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Cell Activation , Is Required for T Lck SH2 Domain of P56 Lad, An Lad, an Adapter Protein Interacting with the SH2 Domain of p56 lck, Is Required for T Cell Activation This information is current as Young Bong Choi, Chan Ki Kim and Yungdae Yun of September 27, 2021. J Immunol 1999; 163:5242-5249; ; http://www.jimmunol.org/content/163/10/5242 Downloaded from References This article cites 54 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/163/10/5242.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 September 27, 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Lad, an Adapter Protein Interacting with the SH2 Domain of p56lck, Is Required for T Cell Activation1,2 Young Bong Choi,*† Chan Ki Kim,* and Yungdae Yun3*† T cell-specific Src family tyrosine kinase, p56lck, plays crucial roles in T cell differentiation, activation, and proliferation. These multiple functions of p56lck are believed to be conducted through the protein-protein interactions with various cellular signaling proteins. To clarify the mechanisms through which p56lck contributes to T cell signaling, we identified the proteins binding to the Src homology 2 (SH2) domain of p56lck through a tyrosine phosphorylation-dependent yeast two-hybrid screening. Subsequent characterization of positive clones revealed the presence of a protein of 366 aa named Lad (Lck-associated adapter protein), which is a potential murine homologue of previously reported TSAd, a T cell-specific adapter protein. Lad contains several protein- protein interaction domains including a zinc-finger motif, an SH2 domain, a proline-rich SH3 binding motif, and several phos- lck photyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56 in vivo and redistributed from Downloaded from cytoplasm to the plasma membrane in a T cell activation-dependent manner. Moreover in T cells, IL-2 promoter activity was enhanced upon coexpression of Lad but was inhibited by the coexpression of antisense Lad RNA. These characteristics of Lad suggest that Lad play an essential role as an adapter protein in p56lck-mediated T cell signaling. The Journal of Immunology, 1999, 163: 5242–5249. 4 lck he best-characterized lymphocyte-specific member of the Like other Src family protein tyrosine kinases (PTK), p56 http://www.jimmunol.org/ Src family tyrosine kinases, p56lck, plays essential roles in consists of five domains: SH1 (Src homology domain 1), SH2, T cell signaling that regulates diverse T cell functions SH3, SH4, and NH unique domain. The SH1 is the enzymatic T 2 such as development, activation, proliferation, and adhesion. domain of PTK that phosphorylates tyrosines on cellular proteins Knock-out mice lacking p56lck show a pronounced thymic at- with catalytic specificity (16). The N-terminal unique domain in- rophy owing to blockade of the progression from CD42CD82 fluences substrate preference without the regulation of intrinsic double negative to CD41CD81 double positive thymocytes (1). kinase activity (17) and regulates interaction with protein tyrosine Transgenic mice harboring a dominant negative form of p56lck are phosphatases (18). The SH4 domain directs p56lck to the plasma defective in allelic exclusion of the pre TCR b-chain gene which membrane by denoting sites for lipidation such as palmitoylation lck permits normal thymic selection (2, 3). Both animal studies indi- or myristoylation (19, 20), which enables p56 to interact with by guest on September 27, 2021 cate that p56lck is important for T cell development (4). GPI-anchored proteins such as CD59 (21). The SH3 domain neg- During Ag-induced T cell activation, p56lck transmits a positive atively regulates the enzymatic activity of p56lck and is dispensable signal by interacting with the CD4/CD8 glycoproteins (5–7). Fur- for cell transformation by activated p56lck (F505) (22, 23). Several thermore, genetic evidence using JCaM1 cells lacking p56lck groups, however, reported that the SH3 domain interacts with sev- shows that p56lck is involved in TCR-mediated cell activation (8). eral cellular signaling proteins including phosphatidylinositol 3-ki- During T cell proliferation by IL-2, p56lck associates with the nase (PI3K) (24, 25), p120 (26), and LckBP1 (27) through their IL-2R b-chain (9) and regulates c-fos/c-jun gene expression (10, proline-rich motifs. The functional significance of the p56lck SH3 11). In addition, p56lck associates with other costimulatory adhe- domain in T cell signaling remains yet to be elucidated. Finally, sion molecules such as 4–1BB (12), CD2 (13), CD44 (14), and the SH2 domain negatively or positively regulates the function of L-selectin (15) to enhance T cell responsiveness. These multiple p56lck (22). In the inactive form of p56lck, the SH2 domain inter- functions of p56lck are believed to be conducted through interac- acts with its own phosphorylated Y505 (pY505), but in the active tion with various cellular signaling proteins. form of p56lck, the SH2 domain interacts with other tyrosine phos- phorylated cellular signaling proteins (28) to transmit a positive signal for T cell activation. The importance of p56lck in T cell activation has been described *Signal Transduction Laboratory Mogam Biotechnology Research Institute, Koosungmyon, Yonginsi, Kyunggido, Korea; and †Department of Molecular Life extensively and both the kinase and the regulatory domains have Science and Center for Cell Signaling Research, Ewha Women’s University, Seo- been shown to be required. A model was established in which, daemungu, Daehyundong 11-1, Seoul, 120-750, Korea. upon engagement with CD4/CD8, the kinase domain of p56lck Received for publication February 10, 1999. Accepted for publication August phosphorylates the z-chain of TCR and provides the binding site 30, 1999. for another kinase, ZAP-70. These successive events lead to the 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 amplification of TCR-mediated signaling (29). On the other hand, with 18 U.S.C. Section 1734 solely to indicate this fact. even though the kinase activity of p56lck is required for full T cell 1 This work was supported in part by grants from the Korea Green Cross Co. and the Ministry of Science and Technology of Korea. 2 The sequence reported in this article will appear in the GenBank database under accession no. U69460. 4 Abbreviations used in this paper: PTK, protein tyrosine kinase; SH1, Src homology 3 Address correspondence and requests to Dr. Y. Yun, Division of Molecular Life domain 1; PI3K, phosphatidylinositol 3-kinase; pY, phosphotyrosine; 59-RACE, 59- Science and Center for Cell Signaling Research, Ewha Women’s University, Seoul, rapid amplification of cDNA ends; Lad, Lck-associated adapter protein; TSAd, T 120-750, Korea. E-mail adddress: [email protected] cell-specific adapter protein; WT, wild type. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 5243 activation, a kinase-independent function of p56lck, mainly medi- grown in DMEM supplemented with 10% FBS and antibiotics. Thymo- ated by the SH2 domain was shown to independently contribute to cytes and splenocytes were isolated by passing mouse thymus and spleen T cell activation (7, 30–32). Subsequent efforts resulted in the through a sieve. Jurkat T cells were activated by cross-linking CD3 and CD4 with corresponding Abs, OKT3 and OKT4 (a gift of Dr. Shin, Har- identification of ZAP-70 (33), CD45 (34, 35), and Sam68 (36) as vard Medical School, Boston, MA), respectively, at a saturated concentra- lck binding partners of the p56 SH2 domain. However, this infor- tion. EL4 cells were activated by CD3 cross-linking with 145-2C11 Ab. mation does not fully explain the contribution of the SH2 domain COS-1 cells were transfected using the standard DEAE-dextran method. to the multiple functions of p56lck in T cells. Abs, immunoprecipitation, and Western blot analysis Here, to understand the mechanisms by which p56lck acts in T cell signaling and the role of the p56lck SH2 domain in this pro- Anti-p56lck Ab was obtained from Transduction Laboratory (Lexington, cess, we identified the binding partners of the p56lck SH2 domain KY) or generated by immunization of rabbits with GST fusion proteins using a tyrosine phosphorylation-dependent yeast two-hybrid sys- encompassing the SH3 and SH2 domain (aa 66–224). Antiphosphotyrosine (pY) Ab (4G10) and anti-SHP Ab were obtained from Upstate Biotech- tem. As a result of the screening, a novel protein of 366 aa that we nology (Lake Placid, NY). Anti-mouse CD3e (145-2C11), anti-CD90 named Lad (Lck adapter) was isolated. Upon T cell activation, Lad (Thy-1) (G7), anti-human CD3 (UCHT1), anti-hamster IgG, and anti- coimmunoprecipitated p56lck, was tyrosine phosphorylated, and mouse IgG were obtained from PharMingen (San Diego, CA). Polyclonal acted as a substrate of p56lck tyrosine kinase. Moreover, overex- antiserum against Lad or GST was raised in rabbits immunized with GST- Lad C terminus (aa 208–366) or GST, respectively. For immunoprecipi- pression of dominant negative Lad blocked the IL-2 promoter- tation or Western blot, cells were lysed with TNE buffer (50 mM Tris (pH driven transcriptional activation following TCR stimulation.
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