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

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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 CD4ϪCD8Ϫ fluences substrate preference without the regulation of intrinsic double negative to CD4ϩCD8ϩ 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 ␤-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 ␤-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 phosphorylated 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 ␨-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; 5Ј-RACE, 5Ј- 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 CD3⑀ (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. 8.0), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4,5 Taken together, these results indicate that Lad plays an essential mM NaF, 25 ␮g/ml aprotinin, 1 mM PMSF, 25 ␮g/ml leupeptin, and 1 lck role as an adapter protein in p56 -mediated T cell signaling. mg/ml BSA) for1honice. All bands analyzed by Western blot were Downloaded from detected using the enhanced chemiluminescence protocol (Amersham, Ar- lington, Heights, IL). Materials and Methods Plasmids Purification of GST fusion proteins and the binding assay All bait plasmids encoding parts of murine p56lck and Lad were generated The GST fusion protein encompassing the SH2 domain of p56lck (aa 123– by cloning the PCR-amplified fragment into pGBT9, a GAL4 DNA bind- 224) or the C terminus of Lad (aa 208–366) was expressed in Escherichia ing domain vector (Clontech, Palo Alto, CA). Primers with EcoRI (5Ј coli and purified as previously described (39). For the binding study, 5 ␮g http://www.jimmunol.org/ primer) or BamHI (3Ј primer) restriction sites were used to facilitate the of GST-p56lck SH2 fusion protein immobilized on glutathione-Sepharose subcloning. The PCR products were digested with EcoRI/BamHI and 4B beads was incubated with Jurkat T cell lysates prepared in TNE buffer cloned into the corresponding site of pGBT9. The mammalian expression for1hat4°C. After washing, samples were analyzed by Western blotting plasmids of p56lck wild type (WT), F505, A273, K154, Lad, Lad-antisense, using the antiserum against GST-LadC. and Lad-SH2 were constructed by PCR amplification and subsequent in- sertion into the EcoRI/XhoI site of pcDNAI/Amp (Invitrogen, San Diego, In vitro kinase assay CA) using the same approach as described above. The point mutants of lck Purified GST-Lad C terminus or GST protein was incubated with 20 U of p56 were generated using a Quick mutagenesis kit (Stratagene, La Jolla, lck CA). The GST-fusion constructs of the SH2 domain of p56lck (aa 123–225) purified p56 (Upstate Biotechnology) in a kinase buffer containing 50 or the Lad C terminus (aa 208–366) were generated by PCR and subcloned mM Tris (pH 7.5), 1 mM DTT, 10 mM MnCl2, and 50 mM NaCl in the

␮ by guest on September 27, 2021 into pGEX-KG (Pharmacia, Piscataway, NJ) using EcoRI or BamHI/EcoRI presence or absence of 100 M ATP. After2hat30°C, the reaction sites, respectively. The pGL3/IL-2-Luc contains the luciferase reporter mixtures were subjected to SDS-PAGE followed by Western blot analysis gene downstream from the IL-2 promoter region, including 548 bp 5Ј of the using anti-pY Ab, 4G10 or anti-GST Ab. transcriptional starting site, and was generated by subcloning a HindIII fragment of pIL-2-CAT (37) into the HindIII site of pGL3-Basic (Promega, Subcellular fractionation Madison, WI). All steps were performed as described (40). Approximately 2 ϫ 107 EL4 Yeast two hybrid screen cells were activated by cross-linking CD3, resuspended in 0.5 ml of hy- potonic solution (25 mM Tris (pH 7.5), 5 mM EDTA, 5 mM EGTA, 250 A cDNA fragment encoding the SH2 and kinase domain (the constitutively mM sucrose, 25 ␮g/ml aprotinin, 1 mM PMSF, and 25 ␮g/ml leupeptin), active form, F505) of mouse p56lck was cloned into pGBT9. The resulting and then subjected to two successive freeze-thaw cycles. The cell suspen- plasmid, Lck SH2K (F505) was used as the bait in the yeast two-hybrid sion was homogenized on ice using a Dounce homogenizer (40 strokes), screens of a murine T cell lymphoma cDNA library cloned into pACT and the salt concentration was adjusted to 150 mM NaCl. Nuclei and other (Clontech). The bait and library DNAs were cotransformed by the lithium debris in the cell lysates were removed by two rounds of centrifugation at acetate method as previously described (38). Seventy-five out of 2 ϫ 106 480 ϫ g for 5 min. The soluble and particulate fractions were separated by transformants grew in the absence of histidine and showed detectable ␤-ga- centrifugation at 100,000 ϫ g for 30 min. Fractionated proteins were re- lactosidase staining within2hofincubation. To eliminate clones binding solved by SDS-PAGE and Western blotted with the corresponding Abs. to the bait in a tyrosine phosphorylation-independent manner, the plasmids from each positive clone were cotransformed with control bait plasmids Luciferase assay encoding SH2 domain only (Lck SH2) or kinase domain only (Lck K A total of 5 ϫ 106 Jurkat T cells were cotransfected with 2.5 ␮g each of (F505)). Approximately 70 clones, which did not bind to either of the two ␮ control baits, were finally selected and subjected to partial sequence de- IL-2-luc along with 2.5 g of pcDNAI/Lad, pcDNAI/Lad-antisense or termination. The sequences revealed that seven clones contained a part of pcDNAI/Lad-SH2 using Superfect (Qiagen, Chatswroth, CA). After incu- the same cDNA encoding a novel protein which we named Lad. bation for 24 h with DNA-Superfect mixtures, cells were activated by incubation on anti-CD3 Ab plates coated with 5 ␮g/ml UCHT1, by treat- cDNA cloning ment of 10 ␮g/ml PHA, or by treatment with 5 ng/ml PMA plus 0.5 ␮g/ml A23187 for 14 h and were harvested. One of the Lad clones isolated from the yeast two hybrid screen was employed as a probe to screen the mouse spleen cDNA library in ␭gt10 (Clon- tech). Three independent clones were obtained and subjected to nucleotide Results sequencing. In addition, 5Ј-RACE was performed using the mouse spleen Identification of Lad as a phosphorylation-dependent binding 5Ј stretch cDNA template (Clontech). From the combination of both ap- partner of the p56lck SH2 domain proaches, the cDNA encompassing ϳ1.6 kb in its entirety was obtained. Based on the previous report that the binding specificity of phos- Cells, activation, and transfection phopeptides to the p56lck SH2 domain overlaps with the substrate specificity of the p56lck kinase domain in a peptide library ap- Jurkat and EL4 cells were obtained from the American Type Culture Col- lck lection (Manassas, VA) and maintained in RPMI 1640 medium supple- proach (16), a part of p56 encompassing the SH2 and kinase mented with 10% FBS, 5 mM 2-ME, and antibiotics. COS-1 cells were domains (SH2K (F505) in Fig. 1A; aa 123–509) was used as a bait. 5244 Lck-BINDING ADAPTER PROTEIN: Lad

In this bait, Tyr505 was substituted for Phe505 to provide the constitutively active kinase form. In this system, presumably, the tyrosine kinase activity of the bait will phosphorylate the binding partner proteins expressed from the cotransformed library, which in turn will bind to the SH2 domain of the bait through pY to give a positive signal. From the screening of a total of 2 ϫ 106 independent colonies of a murine T cell lymphoma cDNA library, 75 strong positives were isolated and tested by retransformation with a series of control plasmids to confirm specificity and tyrosine phosphorylation-dependent binding (data not shown). Partial nucleotide sequencing of these cDNA fragments revealed that seven clones encoded a portion of the same protein that we named Lad for reasons that will be described in the latter part of this paper. The binding specificity of Lad to p56lck was studied further using the various control plasmids described in Fig. 1A. First, even if the cloning vectors for Lad and SH2K (F505) were switched, the interaction was not affected (Fig. 1B). Second, Lad interacted with SH2K (F505), but not with unrelated proteins such as GAL4 bind-

ing domain only, p53 protein, and hepatitis B virus X-gene prod- Downloaded from uct. Third, Lad did not interact with ZAP-70 SH2-p56lck kinase fusion protein (ZAP-70 SH2K (F505)) or PI3K SH2-p56lck kinase fusion protein (PI3K SH2K (F505)) (Fig. 1B), suggesting that Lad may preferentially bind to p56lck SH2 domain. Fourth, Lad did not interact with either the SH2 domain or kinase domain (F505) only

(Fig. 1C). This result supports the notion that the interaction be- http://www.jimmunol.org/ tween SH2K (F505) and Lad involves cooperation between the SH2 domain and the active kinase domain rather than the individ- ual binding to either one domain. Finally, the pY-dependent interaction was demonstrated further using the SH2 mutant (K154), which is incapable of binding to pY and the kinase mutant (A273), which is catalytcally inactive (Fig. 1C). Neither of these mutants showed any binding to Lad. by guest on September 27, 2021 Structural characteristics of Lad Next, a full open reading frame of Lad was obtained through the combination of screening of a mouse lung cDNA library and 5Ј- RACE from the mouse spleen 5Ј stretch cDNA (Fig. 2A). The open reading frame of Lad encodes a 366-aa protein with homology to a T cell-specific adapter protein (TSAd) recently reported as an inducible human protein in activated T cells (41), even though the function of TSAd remained elusive. Lad is potentially a mouse homologue of TSAd and displays several interesting features of a signaling molecule. The structure of Lad and TSAd is conserved except for the presence of a zinc-finger motif in Lad (Fig. 2B). The N terminus of Lad contains a CC-CC class of zinc-finger motif. Considering that some of the zinc finger motifs mediate protein- to-protein interaction, as exemplified by the association of ZPR1 to the cytoplasmic tail of epidermal growth factor receptor (42), the CC-CC motif of Lad may act as an interface for signaling molecules. The central region contains an SH2 domain, which belongs to the 1b class of SH2 domains for which the amino acid at ␤D5 FIGURE 1. Binding properties of Lad to p56lck in a yeast two-hybrid position is tyrosine or phenylalanine (43). The highest identity is system. A, Schematic representation of the baits used in the yeast two- found with the SH2 domains of GAPn, Csk, Grb2, and Src and hybrid assay. B, Bait-specific interaction of Lad. In the swapping experi- falls within the range of 25–35% (data not shown). The proline- ment, Lad was fused to GAL4 BD and SH2K (F505) was fused to the rich motif of Lad exactly matches the consensus sequence transcriptional activation domain (AD) of GAL4. Interaction of Lad with ϩPP␺PXKP (ϩ, basic amino acids; ␺, hydrophobic amino acids) unrelated proteins, such as GAL4 BD only, p53, hepatitis B Virus X-gene (44) preferred by class II cortactin SH3 domains found in cortactin, product (HBV-X), ZAP-70 SH2-p56lck kinase (ZAP-70 SH2K (F505)), and lck HS1, LckBP1, SH3P7, and SH3P8 (27, 45). The C terminus of Lad PI3K SH2-p56 kinase (PI3K SH2K (F505))was tested for control. C, 292 Tyrosine phosphorylation-dependent interaction of Lad to p56lck SH2 contains four potential pY sites, one of which is an NPXpY domain. Binding affinity of SH2K (F505) to Lad was compared with those motif known as the ligand for the pY binding domain (20). Over- of other baits. Binding affinity was scored as ϩϩϩ (deep blue), ϩϩ (in- all, these sequence characteristics suggest that Lad may be an termediate blue), ϩ (pale blue), or Ϫ (white) upon X-Gal (5-bromo-4- adapter protein with several domains possibly involved in protein- chloro-3-indolyl ␤-D-galactoside) staining. protein interactions. The Journal of Immunology 5245

FIGURE 2. Structural characteristics of Lad. A, Comparison of the deduced amino acid sequence of Lad with that of TSAd. The amino acids conserved between Lad and TSAd are marked by as- -Potential protein-protein inter .(ء) terisk action motifs, a zinc-ﬁnger motif, an SH2 domain, a proline-rich SH3 binding motif, and four pY sites are underlined. B, Schematic representation of the Lad structure. Downloaded from http://www.jimmunol.org/

On Northern blot analysis, Lad cDNA detected messages of 1.7 Western blot analysis with this Ab, Lad was detected as a 45-kDa and 4.4 kb in spleen and lung of mouse and of 1.8 and 4.9 kb in protein in EL4 mouse thymoma cells and mouse lymphocytes pu- spleen, thymus, and PBMC of human (Fig. 3A). The size of 1.7-kb riﬁed from thymus and spleen, but not in NIH 3T3 cells (Fig. 3B). message in mouse is consistent with that of assembled cDNA. At Notably, the expression level of Lad protein in thymus was much this stage, the identity of 4.4-kb and 4.9-kb message is not clear. higher than that in spleen. In addition, size of in vitro translated Next, an anti-Lad Ab was raised against the GST fusion protein Lad was identical to that in EL4 cells (Fig. 3C), even though the by guest on September 27, 2021 covering the C terminus of Lad (GST-LadC, aa 208–366). On calculated molecular mass was 40 kDa, indicating the authenticity of the predicted open reading frame.

Lad associates with p56lck upon TCR stimulation To confirm the binding of Lad with p56lck, GST fusion proteins of p56lck SH2 domain bound to glutathione-Sepharose 4B beads were incubated with Jurkat T cell lysates unstimulated/stimulated with anti-CD4 and CD3 mAbs (Fig. 4A). The surface expression of CD3 and CD4 on Jurkat T cells were confirmed by FACS analysis. CD3 and CD4 were detected in ϳ90% and 50% of the cells, respectively, and the total level of tyrosine phosphorylation was enhanced by CD3/CD4 cross-linking compared with that by CD3 cross-linking alone (data not shown). As shown in Fig. 4A, the GST-p56lck SH2 fusion protein precipitated a 52-kDa band, corresponding to the m.w. of TSAd. The amount of precipitated Lad was increased by ϳ3-fold upon T cell activation. Next, the in vivo interaction of Lad to p56lck was analyzed by coimmunoprecipitation studies in Jurkat or EL4 cells. For the employed EL4 cells, we observed that tyrosine phosphorylation of the cellular proteins was rapidly induced upon CD3 cross-linking (data not shown). A mAb (145-2C11) raised against the CD3⑀ of the murine TCR has been shown to induce IL-2 expression in EL4 cells (46, 47) and was employed for stimulation of EL4 cells. FIGURE 3. Expression of Lad. A, Tissue distribution of Lad mRNA. p56lck could be readily detected in the Lad immunoprecipitates of Northern blot analysis was performed with mouse and human Multiple the T cell lysates upon CD3/CD4 cross-linking of Jurkat T cells Tissue Northern blots (Clontech). B, Expression of Lad protein. Western blot analysis was performed with EL4, NIH 3T3, and purified thymocytes (Fig. 4B) or CD3 cross-linking of EL4 cells (Fig. 4C). Conversely, and splenocytes at 5 ϫ 106 cells/lane. C. Molecular weight of Lad. Lad is Lad was readily detected in the Lck immunoprecipitates upon CD3 detected as a 45-kDa protein upon in vitro translation and in T cell lysates. stimulation of EL4 cells (Fig. 4D). These results indicate that Lad lck (Ϫ), control without Lad mRNA; Lad, in vitro translation product of Lad inducibly binds to the SH2 domain of p56 in vivo upon T cell mRNA; EL4, lysates of mouse EL4 T cell. activation through TCR. 5246 Lck-BINDING ADAPTER PROTEIN: Lad Downloaded from

FIGURE 4. In vitro and in vivo interaction of Lad with p56lck. A, Direct binding of Lad to the SH2 domain of p56lck. Jurkat T cell lysates unstimulated http://www.jimmunol.org/ (Ϫ) or stimulated by CD3/CD4 cross-linking (ϩ) were incubated with GST or GST-p56lck SH2 fusion protein. After extensive washing, bound Lad protein was detected by anti-Lad immunoblotting. B, Lad associates with p56lck in vivo. Immunopecipitation with anti-Lad Ab and Western with anti-p56lck Ab. The Lad immunoprecipitates of unstimulated (Ϫ) or CD3/CD4 cross-linked (ϩ) Jurkat T cells were separated on a 10% SDS-polyacrylamide gel and analyzed by anti-p56lck immunoblotting. IP, Ab used for immunoprecipitation. C was identical to B except that EL4 cells were used and stimulated by CD3 cross-linking. EL4 T cell lysates (lysate, third lane) were included as a control. The p56lck coimmunoprecipitating with Lad is marked with an arrowhead. p56lck was detected only when cells were stimulated with anti-CD3 Ab as a band partly overlapping with Ig heavy chain. The amount of immunoprecipitated Lad in each lane is shown in a lower panel. D, Immunoprecipitation with anti-p56lck Ab and Western blotting with anti-Lad Ab. PI, preimmune serum. p56lck was immunoprecipitated from unstimulated (Ϫ) or CD3 cross-linked (ϩ) EL4 T cell lysates and analyzed by Western bolt with anti-Lad Ab. EL4 lck cell lysates (lysate, ﬁfth lane) were included as a control. The amount of immunoprecipitated p56 in each lane is shown in a lower panel. by guest on September 27, 2021

Lad is phosphorylated upon TCR-stimulation tial pY sites was incubated with purified p56lck in the presence or Insomuch as Lad contains several potential tyrosine phosphoryla- absence of ATP (Fig. 5C). Upon immunoblotting with anti-pY, tion sites, we studied the tyrosine phosphorylation status of Lad by GST-LadC was detected as a phosphoprotein. Taken together, these results suggest that Lad is most likely a direct substrate of immunoprecipitation of Lad from EL4 T cell lysates and subse- lck quent immunoblotting with anti-pY Ab (Fig. 5A). Following CD3 p56 upon TCR stimulation. stimulation, tyrosine phosphorylation of Lad was rapidly induced within 10 min and maintained up to 60 min. These results show Lad is required in IL-2 gene expression that Lad is a phosphoprotein and that its phosphorylation is induc- To address the functional significance of Lad in T cell activation, ible through TCR stimulation. In addition, phosphoproteins with we overexpressed Lad in a sense (S) or an antisense (AS) orien- sizes of 52 kDa, 56–58 kDa, and 70–80 kDa were coimmunopre- tation in Jurkat T cells and examined its effect on the IL-2 pro- cipitated with Lad. The size of 56- to58-kDa bands corresponds to moter-driven reporter activity upon T cell stimulation (Fig. 6A). In those of p56lck. Subsequently, to test whether Lad is phosphory- addition, we tested the effect of overexpression of the Lad SH2 lated by p56lck, Lad was coexpressed with p56lck WT, F505, or domain (SH2), a potential dominant negative form. The overex- A273 in COS-1 cells (Fig. 5B). As shown in the first panel of Fig. pression of Lad AS or the SH2 domain resulted in the repression 5B, immunoblotting of total cell extracts with anti-pY Ab dis- of CD3- or PHA-stimulated reporter activity by about 70%, indi- played that a 45-kDa band was heavily tyrosine-phosphorylated by cating the requirement of Lad in TCR-mediated IL-2 gene activa- p56lck (F505), but not by p56lck WT or p56lck A273. The expres- tion (upper and middle panels of Fig. 6A). PMA plus A23187 sion of p56lck and its mutants was confirmed by immunoblotting (PϩI)-stimulated reporter activity, however, was not repressed by with anti-p56lck Ab (Fig. 5B, the second panel). Next, to confirm the overexpression of Lad anti-sense or the SH2 domain (lower that Lad is phosphorylated by p56lck, Lad was immunoprecipitated panel of Fig. 6A). These results indicate that Lad acts in the TCR- with anti-Lad Ab and the immunoprecipitates were analyzed by proximal signaling events upstream of PMA/ionomycin. More- Western blot with anti-pY Ab. As shown in the third panel of Fig. over, without any activation signals, overexpression of Lad itself 5B, Lad was heavily tyrosine-phosphorylated by p56lck (F505), but led to a 7-fold induction of the IL-2 promoter-driven luciferase not by p56lck WT or p56lck (A273). The same blot was reprobed activity, supporting that Lad is involved in events leading to IL-2 with anti-Lad Ab to confirm the amount of precipitated Lad (Fig. gene activation. However, under CD3- or PHA-stimulation condi- 5B, the fourth panel). Finally, to test whether Lad is a direct sub- tions, overexpression of Lad showed only a marginal effect sug- strate of p56lck, we performed in vitro kinase assay in a cell-free gesting that some components of the signaling pathway may be system. Purified GST-LadC (aa 208–366) containing four poten- near saturation. Under the experimental condition, Lad antisense The Journal of Immunology 5247

FIGURE 5. Analysis of Lad phosphorylation. A, Lad is tyrosine phosphorylated upon T cell activation. Lad was immunoprecipitated from EL4 T cell lysates activated by CD3 cross-linking for 0, 10, 30, and 60 min. The immunoprecipitates were subjected to Western blot analysis with anti-pY (4G10) Ab (upper panel) or anti- Lad Ab (lower panel). B, Phosphorylation of Lad by p56lck in COS-1 cells. COS-1 cells were transfected with plasmids expressing Lad only (lane 1), Lad plus p56lck (WT) (lane 2), Lad plus p56lck (F505) (lane 3), and Lad plus p56lck (A273) (lane 4). Total cell extracts were analyzed by Western blot with 4G10 (first panel) and anti-p56lck Ab (second panel). Subse- quently, Lad was immunoprecipitated from Downloaded from COS-1 cell extracts and the immunoprecipitates were analyzed by Western with 4G10 (third panel), or anti-Lad Ab (fourth panel). C, In vitro phosphorylation of Lad by p56lck. Upper panel, purified GST or GST-LadC (Lad C terminus, aa 208–366) was incubated with purified p56lck in ϩ Ϫ the presence ( ) or absence ( ) of ATP. The http://www.jimmunol.org/ kinase reaction mixtures were analyzed with anti- pY immunoblotting. The lower panel shows the amount of loaded GST or GST-LadC as a control. by guest on September 27, 2021 effectively inhibited the expression of Lad protein induced upon involved in the up-regulation of the IL-2 gene expression upon T CD3 stimulation in a dose-dependent manner (Fig. 6B). Taken cell activation; and 5) Lad is redistributed to the plasma membrane together, these results demonstrate that Lad is required for the upon TCR stimulation. These results indicate that Lad physically TCR-mediated signaling pathway leading to IL-2 gene expression. interacts with p56lck upon T cell activation and plays an important role in the p56lck-dependent T cell signaling. Activation-dependent plasma membrane localization of Lad Insomuch as Lad is directly phosphorylated by p56lck in intact To examine the subcellular localization of Lad, we prepared the cells and by purified p56lck in vitro (Fig. 5, B and C), it is most cytoplasmic and particulate fractions from CD3-stimulated EL4 likely that the tyrosine phosphorylation of Lad upon T cell acti- cells and studied the distribution of Lad by Western blot analysis vation is mediated by p56lck. However, we do not exclude the (Fig. 7). In the absence of stimulation, the majority of Lad was possibility that the initial tyrosine phosphorylation is mediated by detected in the cytoplasmic fraction. Upon stimulation by CD3 other tyrosine kinases (e.g., Csk, ZAP-70, Tec family, and p59fyn) cross-linking, the level of cytoplasmic Lad gradually diminished known to be involved in T cell activation. Upon translocation to and was almost nondetectable after 2 h, whereas the level of par- the plasma membrane, the SH2 domain, proline-rich motif or zinc- ticulate Lad gradually increased during that time. On the other finger motif of Lad may recruit additional signaling molecules to hand, the levels of SHP-1 and Thy-1, employed as controls, were the p56lck/TCR complex leading to the amplification of activation consistent throughout the period (Fig. 7). In addition, redistribution signals. of Lad to the plasma membrane was also observed in the immu- Four potential pY sites are concentrated in the C terminus of nofluorescence assay with anti-Lad Ab (data not shown). The ob- Lad. Notably, the NPXpY292 and NXpY317 motifs match the se- served redistribution to the plasma membrane provides Lad with quences known to be recognized by pY binding domains, which lck an opportunity to interact with p56 upon TCR engagement and are found in signaling proteins such as Shc and IRS-1 (NPXpY), lck supports the model that Lad acts as a partner of p56 at the mem- or Cbl (NxpY) (48, 49). However, in our preliminary experiments, brane-proximal signaling pathway of TCR activation. association of Shc to Lad was not detected upon T cell activation (data not shown). On the other hand, all four pY motifs Discussion (pY275TSP, pY292QEP, pY302AMG, and pY317AEV) may serve as In this report, we have described the molecular cloning and char- substrates for various SH2 domains. The pYXXP motif is pre- acterization of a p56lck-binding protein, Lad. We have found that: ferred by the SH2 domains of Crk, PLC ␥1 and c-Abl, and the 1) Lad is expressed in T cells; 2) Lad binds to the SH2 domain of pYXXV motif is preferred by the SH2 domains of Src family p56lck in vitro and in vivo in a T cell activation-dependent manner; tyrosine kinases (43). Consistent with this possibility, we have 3) Lad is tyrosine phosphorylated upon TCR stimulation; 4) Lad is preliminarily observed that the SH2 domains of Grb-2 and PLC-␥1 5248 Lck-BINDING ADAPTER PROTEIN: Lad

FIGURE 7. Redistribution of Lad to the plasma membrane upon T cell Downloaded from stimulation. EL4 cells were stimulated by CD3 cross-linking for 0, 10, 60, 120, and 180 min. The cytoplasmic and particulate fractions were obtained as described in Materials and Methods and were subjected to Western blot analysis with anti-Lad Abs. As controls for the amount of protein in each lane of the cytoplasmic and particulate fractions, the levels of SHP and Thy-1, respectively, were analyzed by reprobing the same blots. The experiments were each performed three times with reproducible results. http://www.jimmunol.org/

tion of early thymocyte maturation (4). The detection of Lad protein in primary thymocytes (Fig. 3B) is consistent with its role during development. On the other hand, Lad may mediate the IL- 2-dependent signal leading to T cell proliferation, because p56lck is ␤ coupled to the -chain of the IL-2 receptor (9). by guest on September 27, 2021 Many of the nonreceptor type protein tyrosine kinases (PTK) contain SH2 domains. According to a processive phosphorylation model suggested by Songyang (54), the SH2-containing PTKs selectively phosphorylate tyrosine residues recognized by their own or related SH2 domains. Based on this information, in our yeast two-hybrid system, the kinase domain of p56lck was included in FIGURE 6. Requirement of Lad for the TCR-mediated activation of the bait to allow for the phosphorylation of the binding partners of IL-2 promoter. A, IL-2-Luc reporter was cotransfected into Jurkat T cells its own SH2 domain. The cloning of Lad and conﬁrmation of its lck along with pcDNA/Lad (sense, S), pcDNA/Lad-antisense (AS), pcDNA/ binding to p56 in vivo dictates that the established yeast two- Lad-SH2 (SH2) or a control (Con) plasmid. Twenty-four hours after trans- hybrid screening system will be useful for identifying the partners fection, cells were incubated in the presence or absence of stimulation by of the SH2 domains of other kinases including other Src family PHA (10 ␮g/ml; upper panel), anti-CD3 (5 ␮g/ml; middle panel), and tyrosine kinases, c-Abl, c-Fes, Csk, Syk, and the Tec family of PMA plus A23187 (5 ng/ml and 0.5 ␮g/ml, respectively; lower panel). kinases. After 14 h, cells were assayed for luciferase activity. All the experiments were performed in triplicate and the SDs are indicated as bars. B, After transfection of 2.5 ␮gor5␮g of pcDNA/Lad-antisense (AS), the expres- Acknowledgments sion of Lad was analyzed by Western blot with anti-Lad Ab. As controls We thank other members of this laboratory for materials and helpful dis- for the amount of protein in each lane, the levels of SHP1 were analyzed cussions, and especially H. Kang for critical reading of the manuscript. by reprobing the same blot. Thanks are extended to Dr. Jaegyoon Shin for various materials. bind Lad in our tyrosine phosphorylation-dependent yeast two- References hybrid system (data not shown). 1. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette, et al. 1992. In addition to a role in T cell activation, Lad may function in Profound block in thymocyte development in mice lacking p56lck. Nature 357: activated/memory T cells. The identiﬁcation of TSAd, a potential 161. human homologue of Lad, as an inducible protein in activated T 2. Anderson, S. J., S. D. Levin, and R. M. Perlmutter. 1993. Protein tyrosine kinase p56lck controls allelic exclusion of T-cell receptor ␤-chain genes. Nature 365: cells (41) supports the role of Lad in activated T cells. In addition 552. lck to this, p56 was shown to be associated with CD44 or CD26, 3. Hashimoto, K., S. J. Sohn, S. D. Levin, T. Tada, R. M. Perlmutter, and markers of activated/memory T cells (14, 50–53). Additionally, T. Nakayama. 1996. Requirement for p56lck tyrosine kinase activation in T cell receptor-mediated thymic selection. J. Exp. Med. 184:931. Lad may function in early T cell development insomuch as inac- 4. Lowell, C. A., and P. Soriano. 1996. Knockouts of Src-family kinases: stiff bones, tivation of p56lck function in transgenic mice results in the disrup- wimpy T cells, and bad memories. Genes Dev. 10:1845. The Journal of Immunology 5249

5. Turner, J. M., M. H. Brodsky, B. A. Irving, S. D. Levin, R. M. Perlmutter, and 29. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. D. R. Littman. 1990. Interaction of the unique N-terminal region of tyrosine Sequential interactions of the TCR with two distinct cytoplasmic tyrosine ki- kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine nases. Science 263:1136. motifs. Cell 60:755. 30. Xu, H., and D. R. Littman. 1993. A kinase-independent function of Lck in po- 6. Glaichenhaus, N., N. Shastri, D. R. Littman, and J. M. Turner. 1991. Requirement tentiating antigen-specific T cell activation. Cell 74:633. for association of p56lck with CD4 in antigen-specific signal transduction in T 31. Straus, D. B., A. C. Chan, B. Patai, and A. Weiss. 1996. SH2 domain function is cells. Cell 64:511. essential for the role of the Lck tyrosine kinase in T cell receptor signal trans- 7. Collins, T. L., and S. J. Burakoff. 1993. Tyrosine kinase activity of CD4-asso- duction. J. Biol. Chem. 271:9976. ciated p56lck may not be required for CD4-dependent T-cell activation. Proc. 32. Lewis, L. A., C. D. Chung, J. Chen, J. R. Parnes, M. Moran, V. P. Patel, and Natl. Acad. Sci. USA 90:11885. M. C. Miceli. 1997. The Lck SH2 phosphotyrosine binding site is critical for 8. Straus, D. B., and A. Weiss. 1992. Genetic evidence for the involvement of the efficient TCR-induced processive tyrosine phosphorylation of the ␨-chain and lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell IL-2 production. J. Immunol. 159:2292. 70:585. 33. Duplay, P., M. Thome, F. Herve, and O. Acuto. 1994. p56lck interacts via its Src 9. Hatakeyama, M., T. Kono, N. Kobayashi, A. Kawahara, S. D. Levin, homology 2 domain with the ZAP-70 kinase. J. Exp. Med. 179:1163. R. M. Perlmutter, and T. Taniguchi. 1991. Interaction of the IL-2 receptor with 34. Lee, J. M., M. Fournel, A. Veillette, and P. E. Branton. 1996. Association of lck the src-family kinase p56 : identification of novel intermolecular association. CD45 with Lck and components of the Ras signaling pathway in pervanadate- Science 252:1523. treated mouse T-cell lines. Oncogene 12:253. 10. Minami, Y., T. Kono, K. Yamada, N. Kobayashi, A. Kawahara, R. M. Perlmutter, 35. Ng, D. H., J. D. Watts, R. Aebersold, and P. Johnson. 1996. Demonstration of a lck and T. Taniguchi. 1993. Association of p56 with IL-2 receptor ␤ chain is direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro. lck critical for the IL-2-induced activation of p56 . EMBO J. 12:759. J. Biol. Chem. 271:1295. 11. Miyazaki, T., Z. J. Liu, A. Kawahara, Y. Minami, K. Yamada, Y. Tsujimoto, 36. Fusaki, N., A. Iwamatsu, M. Iwashima, and J. i. Fujisawa. 1997. Interaction E. L. Barsoumian, R. M. Perlmutter, and T. Taniguchi. 1995. Three distinct IL-2 between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling pathways mediated by bcl-2, c-myc, and lck cooperate in hematopoietic signaling. J. Biol. Chem. 272:6214. cell proliferation. Cell 81:223. 37. Williams, T. M., J. E. Burlein, S. Ogden, L. J. Kricka, and J. A. Kant. 1989. 12. Kim, Y. J., K. E. Pollok, Z. Zhou, A. Shaw, J. B. Bohlen, M. Fraser, and Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 B. S. Kwon. 1993. Novel T cell antigen 4–1BB associates with the protein ty- Downloaded from lck gene promoter. Anal. Biochem. 176:28. rosine kinase p56 . J. Immunol. 151:1255. 38. Gietz, D., A. St. Jean, R. A. Woods, and R. H. Schiestl. 1992. Improved method 13. Bell, G. M., J. Fargnoli, J. B. Bolen, L. Kish, and J. B. Imboden. 1996. The SH3 lck for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20: domain of p56 binds to proline-rich sequences in the cytoplasmic domain of 1425. CD2. J. Exp. Med. 183:169. 39. Joung, I., T. Kim, L. A. Stolz, G. Payne, D. G. Winkler, C. T. Walsh, 14. Taher, T. E., L. Smit, A. W. Griffioen, E. J. Schilder-Tol, J. Borst, and S. T. Pals. J. L. Strominger, and J. Shin. 1995. Modification of Ser59 in the unique N- 1996. Signaling through CD44 is mediated by tyrosine kinases: association with lck lck terminal region of tyrosine kinase p56 regulates specificity of its Src homology p56 in T lymphocytes. J. Biol. Chem. 271:2863. 2 domain. Proc. Natl. Acad. Sci. USA 92:5778.

15. Brenner, B., E. Gulbins, K. Schlottmann, U. Koppenhoefer, G. L. Busch, 40. Kabouridis, P. S., A. I. Magee, and S. C. Ley. 1997. S-acylation of Lck protein http://www.jimmunol.org/ B. Walzog, M. Steinhausen, K. M. Coggeshall, O. Linderkamp, and F. Lang. lck tyrosine kinase is essential for its signaling function in T lymphocytes. EMBO J. 1996. L-selectin activates the Ras pathway via the tyrosine kinase p56 . Proc. 16:4983. Natl. Acad. Sci. USA 93:15376. 41. Spurkland, A., J. E. Brinchmann, G. Markussen, F. Pedeutour, E. Munthe, T. Lea, 16. Zhou, S., R., K. L. Carraway, M. J. Eck, S. C. Harrison, R. A. Feldman, F. Vartdal, and H. C. Aasheim. 1998. Molecular cloning of a T cell-specific M. Mohammadi, J. Schlessinger, S. R. Hubbard, D. P. Smith, C. Eng, et al. 1995. adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. SH3 and phosphotyrosine binding sites. J. Biol. Chem. 273:4539. Nature 373:536. 42. Galcheva-Gargova, Z., K. N. Konstantinov, I. H. Wu, F. G. Klier, T. Barrett, and 17. Carrera, A. C., H. Paradis, L. R. Borlado, T. M. Roberts, and C. Martinez. 1995. R. J. Davis. 1996. Binding of zinc finger protein ZPR1 to the epidermal growth Lck unique domain influences Lck specificity and biological function. J. Biol. factor receptor. Science 272:1797. Chem. 270:3385. 43. Songyang, Z., S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, 18. Gervais, F. G., and A. Veillette. 1995. The unique amino-terminal domain of lck F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, et al. 1993. SH2 domains

p56 regulates interactions with tyrosine protein phosphatases in T lymphocytes. by guest on September 27, 2021 recognize specific phosphopeptide sequences. Cell 72:767. Mol. Cell. Biol. 15:2393. 19. Paige, L. A., M. J. Nadler, M. L. Harrison, J. M. Cassady, and R. L. Geahlen. 44. Sparks, A. B., J. E. Rider, N. G. Hoffman, D. M. Fowlkes, L. A. Quillam, and lck B. K. Kay. 1996. Distinct ligand preferences of Src homology 3 domains from 1993. Reversible palmitoylation of the protein-tyrosine kinase p56 . J. Biol. ␥ Chem. 268:8669. Src, Yes, Abl, Cortactin, p53bp2, PLC , Crk, and Grb2. Proc. Natl. Acad. Sci. 20. Kavanaugh, W. M., C. W. Turck, and L. T. Williams. 1995. PTB domain binding USA 93:1540. to signaling proteins through a sequence motif containing phosphotyrosine. Sci- 45. Sparks, A. B., N. G. Hoffman, S. J. McConnell, D. M. Fowlkes, and B. K. Kay. ence 268:1177. 1996. Cloning of ligand targets: systematic isolation of SH3 domain-containing 21. Shenoy-Scaria, A. M., L. K. Gauen, J. Kwong, A. S. Shaw, and D. M. Lublin. proteins. Nat. Biotechnol. 14:741. 46. Rayter, S. I., M. Woodrow, S. C. Lucas, D. A. Cantrell, and J. Downward. 1992. 1993. Palmitylation of an amino-terminal cysteine motif of protein tyrosine ki- ras nases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol- p21 mediates control of IL-2 gene promoter function in T cell activation. anchored proteins. Mol. Cell. Biol. 13:6385. EMBO J. 11:4549. ras 22. Veillette, A., L. Caron, M. Fournel, and T. Pawson. 1992. Regulation of the 47. Woodrow, M. A., S. Rayter, J. Downward, and D. A. Cantrell. 1993. p21 enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck by function is important for T cell antigen receptor and protein kinase C regulation the noncatalytic SH2 and SH3 domains. Oncogene 7:971. of nuclear factor of activated T cells. J. Immunol. 150:3853. 23. Reynolds, P. J., T. R. Hurley, and B. M. Sefton. 1992. Functional analysis of the 48. Wolf, G., T. Trub, E. Ottinger, L. Groninga, A. Lynch, M. F. White, SH2 and SH3 domains of the lck tyrosine protein kinase. Oncogene 7:1949. M. Miyazaki, J. Lee, and S. E. Shoelson. 1995. PTB domains of IRS-1 and Shc 24. Prasad, K. V., R. Kapeller, O. Janssen, H. Repke, J. S. Duke-Cohan, have distinct but overlapping binding specificities. J. Biol. Chem. 270:27407. L. C. Cantley, and C. E. Rudd. 1993. Phosphatidylinositol (PI) 3-kinase and PI 49. Lupher, M. L. Jr., Z. Songyang, S. E. Shoelson, L. C. Cantley, and H. Band. 1997. 4-kinase binding to the CD4–p56lck complex: the p56lck SH3 domain binds to PI The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds 3-kinase but not PI 4-kinase. Mol. Cell. Biol. 13:7708. to the Tyr292 negative regulatory phosphorylation site of ZAP-70. J. Biol. Chem. 25. Vogel, L. B., and D. J. Fujita. 1993. The SH3 domain of p56lck is involved in 272:33140. binding to phosphatidylinositol 3Ј-kinase from T lymphocytes. Mol. Cell. Biol. 50. Ulmer, A. J., T. Mattern, and H. D. Flad. 1992. Expression of CD26 (dipeptidyl 13:7408. peptidase IV) on memory and naive T lymphocytes. Scand. J. Immunol. 35:551. 26. Reedquist, K. A., T. Fukazawa, B. Druker, G. Panchamoorthy, S. E. Shoelson, 51. Hegen, M., J. Kameoka, R. P. Dong, S. F. Schlossman, and C. Morimoto. 1997. and H. Band. 1994. Rapid T-cell receptor-mediated tyrosine phosphorylation of Cross-linking of CD26 by antibody induces tyrosine phosphorylation and acti- p120, an Fyn/Lck Src homology 3 domain-binding protein. Proc. Natl. Acad. Sci. vation of mitogen-activated protein kinase. Immunology 90:257. USA 91:4135. 52. Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annu. Rev. 27. Takemoto, Y., M. Furuta, X. K. Li, W. J. Strong-Sparks, and Y. Hashimoto. Immunol. 16:201. 1995. LckBP1, a proline-rich protein expressed in haematopoietic lineage cells, 53. Ilangumaran, S., A. Briol, and D. C. Hoessli. 1998. CD44 selectively associates directly associates with the SH3 domain of protein tyrosine kinase p56lck. EMBO with active Src family protein tyrosine kinases Lck and Fyn in glycosphingolipid- J. 14:3403. rich plasma membrane domains of human peripheral blood lymphocytes. Blood 28. Peri, K. G., F. G. Gervais, R. Weil, D. Davidson, G. D. Gish, and A. Veillette. 91:3901. 1993. Interactions of the SH2 domain of lymphocyte-specific tyrosine protein 54. Zhou, S., and L. C. Cantley. 1995. Recognition and specificity in protein tyrosine kinase p56lck with phosphotyrosine-containing proteins. Oncogene 8:2765. kinase-mediated signaling. Trends Biochem. Sci. 20:470.