8 was deleted, that is, the Myc or similarly fused to maltose-binding protein or the transmembrane and epitopes are between the CD8 signal peptide and transmembrane domain. ectodomains of CD8. The PxxDY motif is underlined and the ITAM ty- Details regarding all plasmid constructs are available upon request. 293FT cells were used for transfections; they were maintained in DMEM high rosine residues are in boldface type. glucose supplemented with 10% FBS and 2 mM glutamine. A standard calcium phosphate precipitation method was used for all transient trans- Analysis of mRNA expression fections. Typically, 2 ␮g of pEBBGST plasmids, 3 ␮g of pLck-DA, and 6 ␮g of plasmid for CD3␧ were used per a 10-cm dish. Immunoprecipita- Total RNA was isolated from 107 cells using GenElute Mammalian Total tions, electrophoresis, and immunoblotting were done by standard methods RNA Miniprep kit (Sigma-Aldrich). A total of 2.5-␮g samples were first (14). Cells were lysed in 1% Nonidet P-40 (14) buffer containing 50 mM treated with RNase-free DNase I (Fermentas UAB) to remove any con- octylglucopyranoside (Sigma-Aldrich). The human SH3 phage library and taminating DNA, and then either reverse-transcribed
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