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The Journal of Immunology, 1999, 163: 5242-5249.
Copyright © 1999 by The American Association of Immunologists

Lad, an Adapter Protein Interacting with the SH2 Domain of p56lck, Is Required for T Cell Activation1 ,2

Young Bong Choi*,{dagger}, Chan Ki Kim* and Yungdae Yun3,*,{dagger}

* Signal Transduction Laboratory Mogam Biotechnology Research Institute, Koosungmyon, Yonginsi, Kyunggido, Korea; and {dagger} Department of Molecular Life Science and Center for Cell Signaling Research, Ewha Women’s University, Seodaemungu, Daehyundong 11-1, Seoul, 120-750, Korea.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 phosphotyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56lck in vivo and redistributed 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The best-characterized lymphocyte-specific member of the Src family tyrosine kinases, p56lck, plays essential roles in T cell signaling that regulates diverse T cell functions such as development, activation, proliferation, and adhesion.

Knock-out mice lacking p56lck show a pronounced thymic atrophy owing to blockade of the progression from CD4-CD8- double negative to CD4+CD8+ double positive thymocytes (1). Transgenic mice harboring a dominant negative form of p56lck are defective in allelic exclusion of the pre TCR ß-chain gene which permits normal thymic selection (2, 3). Both animal studies indicate that p56lck is important for T cell development (4).

During Ag-induced T cell activation, p56lck transmits a positive signal by interacting with the CD4/CD8 glycoproteins (5, 6, 7). Furthermore, genetic evidence using JCaM1 cells lacking p56lck shows that p56lck is involved in TCR-mediated cell activation (8). During T cell proliferation by IL-2, p56lck associates with the IL-2R ß-chain (9) and regulates c-fos/c-jun gene expression (10, 11). In addition, p56lck associates with other costimulatory adhesion molecules such as 4–1BB (12), CD2 (13), CD44 (14), and L-selectin (15) to enhance T cell responsiveness. These multiple functions of p56lck are believed to be conducted through interaction with various cellular signaling proteins.

Like other Src family protein tyrosine kinases (PTK),4 p56lck consists of five domains: SH1 (Src homology domain 1), SH2, SH3, SH4, and NH2 unique domain. The SH1 is the enzymatic domain of PTK that phosphorylates tyrosines on cellular proteins with catalytic specificity (16). The N-terminal unique domain influences substrate preference without the regulation of intrinsic kinase activity (17) and regulates interaction with protein tyrosine phosphatases (18). The SH4 domain directs p56lck to the plasma membrane by denoting sites for lipidation such as palmitoylation or myristoylation (19, 20), which enables p56lck to interact with GPI-anchored proteins such as CD59 (21). The SH3 domain negatively regulates the enzymatic activity of p56lck and is dispensable for cell transformation by activated p56lck (F505) (22, 23). Several groups, however, reported that the SH3 domain interacts with several cellular signaling proteins including phosphatidylinositol 3-kinase (PI3K) (24, 25), p120 (26), and LckBP1 (27) through their proline-rich motifs. The functional significance of the p56lck SH3 domain in T cell signaling remains yet to be elucidated. Finally, the SH2 domain negatively or positively regulates the function of p56lck (22). In the inactive form of p56lck, the SH2 domain interacts with its own phosphorylated Y505 (pY505), but in the active 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 extensively and both the kinase and the regulatory domains have been shown to be required. A model was established in which, upon engagement with CD4/CD8, the kinase domain of p56lck phosphorylates the {zeta}-chain of TCR and provides the binding site for another kinase, ZAP-70. These successive events lead to the amplification of TCR-mediated signaling (29). On the other hand, even though the kinase activity of p56lck is required for full T cell activation, a kinase-independent function of p56lck, mainly mediated by the SH2 domain was shown to independently contribute to T cell activation (7, 30, 31, 32). Subsequent efforts resulted in the identification of ZAP-70 (33), CD45 (34, 35), and Sam68 (36) as binding partners of the p56lck SH2 domain. However, this information does not fully explain the contribution of the SH2 domain to the multiple functions of p56lck in T cells.

Here, to understand the mechanisms by which p56lck acts in T cell signaling and the role of the p56lck SH2 domain in this process, we identified the binding partners of the p56lck SH2 domain using a tyrosine phosphorylation-dependent yeast two-hybrid system. As a result of the screening, a novel protein of 366 aa that we named Lad (Lck adapter) was isolated. Upon T cell activation, Lad coimmunoprecipitated p56lck, was tyrosine phosphorylated, and acted as a substrate of p56lck tyrosine kinase. Moreover, overexpression of dominant negative Lad blocked the IL-2 promoter-driven transcriptional activation following TCR stimulation. Taken together, these results indicate that Lad plays an essential role as an adapter protein in p56lck-mediated T cell signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

All bait plasmids encoding parts of murine p56lck and Lad were generated by cloning the PCR-amplified fragment into pGBT9, a GAL4 DNA binding domain vector (Clontech, Palo Alto, CA). Primers with EcoRI (5' primer) or BamHI (3' primer) restriction sites were used to facilitate the subcloning. The PCR products were digested with EcoRI/BamHI and cloned into the corresponding site of pGBT9. The mammalian expression plasmids of p56lck wild type (WT), F505, A273, K154, Lad, Lad-antisense, and Lad-SH2 were constructed by PCR amplification and subsequent insertion into the EcoRI/XhoI site of pcDNAI/Amp (Invitrogen, San Diego, CA) using the same approach as described above. The point mutants of p56lck were generated using a Quick mutagenesis kit (Stratagene, La Jolla, CA). The GST-fusion constructs of the SH2 domain of p56lck (aa 123–225) or the Lad C terminus (aa 208–366) were generated by PCR and subcloned into pGEX-KG (Pharmacia, Piscataway, NJ) using EcoRI or BamHI/EcoRI sites, respectively. The pGL3/IL-2-Luc contains the luciferase reporter gene downstream from the IL-2 promoter region, including 548 bp 5' of the transcriptional starting site, and was generated by subcloning a HindIII fragment of pIL-2-CAT (37) into the HindIII site of pGL3-Basic (Promega, Madison, WI).

Yeast two hybrid screen

A cDNA fragment encoding the SH2 and kinase domain (the constitutively active form, F505) of mouse p56lck was cloned into pGBT9. The resulting plasmid, Lck SH2K (F505) was used as the bait in the yeast two-hybrid screens of a murine T cell lymphoma cDNA library cloned into pACT (Clontech). The bait and library DNAs were cotransformed by the lithium acetate method as previously described (38). Seventy-five out of 2 x 106 transformants grew in the absence of histidine and showed detectable ß-galactosidase staining within 2 h of incubation. To eliminate clones binding to the bait in a tyrosine phosphorylation-independent manner, the plasmids from each positive clone were cotransformed with control bait plasmids encoding SH2 domain only (Lck SH2) or kinase domain only (Lck K (F505)). Approximately 70 clones, which did not bind to either of the two control baits, were finally selected and subjected to partial sequence determination. The sequences revealed that seven clones contained a part of the same cDNA encoding a novel protein which we named Lad.

cDNA cloning

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 {lambda}gt10 (Clontech). Three independent clones were obtained and subjected to nucleotide sequencing. In addition, 5'-RACE was performed using the mouse spleen 5' stretch cDNA template (Clontech). From the combination of both approaches, the cDNA encompassing ~1.6 kb in its entirety was obtained.

Cells, activation, and transfection

Jurkat and EL4 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% FBS, 5 mM 2-ME, and antibiotics. COS-1 cells were grown in DMEM supplemented with 10% FBS and antibiotics. Thymocytes and splenocytes were isolated by passing mouse thymus and spleen 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, Harvard Medical School, Boston, MA), respectively, at a saturated concentration. EL4 cells were activated by CD3 cross-linking with 145-2C11 Ab. COS-1 cells were transfected using the standard DEAE-dextran method.

Abs, immunoprecipitation, and Western blot analysis

Anti-p56lck Ab was obtained from Transduction Laboratory (Lexington, KY) or generated by immunization of rabbits with GST fusion proteins encompassing the SH3 and SH2 domain (aa 66–224). Antiphosphotyrosine (pY) Ab (4G10) and anti-SHP Ab were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-mouse CD3{epsilon} (145-2C11), anti-CD90 (Thy-1) (G7), anti-human CD3 (UCHT1), anti-hamster IgG, and anti-mouse IgG were obtained from PharMingen (San Diego, CA). Polyclonal antiserum against Lad or GST was raised in rabbits immunized with GST-Lad C terminus (aa 208–366) or GST, respectively. For immunoprecipitation or Western blot, cells were lysed with TNE buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 25 µg/ml aprotinin, 1 mM PMSF, 25 µg/ml leupeptin, and 1 mg/ml BSA) for 1 h on ice. All bands analyzed by Western blot were detected using the enhanced chemiluminescence protocol (Amersham, Arlington, Heights, IL).

Purification of GST fusion proteins and the binding assay

The GST fusion protein encompassing the SH2 domain of p56lck (aa 123–224) or the C terminus of Lad (aa 208–366) was expressed in Escherichia coli and purified as previously described (39). For the binding study, 5 µg of GST-p56lck SH2 fusion protein immobilized on glutathione-Sepharose 4B beads was incubated with Jurkat T cell lysates prepared in TNE buffer for 1 h at 4°C. After washing, samples were analyzed by Western blotting using the antiserum against GST-LadC.

In vitro kinase assay

Purified GST-Lad C terminus or GST protein was incubated with 20 U of purified p56lck (Upstate Biotechnology) in a kinase buffer containing 50 mM Tris (pH 7.5), 1 mM DTT, 10 mM MnCl2, and 50 mM NaCl in the presence or absence of 100 µM ATP. After 2 h at 30°C, the reaction mixtures were subjected to SDS-PAGE followed by Western blot analysis using anti-pY Ab, 4G10 or anti-GST Ab.

Subcellular fractionation

All steps were performed as described (40). Approximately 2 x 107 EL4 cells were activated by cross-linking CD3, resuspended in 0.5 ml of hypotonic solution (25 mM Tris (pH 7.5), 5 mM EDTA, 5 mM EGTA, 250 mM sucrose, 25 µg/ml aprotinin, 1 mM PMSF, and 25 µg/ml leupeptin), and then subjected to two successive freeze-thaw cycles. The cell suspension was homogenized on ice using a Dounce homogenizer (40 strokes), and the salt concentration was adjusted to 150 mM NaCl. Nuclei and other debris in the cell lysates were removed by two rounds of centrifugation at 480 x g for 5 min. The soluble and particulate fractions were separated by centrifugation at 100,000 x g for 30 min. Fractionated proteins were resolved by SDS-PAGE and Western blotted with the corresponding Abs.

Luciferase assay

A total of 5 x 106 Jurkat T cells were cotransfected with 2.5 µg each of IL-2-luc along with 2.5 µg of pcDNAI/Lad, pcDNAI/Lad-antisense or pcDNAI/Lad-SH2 using Superfect (Qiagen, Chatswroth, CA). After incubation for 24 h with DNA-Superfect mixtures, cells were activated by incubation on anti-CD3 Ab plates coated with 5 µg/ml UCHT1, by treatment of 10 µg/ml PHA, or by treatment with 5 ng/ml PMA plus 0.5 µg/ml A23187 for 14 h and were harvested.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of Lad as a phosphorylation-dependent binding partner of the p56lck SH2 domain

Based on the previous report that the binding specificity of phosphopeptides to the p56lck SH2 domain overlaps with the substrate specificity of the p56lck kinase domain in a peptide library approach (16), a part of p56lck encompassing the SH2 and kinase domains (SH2K (F505) in Fig. 1GoA; aa 123–509) was used as a bait. 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 x 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.



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  		FIGURE 1. Binding properties of Lad to p56lck in a yeast two-hybrid system. A, Schematic representation of the baits used in the yeast two-hybrid assay. B, Bait-specific interaction of Lad. In the swapping experiment, Lad was fused to GAL4 BD and SH2K (F505) was fused to the transcriptional activation domain (AD) of GAL4. Interaction of Lad with unrelated proteins, such as GAL4 BD only, p53, hepatitis B Virus X-gene product (HBV-X), ZAP-70 SH2-p56lck kinase (ZAP-70 SH2K (F505)), and PI3K SH2-p56lck kinase (PI3K SH2K (F505))was tested for control. C, Tyrosine phosphorylation-dependent interaction of Lad to p56lck SH2 domain. Binding affinity of SH2K (F505) to Lad was compared with those of other baits. Binding affinity was scored as +++ (deep blue), ++ (intermediate blue), + (pale blue), or - (white) upon X-Gal (5-bromo-4-chloro-3-indolyl ß-D-galactoside) staining.

 
The binding specificity of Lad to p56lck was studied further using the various control plasmids described in Fig. 1GoA. First, even if the cloning vectors for Lad and SH2K (F505) were switched, the interaction was not affected (Fig. 1GoB). Second, Lad interacted with SH2K (F505), but not with unrelated proteins such as GAL4 binding domain only, p53 protein, and hepatitis B virus X-gene product. 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. 1GoB), 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. 1GoC). This result supports the notion that the interaction between SH2K (F505) and Lad involves cooperation between the SH2 domain and the active kinase domain rather than the individual 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. 1GoC). Neither of these mutants showed any binding to Lad.

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 5'-RACE from the mouse spleen 5' stretch cDNA (Fig. 2GoA). 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. 2GoB). 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 ßD5 position is tyrosine or phenylalanine (43). The highest identity is found with the SH2 domains of GAPn, Csk, Grb2, and Src and falls within the range of 25–35% (data not shown). The proline-rich motif of Lad exactly matches the consensus sequence +PP{psi}PXKP (+, basic amino acids; {psi}, hydrophobic amino acids) (44) preferred by class II cortactin SH3 domains found in cortactin, HS1, LckBP1, SH3P7, and SH3P8 (27, 45). The C terminus of Lad contains four potential pY sites, one of which is an NPXpY292 motif known as the ligand for the pY binding domain (20). Overall, these sequence characteristics suggest that Lad may be an adapter protein with several domains possibly involved in protein-protein interactions.



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  		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 asterisk (*). Potential protein-protein interaction 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.

 
On Northern blot analysis, Lad cDNA detected messages of 1.7 and 4.4 kb in spleen and lung of mouse and of 1.8 and 4.9 kb in spleen, thymus, and PBMC of human (Fig. 3GoA). The size of 1.7-kb message in mouse is consistent with that of assembled cDNA. At this stage, the identity of 4.4-kb and 4.9-kb message is not clear.



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  		FIGURE 3. Expression of Lad. A, Tissue distribution of Lad mRNA. Northern blot analysis was performed with mouse and human Multiple Tissue Northern blots (Clontech). B, Expression of Lad protein. Western blot analysis was performed with EL4, NIH 3T3, and purified thymocytes and splenocytes at 5 x 106 cells/lane. C. Molecular weight of Lad. Lad is detected as a 45-kDa protein upon in vitro translation and in T cell lysates. (-), control without Lad mRNA; Lad, in vitro translation product of Lad mRNA; EL4, lysates of mouse EL4 T cell.

 
Next, an anti-Lad Ab was raised against the GST fusion protein covering the C terminus of Lad (GST-LadC, aa 208–366). On Western blot analysis with this Ab, Lad was detected as a 45-kDa protein in EL4 mouse thymoma cells and mouse lymphocytes purified from thymus and spleen, but not in NIH 3T3 cells (Fig. 3GoB). Notably, the expression level of Lad protein in thymus was much higher than that in spleen. In addition, size of in vitro translated Lad was identical to that in EL4 cells (Fig. 3GoC), even though the 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. 4GoA). The surface expression of CD3 and CD4 on Jurkat T cells were confirmed by FACS analysis. CD3 and CD4 were detected in ~90% 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. 4GoA, 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 ~3-fold upon T cell activation.



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  		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 (-) or stimulated by CD3/CD4 cross-linking (+) 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 (-) or CD3/CD4 cross-linked (+) 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 (-) or CD3 cross-linked (+) EL4 T cell lysates and analyzed by Western bolt with anti-Lad Ab. EL4 cell lysates (lysate, fifth lane) were included as a control. The amount of immunoprecipitated p56lck in each lane is shown in a lower panel.

 
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{epsilon} 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. p56lck could be readily detected in the Lad immunoprecipitates of the T cell lysates upon CD3/CD4 cross-linking of Jurkat T cells (Fig. 4GoB) or CD3 cross-linking of EL4 cells (Fig. 4GoC). Conversely, Lad was readily detected in the Lck immunoprecipitates upon CD3 stimulation of EL4 cells (Fig. 4GoD). These results indicate that Lad inducibly binds to the SH2 domain of p56lck in vivo upon T cell activation through TCR.

Lad is phosphorylated upon TCR-stimulation

Insomuch as Lad contains several potential tyrosine phosphorylation sites, we studied the tyrosine phosphorylation status of Lad by immunoprecipitation of Lad from EL4 T cell lysates and subsequent immunoblotting with anti-pY Ab (Fig. 5GoA). Following CD3 stimulation, tyrosine phosphorylation of Lad was rapidly induced within 10 min and maintained up to 60 min. These results show that Lad is a phosphoprotein and that its phosphorylation is inducible through TCR stimulation. In addition, phosphoproteins with sizes of 52 kDa, 56–58 kDa, and 70–80 kDa were coimmunoprecipitated with Lad. The size of 56- to58-kDa bands corresponds to those of p56lck. Subsequently, to test whether Lad is phosphorylated by p56lck, Lad was coexpressed with p56lck WT, F505, or A273 in COS-1 cells (Fig. 5GoB). As shown in the first panel of Fig. 5GoB, immunoblotting of total cell extracts with anti-pY Ab displayed that a 45-kDa band was heavily tyrosine-phosphorylated by p56lck (F505), but not by p56lck WT or p56lck A273. The expression of p56lck and its mutants was confirmed by immunoblotting with anti-p56lck Ab (Fig. 5GoB, the second panel). Next, to confirm that Lad is phosphorylated by p56lck, Lad was immunoprecipitated with anti-Lad Ab and the immunoprecipitates were analyzed by Western blot with anti-pY Ab. As shown in the third panel of Fig. 5GoB, Lad was heavily tyrosine-phosphorylated by p56lck (F505), but not by p56lck WT or p56lck (A273). The same blot was reprobed with anti-Lad Ab to confirm the amount of precipitated Lad (Fig. 5GoB, the fourth panel). Finally, to test whether Lad is a direct substrate of p56lck, we performed in vitro kinase assay in a cell-free system. Purified GST-LadC (aa 208–366) containing four potential pY sites was incubated with purified p56lck in the presence or absence of ATP (Fig. 5GoC). Upon immunoblotting with anti-pY, GST-LadC was detected as a phosphoprotein. Taken together, these results suggest that Lad is most likely a direct substrate of p56lck upon TCR stimulation.



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  		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). Subsequently, Lad was immunoprecipitated 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 the presence (+) or absence (-) of ATP. The kinase reaction mixtures were analyzed with anti-pY immunoblotting. The lower panel shows the amount of loaded GST or GST-LadC as a control.

 
Lad is required in IL-2 gene expression

To address the functional significance of Lad in T cell activation, we overexpressed Lad in a sense (S) or an antisense (AS) orientation in Jurkat T cells and examined its effect on the IL-2 promoter-driven reporter activity upon T cell stimulation (Fig. 6GoA). In addition, we tested the effect of overexpression of the Lad SH2 domain (SH2), a potential dominant negative form. The overexpression of Lad AS or the SH2 domain resulted in the repression of CD3- or PHA-stimulated reporter activity by about 70%, indicating the requirement of Lad in TCR-mediated IL-2 gene activation (upper and middle panels of Fig. 6GoA). PMA plus A23187 (P+I)-stimulated reporter activity, however, was not repressed by the overexpression of Lad anti-sense or the SH2 domain (lower panel of Fig. 6GoA). These results indicate that Lad acts in the TCR-proximal signaling events upstream of PMA/ionomycin. Moreover, without any activation signals, overexpression of Lad itself led to a 7-fold induction of the IL-2 promoter-driven luciferase activity, supporting that Lad is involved in events leading to IL-2 gene activation. However, under CD3- or PHA-stimulation conditions, overexpression of Lad showed only a marginal effect suggesting that some components of the signaling pathway may be near saturation. Under the experimental condition, Lad antisense effectively inhibited the expression of Lad protein induced upon CD3 stimulation in a dose-dependent manner (Fig. 6GoB). Taken together, these results demonstrate that Lad is required for the TCR-mediated signaling pathway leading to IL-2 gene expression.



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  		FIGURE 6. Requirement of Lad for the TCR-mediated activation of IL-2 promoter. A, IL-2-Luc reporter was cotransfected into Jurkat T cells along with pcDNA/Lad (sense, S), pcDNA/Lad-antisense (AS), pcDNA/Lad-SH2 (SH2) or a control (Con) plasmid. Twenty-four hours after transfection, cells were incubated in the presence or absence of stimulation by PHA (10 µg/ml; upper panel), anti-CD3 (5 µg/ml; middle panel), and PMA plus A23187 (5 ng/ml and 0.5 µg/ml, respectively; lower panel). 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 µg or 5 µg of pcDNA/Lad-antisense (AS), the expression of Lad was analyzed by Western blot with anti-Lad Ab. As controls for the amount of protein in each lane, the levels of SHP1 were analyzed by reprobing the same blot.

 
Activation-dependent plasma membrane localization of Lad

To examine the subcellular localization of Lad, we prepared the cytoplasmic and particulate fractions from CD3-stimulated EL4 cells and studied the distribution of Lad by Western blot analysis (Fig. 7Go). In the absence of stimulation, the majority of Lad was detected in the cytoplasmic fraction. Upon stimulation by CD3 cross-linking, the level of cytoplasmic Lad gradually diminished and was almost nondetectable after 2 h, whereas the level of particulate Lad gradually increased during that time. On the other hand, the levels of SHP-1 and Thy-1, employed as controls, were consistent throughout the period (Fig. 7Go). In addition, redistribution of Lad to the plasma membrane was also observed in the immunofluorescence assay with anti-Lad Ab (data not shown). The observed redistribution to the plasma membrane provides Lad with an opportunity to interact with p56lck upon TCR engagement and supports the model that Lad acts as a partner of p56lck at the membrane-proximal signaling pathway of TCR activation.



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  		FIGURE 7. Redistribution of Lad to the plasma membrane upon T cell 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have described the molecular cloning and characterization of a p56lck-binding protein, Lad. We have found that: 1) Lad is expressed in T cells; 2) Lad binds to the SH2 domain of p56lck in vitro and in vivo in a T cell activation-dependent manner; 3) Lad is tyrosine phosphorylated upon TCR stimulation; 4) Lad is involved in the up-regulation of the IL-2 gene expression upon T cell activation; and 5) Lad is redistributed to the plasma membrane upon TCR stimulation. These results indicate that Lad physically interacts with p56lck upon T cell activation and plays an important role in the p56lck-dependent T cell signaling.

Insomuch as Lad is directly phosphorylated by p56lck in intact cells and by purified p56lck in vitro (Fig. 5Go, B and C), it is most likely that the tyrosine phosphorylation of Lad upon T cell activation is mediated by p56lck. However, we do not exclude the possibility that the initial tyrosine phosphorylation is mediated by other tyrosine kinases (e.g., Csk, ZAP-70, Tec family, and p59fyn) known to be involved in T cell activation. Upon translocation to the plasma membrane, the SH2 domain, proline-rich motif or zinc-finger motif of Lad may recruit additional signaling molecules to the p56lck/TCR complex leading to the amplification of activation signals.

Four potential pY sites are concentrated in the C terminus of Lad. Notably, the NPXpY292 and NXpY317 motifs match the sequences known to be recognized by pY binding domains, which are found in signaling proteins such as Shc and IRS-1 (NPXpY), or Cbl (NxpY) (48, 49). However, in our preliminary experiments, association of Shc to Lad was not detected upon T cell activation (data not shown). On the other hand, all four pY motifs (pY275TSP, pY292QEP, pY302AMG, and pY317AEV) may serve as substrates for various SH2 domains. The pYXXP motif is preferred by the SH2 domains of Crk, PLC {gamma}1 and c-Abl, and the pYXXV motif is preferred by the SH2 domains of Src family tyrosine kinases (43). Consistent with this possibility, we have preliminarily observed that the SH2 domains of Grb-2 and PLC-{gamma}1 bind Lad in our tyrosine phosphorylation-dependent yeast two-hybrid system (data not shown).

In addition to a role in T cell activation, Lad may function in activated/memory T cells. The identification of TSAd, a potential human homologue of Lad, as an inducible protein in activated T cells (41) supports the role of Lad in activated T cells. In addition to this, p56lck was shown to be associated with CD44 or CD26, markers of activated/memory T cells (14, 50, 51, 52, 53). Additionally, Lad may function in early T cell development insomuch as inactivation of p56lck function in transgenic mice results in the disruption of early thymocyte maturation (4). The detection of Lad protein in primary thymocytes (Fig. 3GoB) 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 ß-chain of the IL-2 receptor (9).

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 the bait to allow for the phosphorylation of the binding partners of its own SH2 domain. The cloning of Lad and confirmation of its binding to p56lck in vivo dictates that the established yeast two-hybrid screening system will be useful for identifying the partners of the SH2 domains of other kinases including other Src family tyrosine kinases, c-Abl, c-Fes, Csk, Syk, and the Tec family of kinases.


   Acknowledgments
 
We thank other members of this laboratory for materials and helpful discussions, and especially H. Kang for critical reading of the manuscript. Thanks are extended to Dr. Jaegyoon Shin for various materials.


   Footnotes
 
1 This work was supported in part by grants from the Korea Green Cross Co. and the Ministry of Science and Technology of Korea. Back

2 The sequence reported in this article will appear in the GenBank database under accession no. U69460. Back

3 Address correspondence and requests to Dr. Y. Yun, Division of Molecular Life Science and Center for Cell Signaling Research, Ewha Women’s University, Seoul, 120-750, Korea. E-mail address: Back

4 Abbreviations used in this paper: PTK, protein tyrosine kinase; SH1, Src homology domain 1; PI3K, phosphatidylinositol 3-kinase; pY, phosphotyrosine; 5'-RACE, 5'-rapid amplification of cDNA ends; Lad, Lck-associated adapter protein; TSAd, T cell-specific adapter protein; WT, wild type. Back

Received for publication February 10, 1999. Accepted for publication August 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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