INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The role of adapter proteins in signaling is increasingly appreciated (34). Adapters lack catalytic activity but nucleate signaling complexes and mediate intermolecular interactions, leading to increased speed and precision of signaling. SLP-76 is a cell-type-specific adapter protein that is expressed in T lymphocytes, NK cells, platelets, and myeloid cells of the granulocyte and monocyte lineage (6, 24, 35, 43). In these cell types, SLP-76 is required for signaling by ITAM (immunoreceptor tyrosine-based activation motif)-containing receptors, including the T-cell antigen receptor (TCR), the pre-TCR, the high-affinity immunoglobulin E (IgE) receptor, and the platelet collagen receptor (5, 7, 17, 36, 37, 64). In B cells, an analogous adapter, BLNK/SLP-65, is required for signaling by the ITAM-containing B-cell receptor (BCR) (16, 22, 58).

The primary structure of SLP-76 includes three domains capable of mediating intermolecular interactions: an N-terminal acidic domain containing three tyrosine phosphorylation sites, a central proline-rich region, and a C-terminal Src homology 2 (SH2) domain (14, 24). Overexpression of SLP-76 augments TCR-induced transcriptional responses (31, 54, 62) and affects regulation of the actin cytoskeleton (55). Interestingly, all three domains of SLP-76 are required for augmentation of TCR-induced transcriptional responses by overexpressed SLP-76 (14, 33, 54), suggesting that multiple protein-protein interactions play a role in SLP-76 function.

Characterization of the SLP-76-deficient T-cell line J14 revealed that SLP-76 is required to couple TCR-induced tyrosine kinase activity to the tyrosine phosphorylation and activation of phospholipase C-1 (PLC-1) (64). PLC-1 catalyzes the formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, which, respectively, trigger calcium flux and contribute to protein kinase C and Ras activation. In addition, PLC- contributes to the activation of Erk (11, 18). These key signaling events are required for activation of NFAT (nuclear factor of activated T cells), a regulator of interleukin-2 transcription (41, 60). Accordingly, in SLP-76-deficient J14 cells, impaired PLC-1 activation is associated with reduced TCR-induced calcium flux and Erk activation and impaired TCR-induced transcriptional responses (64).

PLC-1 activity is regulated by tyrosine phosphorylation (4, 42). All PLC family members contain two domains, designated X and Y, which fold together to form the catalytic site. In the PLC- subfamily (PLC-1 and PLC-2), the X and Y domains are separated by two SH2 domains and one SH3 domain, bounded by a split pleckstrin homology domain (4, 42). Evidence suggests that this SH region negatively regulates the basal activity of the PLC-1 holoenzyme (19, 20). The tyrosine phosphorylation sites of PLC-1 have been identified as residues 771, 783, and 1254. Of these sites, tyrosines 783 and 1254 are required for activation of PLC-1 (25), although tyrosine kinase-independent mechanisms may also contribute to PLC- activation (48). Interestingly, tyrosine 783 is located within the SH region, between the second SH2 and the SH3 domains.

Three families of tyrosine kinases are required for antigen receptor-induced tyrosine phosphorylation and activation of PLC-1: a Src family kinase, a Syk family kinase, and a Tec family kinase (reviewed in reference 4). In T cells, these kinases are coupled to PLC-1 activation by two cell-type-specific adapter proteins: SLP-76, and a membrane-anchored, inducibly tyrosine-phosphorylated adapter protein called LAT (15, 64, 66, 67). The Src family kinase is required for phosphorylation of receptor ITAMs and contributes to the activation of the Syk and Tec family kinases (38, 65). The Syk and Tec family kinases may make a dual contribution to PLC- activation, by phosphorylating the SLP-76 and LAT adapter proteins and by phosphorylating PLC-1 itself. Following TCR stimulation, the N-terminal SH2 domain of PLC-1 binds to tyrosine-phosphorylated LAT (50, 56, 67). Whereas LAT is required for tyrosine phosphorylation and activation of PLC-1 (15, 66), binding to LAT is not sufficient for optimal tyrosine phosphorylation and activation of PLC-1 in the absence of SLP-76 (64). SLP-76 is indirectly recruited to the LAT complex by the Grb2 family adapter protein Gads, which binds to both SLP-76 and LAT (1, 29). It is likely that SLP-76 acts as part of a complex with LAT to influence tyrosine phosphorylation of PLC-1.

Many different signaling proteins are known to interact with SLP-76; however, their contribution to SLP-76-mediated activation of PLC-1 has not been studied. Tyrosines 113 and 128 of SLP-76 mediate the TCR-inducible association of SLP-76 with the Nck adapter protein (55, 63) and with Vav, a hematopoietic cell-specific exchange factor for Rho family GTPases (13, 40, 53, 62). A short stretch (amino acids 224 to 265) within the proline-rich domain of SLP-76 was originally identified as a Grb2-binding site (31) but is now known to bind with higher affinity to Gads (1, 29) (also known as Grb40, GrpL, GRID, Mona, and Grap-2), a hematopoietically expressed, Grb2-related adapter protein (1, 2, 12, 26, 29, 39). In addition, two tyrosine kinases critical for TCR signaling, Lck and Itk, can associate via their SH3 domains with proline-rich motifs found in SLP-76 (3, 44), while the SH2 domain of Itk has been further suggested to bind to N-terminal tyrosine phosphorylation sites in SLP-76 (52). Finally, the C-terminal SH2 domain of SLP-76 interacts with another adapter protein, SLAP-130/Fyb (8, 32). The relative functional importance of each of these interactions remains to be sorted out.

In this study, we take a genetic approach to identifying the functionally essential domains of SLP-76 and the functionally important SLP-76-interacting proteins, by identifying the regions of SLP-76 that are required to reconstitute TCR signaling in a SLP-76-deficient T-cell line. Our studies have identified a new functional domain within the proline-rich region of SLP-76, comprised of amino acids 157 to 223, which associates with the SH3 domain of PLC-1 and is required for TCR-mediated activation of PLC-1. We propose that binding of the SH3 domain of PLC-1 to SLP-76 is required for optimal tyrosine phosphorylation and activation of PLC-1.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. The mutant alleles of SLP-76 used in this study are depicted in Fig. 1B. All alleles were Flag tagged at the N terminus and were subcloned as SalI-XbaI fragments into pEFBos (30) for use in transient transfections. The wild-type, N, Y3F, Gads, SH2, and SH2^mut alleles of SLP-76 have been previously described (14, 33) and were generously provided by Gary Koretzky. Additional mutants were created, using standard PCR techniques to precisely remove codons for the deleted residues, indicated in Fig. 1. All PCR-generated constructs were verified by sequencing. For stable transfections, SLP-76 alleles were subcloned into the pAWneo3' expression vector, which bears a G418 resistance selectable marker and allows for more physiologic expression levels. The NFAT-luciferase reporter construct, in which the expression of luciferase is driven by multiple copies of the NFAT DNA-binding element, was a gift from G. Crabtree, Stanford University.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   SLP-76 expression constructs used in this study. (A) The major functional domains of SLP-76, as defined in references 14 and 33. (B) Diagrammatic depiction of the SLP-76 mutant constructs used in this study. All constructs are Flag tagged at the N terminus. Names of the constructs are shown at the left, and the residues mutated or deleted are shown at the right.

GST fusion proteins. Glutathione S-transferase (GST) fusion proteins were expressed in bacteria and purified on glutathione-agarose (Sigma) by standard procedures. A bacterial expression construct encoding GST fused to the second SH3 domain of Nck was provided by Bruce Mayer (University of Connecticut). The rat PLC-1 cDNA was generously provided by Graham Carpenter (Vanderbilt University). Sequences encoding residues 538 to 851 (two SH2 and one SH3 domains) and residues 790 to 851 (the SH3 domain) of rat PLC-1 and were amplified by PCR and subcloned into pGEX2TK (Amersham Pharmacia Biotech) to generate GST-PLC-1 SH223 and GST-PLC-1 SH3, respectively.

Cell culture and transfections. The SLP-76-deficient cell line J14 and its SLP-76-reconstituted derivative J14-76-11 have been previously described (64). Additional derivatives were obtained by stably transfecting J14 with mutant alleles of SLP-76 using the pAWneo3' expression vector. At least four clones expressing similar levels of surface TCR and transfected SLP-76 were chosen for analysis. All cells were maintained in RPMI 1640 medium supplemented with 5% fetal calf serum, penicillin, streptomycin, and glutamine. In addition, growth media for stably transfected derivatives of J14 contained G418 (2 mg/ml), of which was washed out 2 days prior to use of the cells for experiments. Transient transfections were performed by electroporation using a Gene Pulser (Bio-Rad Laboratories, Hercules, Calif.), at a setting of 250 V and 960 µF, in cuvettes containing 2 × 10⁷ cells in 0.4 ml serum-free RPMI 1640 and the amount and type of DNA as indicated in the figure legends. Following transfection, cells were incubated for 20 h in RPMI 1640 containing 10% fetal calf serum and processed as indicated for each experiment.

Antibodies. The monoclonal antibody C305 (specific for the Jurkat Ti  chain) (57) was used for anti-TCR stimulations. M2 (anti-Flag epitope) was obtained from Sigma. Anti-PLC-1 mixed monoclonal antibodies and antiphosphotyrosine monoclonal antibody 4G10 were purchased from Upstate Biotechnology, Inc. Polyclonal anti-PLC-1 was purchased from Santa Cruz Biotechnology. Polyclonal anti-Nck antiserum was provided by Joseph Schlessinger (New York University). Sheep anti-human SLP-76 was provided by Gary Koretzky (University of Pennsylvania). Anti-phospho-ERK antibodies were purchased from New England Biolabs. Polyclonal anti-Erk antiserum was obtained from Zymed. Anti-Gads was obtained from C. Jane McGlade (University of Toronto).

Luciferase assays. Cells were transiently cotransfected with 20 µg of an NFAT luciferase reporter plasmid along with the plasmids indicated in figure legends; 20 to 24 h later, cells were aliquoted into a 96-well cell culture dish at 10⁵ cells/well and stimulated for 6 h at 37°C with various stimuli. Cells were lysed and assayed for luciferase activity as previously described (61). To correct for variations in transfection efficiency, the NFAT luciferase activity obtained upon receptor stimulation was normalized to the activity obtained upon treatment with phorbol myristate acetate (PMA; 20 to 50 ng/ml) plus ionomycin (1 µM).

Immunoprecipitations and GST pull-downs. Cells were washed in phosphate-buffered saline containing Ca²⁺ and Mg²⁺ (PBS), preheated to 37°C for 10 min, and either mock stimulated with PBS or stimulated for various times with C305 (anti-TCR). Cells were then collected and lysed at 2 × 10⁸ cells/ml in cold lysis buffer containing 1% Nonidet P-40, 10 mM Tris (pH 7.6), and 150 mM NaCl supplemented with protease and phosphatase inhibitors as described elsewhere (51), as well as 50 mM NaF, 50 mM -glycerol phosphate (pH 7.5), and 20 mM sodium pyrophosphate (pH 7.5). After 15 min at 4°C, lysates were centrifuged at 4°C in a microcentrifuge at 13,000 rpm for 10 min and then in a Beckman Optima miniultracentrifuge at 100,000 × g for 11 min. Supernatants were collected and used for affinity purification on antibody- or GST fusion protein-coated beads, as described below.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Functional analysis of SLP-76 domain mutants on a SLP-76-null genetic background. Previous studies have defined three functional domains within SLP-76: an N-terminal acidic domain containing three tyrosine phosphorylation sites, a small segment of the proline-rich domain responsible for binding to Gads, and a C-terminal SH2 domain (Fig. 1A). Interestingly, all three domains are required for augmentation of TCR signaling by overexpressed SLP-76 (14, 33, 54). However, the cells used for overexpression studies express endogenous, wild-type SLP-76, which can provide compensatory function in trans. As a result, these studies could not address the structural requirements for the normal, physiologic function of SLP-76.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Partial reconstitution of TCR-induced NFAT activation by domain-inactivated mutants of SLP-76. (A and B) The SLP-76-deficient cell line J14 was transiently cotransfected with 20 µg of vector, wild-type (WT) SLP-76, or the indicated SLP-76 mutant constructs, along with an NFAT-luciferase reporter plasmid (20 µg). Twenty hours later, the cells were stimulated for 6 h in tissue culture medium alone (unstimulated) or with soluble monoclonal antibody to TCR (C305) (A), immobilized anti-TCR (B), or a combination of PMA (50 ng/ml) and ionomycin (1 µM). Cells were then lysed and assayed for luciferase activity. The results are expressed as a fraction of the activity obtained upon stimulation with PMA and ionomycin. Results from up to seven experiments for each SLP-76 construct were averaged. Error bars indicate standard deviations, and numbers directly above the error bars indicate the number of experiments performed for each construct. (B) The SLP-76 constructs were expressed at roughly equivalent levels, as shown in this anti-SLP-76 blot of lysates from a representative experiment.

Identification of an additional, essential functional domain within the proline-rich domain of SLP-76. The panel of mutants tested above did not include extensive mutation of the central, proline-rich domain of SLP-76 (with the exception of the Gads-binding site deletion). We considered the possibility that an important functional domain of SLP-76 might be found within this large proline-rich region. To test this possibility, we arbitrarily defined four subdomains (P-I through P-IV), which encompass the entire proline-rich domain, exclusive of the 21 amino acid Gads mutation. Mutants bearing deletions of these subdomains were constructed (Fig. 1B, constructs 6 through 9) and tested for reconstitution of TCR-mediated NFAT activation in J14. These assays revealed that the P-I region (encompassing residues 157 to 223 of SLP-76) is essential for SLP-76 function (Fig. 3A). By contrast, P-III and P-IV are dispensable, while P-II has an intermediate effect on SLP-76 function. All mutants were expressed at roughly equivalent levels (Fig. 3A, right).

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Identification of an additional functional domain within the proline-rich region of SLP-76. (A) Additional SLP-76 mutants, bearing deletions that span the proline-rich domain of SLP-76, were tested for the ability to reconstitute TCR-induced NFAT activation in the SLP-76-deficient T-cell line J14 as in Fig. 2. One of four representative experiments is shown. The P constructs were expressed at a level similar to or higher than that of wild-type (WT) SLP-76, as demonstrated by blotting with an anti-Flag antibody (right). (B) NFAT assay of SLP-76 mutants bearing C-terminal deletions. One of four representative experiments is shown. All SLP-76 constructs were expressed at roughly equivalent levels, as shown in the anti-Flag blot (right).

Effect of SLP-76 domain mutations on TCR-induced signaling through the PLC-1/calcium and Erk pathways. Our initial structure-function analysis (see above) was based on transient transfection of J14 cells, a technique that produces a high level of expression in a small fraction of the transfected cells. To test the functional competence of key SLP-76 mutants at physiologic levels of expression, J14 cells were stably transfected with four mutant constructs, chosen to individually inactivate each of the four functional domains of SLP-76: P-1 (missing the P-I domain). Y3F (three tyrosine phosphorylation sites mutated to phenylalanine), Gads (missing the Gads-binding domain), and SH2^mut (bearing an inactivating mutation in the SH2 domain). Multiple stably transfected clones were chosen that expressed surface TCR and a level of SLP-76 roughly equivalent to those of wild-type Jurkat cells and the previously described, wild-type reconstituted cell line J14-76-11 (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   TCR responses in J14 cells stably reconstituted with mutant forms of SLP-76. J14 cells were stably transfected with the indicated SLP-76 alleles. Clones expressing roughly equivalent levels of surface TCR and of transfected SLP-76 were chosen and tested for the ability to respond to anti-TCR stimulation. Two independent clones were analyzed for each construct, with similar results. (A) The indicated clones were transiently transfected with an NFAT-luciferase reporter plasmid, stimulated with medium, immobilized monoclonal antibody to TCR (C305), or immobilized anti-TCR plus PMA (20 ng/ml) and assayed for luciferase activity as in Fig. 2. WT, wild type. (B) The indicated cell lines were stimulated for 1 min at 37°C with anti-TCR (C305) or mock stimulated with PBS and lysed, and lysates from 2 × 107 cells were immunoprecipitated (IP) with anti-PLC-1 antibody. Immunoprecipitates were separated by SDS-PAGE and probed by Western blotting with antiphosphotyrosine (anti-ptyr; 4G10, top) and anti-PLC-1 (bottom). The experiment was performed a total of six times, and representative results are shown. No reproducible differences were seen between the different mutant-reconstituted cell lines. (C) Cells were stimulated for 2 min at 37°C and lysed as in panel B, and lysates from 2 × 107 cells were immunoprecipitated with anti-Vav antibody. Immunoprecipitates were separated by SDS-PAGE and probed by Western blotting with antiphosphotyrosine (4G10; top) and anti-Vav (bottom). (D) The indicated cell lines were metabolically labeled with [3H]myoinositol, preincubated for 20 min at 37°C in the presence of 20 mM LiCl, and stimulated with anti-TCR. Total accumulation of inositol phosphates was measured as described elsewhere (21). (E) The indicated cell lines were stimulated for 10 min with anti-TCR and lysed, and the lysates were analyzed by Western blotting for total Erk or for phospho-Erk.

Effect of the P-I domain deletion on SLP-76-mediated protein-protein interactions. Of the domains that we have identified as critical for SLP-76 function, the P-I domain is newly defined. We sought to establish whether the P-I domain plays primarily a structural role, enabling proper function of the other domains of SLP-76, or whether it directly mediates protein-protein interactions. To address this question, we examined the function of other SLP-76 domains within the context of the P-1 mutant. Flag-tagged SLP-76 was immunoprecipitated from the stable transfectants described above, before or after stimulation of the TCR. An antiphosphotyrosine blot of the immunoprecipitated proteins demonstrated that the P-1 mutant is inducibly tyrosine phosphorylated following TCR stimulation, as are the wild-type, Gads, and SH2^mut proteins (Fig. 5). Thus, these mutations do not grossly disrupt the overall conformation of SLP-76 or its ability to interact normally with TCR-activated tyrosine kinases. Likewise, the constitutive interaction of SLP-76 with Gads is not disrupted by the P-1, Y3F, or SH2^mut mutation (Fig. 5), again suggesting that these mutations do not grossly disrupt the overall structure of SLP-76.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Effect of the P-I domain deletion on SLP-76 tyrosine phosphorylation and intermolecular interactions. J14 cells, stably transfected with the indicated SLP-76 alleles, were stimulated for 2 min with anti-TCR (C305) or mock stimulated with PBS and lysed, and lysates from 2 × 107 cells were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were separated by SDS-PAGE and probed by Western blotting with anti-Flag, antiphosphotyrosine (anti-ptyr; 4G10) and anti-Gads, as shown. Two independent stable transfectants were analyzed for each construct; a representative experiment is shown. WT, wild type.

Basal and inducible interaction of SLP-76 with PLC-1. We next considered the possibility that the P-I domain may bind to the SH3 domain of PLC-1. While some published data is suggestive of an association between SLP-76 and PLC-1 (24, 55), it has not been conclusively demonstrated. We used an anti-Flag antibody to immunoprecipitate SLP-76-associated proteins from J14-76-11 (J14, stably reconstituted with Flag-tagged, wild-type SLP-76) and probed the immunoprecipitates for PLC-1. We found an inducible association between SLP-76 and PLC-1, which was as striking as the well-established, inducible association between SLP-76 and Nck (Fig. 6A, lanes 1 and 2). To control for nonspecific adsorption of proteins to the anti-Flag beads, we performed an anti-Flag immunoprecipitation from Jurkat cells, which express untagged SLP-76. In this case, we did not observe significant PLC-1 in the immunoprecipitates (Fig. 6A, lane 3).

View larger version (24K): [in this window] [in a new window]   FIG. 6.   Association of SLP-76 with PLC-1. (A) Lysates from stimulated or unstimulated cells were immunoprecipitated (IP) with anti-Flag and analyzed by Western blotting as in Fig. 5. J14-76-11 cells were used in lanes 1 and 2. In lane 3, Jurkat cells were used to control for nonspecific adsorption to the anti-Flag beads. Immunoprecipitates were separated by SDS-PAGE and probed by Western blotting with anti-Flag, anti-PLC-1, and anti-Nck, as shown. (B) Time course of the association of PLC-1 with SLP-76. J14-76-11 (lanes 1 to 6) and Jurkat (lane 7) cells were stimulated with anti-TCR for the indicated times, lysed, and immunoprecipitated with anti-Flag as above. Following SDS-PAGE and Western blotting, immunoprecipitates were probed with anti-PLC-1, anti-Gads, and anti-Flag, as shown. Subsequently, the portion of the blot corresponding to Flag-tagged SLP-76 was stripped and reprobed with antiphosphotyrosine (anti-ptyr; 4G10). (C) Anti-Flag immunoprecipitates from the experiment depicted in Fig. 5 were separated by SDS-PAGE and probed by Western blotting with anti-PLC-1.

Direct interaction of the PLC-1 SH3 domain with SLP-76, mediated by the P-1 domain of SLP-76. To further test our hypothesis that the SH3 domain of PLC-1 associates with the P-I domain of SLP-76, we examined the ability of the PLC-1 SH3 domain to bind SLP-76 in vitro. A GST-PLC-1 SH3 fusion protein specifically bound to SLP-76. By contrast, SLP-76 did not bind to GST alone, nor did it bind to a control GST fusion protein containing the second SH3 domain of human Nck (Fig. 7A). These results demonstrate that the PLC-1 SH3 can bind to SLP-76. A larger fusion protein, encompassing the two SH2 domains and the SH3 domain of PLC-1 (SH223), did not show increased binding to SLP-76 relative to the SH3 domain alone, nor was the interaction affected by anti-TCR stimulation (Fig. 7B). This result provides further confirmation that the PLC-1 SH2 domains do not interact with the sites on SLP-76 that are tyrosine phosphorylated upon TCR stimulation in vivo. To better define the domains of SLP-76 that interact with PLC-1, we tested the in vitro interaction of the PLC-1 fusion proteins with mutant forms of SLP-76 (Fig. 7C). Our results show that the P-I domain is the only domain required for the interaction of SLP-76 with the PLC-1 SH3 domain (Fig. 7C, top left) and with the SH223 construct (top right). Taken together, our results demonstrate that the SH2 domains of PLC-1 do not significantly bind to the in vivo-phosphorylated tyrosine phosphorylation sites on SLP-76, whereas the SH3 domain of PLC-1 binds to a site within the P-I domain of SLP-76. This interaction is responsible for the basal association of SLP-76 with PLC-1 and is likely to be the basis for the functional importance of this domain.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Binding of the PLC-1 SH3 domain to the SLP-76 P-1 domain. GST fusion proteins were used to affinity purify associating proteins from cell lysates as described in Materials and Methods. Associating proteins or total cell lysates were separated by SDS-PAGE and probed with an anti-Flag antibody to detect SLP-76. (A) Equal amounts of GST alone, GST fused to the second SH3 domain of Nck, and GST fused to the PLC-1 SH3 domain were used to pull down proteins from lysates of J14-76-11 cells, which express Flag-tagged SLP-76. (B) J14-76-11 cells were stimulated for 2 min with anti-TCR (C305) or mock stimulated with PBS and lysed. Equal amounts of GST alone, GST-PLC-1 SH3 or GST fused to the two SH2 domains and SH3 domain of PLC-1 (PLC-1 SH223) were used to pull down SLP-76 from the lysates as in panel A. (C) J14 cells, stably transfected with the indicated SLP-76 alleles, were stimulated with anti-TCR (C305) or mock stimulated and lysed as above. Equal amounts of GST-PLC-1 SH3 (left) or GST-PLC-1 SH223 (right) were used to pull down proteins from the lysates. Associating proteins (top) or total cell lysates (bottom) were separated by SDS-PAGE and probed with an anti-Flag antibody to detect SLP-76. WT, wild type.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

SLP-76 plays an essential role in coupling ITAM-containing receptors (including the TCR, the high-affinity IgE receptor, and the platelet collagen receptor) to tyrosine phosphorylation and activation of PLC-1 and PLC-2 and to subsequent calcium flux (5, 17, 37, 64). Nonetheless, the mechanisms by which SLP-76 influences PLC- tyrosine phosphorylation and activation have not been elucidated. In this study, we used mutational analysis to identify the domains of SLP-76 required for PLC-1 activation, taking advantage of the SLP-76-deficient J14 cells as an ideal genetic background for structure-function analysis of SLP-76. Our studies resulted in the identification of a previously unrecognized, essential functional domain within the proline-rich domain of SLP-76. Further, we have identified an association between this domain and the SH3 domain of PLC-1.

Using the NFAT activation assays as a biologically relevant readout for SLP-76 function, we initially used transient transfection assays to test the requirement for previously defined functional domains of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding domain, and the SH2 domain (14, 33). These experiments confirmed that each of these domains contributes to SLP-76 function. Nevertheless, we were surprised to find that none of the above domains is absolutely required for TCR-mediated NFAT activation. Assuming that SLP-76 must interact with one or more essential effector proteins in order to mediate activation of PLC-1 and NFAT, how can one explain that individual mutation of each of the known protein-protein interaction domains of SLP-76 fails to completely abrogate SLP-76 function? One possibility is that the best-characterized SLP-76-interacting proteins, including Vav, Nck, Gads, and SLAP-130/Fyb, which associate with the previously defined functional domains of SLP-76, may be dispensable for SLP-76-mediated signaling events leading to NFAT activation. Another possibility is that SLP-76 may form a multivalent interaction with a critical effector protein, and that this interaction is weakened but not completely disrupted by individual mutation of each interaction site. However, we considered the alternate possibility, that an important effector of SLP-76 may bind to a site not encompassed by the above mutations.

These considerations led us to examine other regions of SLP-76, revealing a requirement for residues 157 to 223 to mediate TCR-induced activation of Erk, PLC-1, and NFAT. We have designated this region the P-I domain. Deletion of the P-I domain does not grossly disrupt the overall conformation of SLP-76, as the P-I deletion protein is inducibly tyrosine phosphorylated in response to TCR stimulation, binds to Gads, and binds inducibly to PLC-1. We therefore concluded that the P-I domain is likely to mediate a functionally important interaction with an effector protein and that this interaction is required for optimal activation of PLC-1.

The 67-amino-acid P-I domain is extremely proline rich, containing a total of 20 proline residues. Not surprisingly, it has been found to associate with a number of SH3 domains, including the SH3 domains of Lck (44), Itk (3), and PLC-1 (this study). Several considerations led us to conclude that neither Lck nor Itk is the physiologically relevant effector of the SLP-76 P-I domain. In carefully controlled immunoprecipitation experiments, we detected association of full-length SLP-76 with PLC-1 but not with Lck or Itk. Indeed, Bunnell et al. (3) have demonstrated only a modest interaction between full-length SLP-76 and Itk, using proteins overexpressed in a baculoviral system. In the context of total cellular proteins, this modest interaction may be competed off by other, more physiologic interactions. In addition, a deletion of 22 amino acids within the P-I domain (181 to 203), which removes the putative binding sites of Lck and Itk, does not impair SLP-76 function (data not shown). Finally, studies from other laboratories suggest that the SH3 domains of Lck and Tec family kinases do not play essential roles in the activation of PLC- and calcium flux, whereas the SH3 domain of PLC-1 is required. Lck-deficient J.CaM1.6 cells, reconstituted with SH3-mutated Lck, show normal activation of PLC-1 and normal calcium flux (10). Likewise, overexpression of an SH3-deletion mutant of the Tec family kinase Btk (the B-cell homologue of Itk) leads to augmented BCR-induced calcium flux (46), a result that would not be expected if the SH3 domain of Tec family kinases played a major role in mediating activation of PLC-. In contrast, the SH3 domain of PLC-1 is required for optimal inositol phosphate production and NFAT activation following stimulation of the BCR in B cells (9). These considerations suggest that while both Lck and Itk are required for optimal TCR-induced calcium flux (28, 45, 51), their effect on calcium flux is not mediated by binding of their SH3 domains to the P-1 domain of SLP-76. Rather, the physiologically important effector of the P-1 domain appears to be PLC-1.

The association between SLP-76 and PLC-1 has two components: a basal component and a TCR-inducible component. These components are largely independent of each other and are mediated by different domains of SLP-76. The basal component depends on the P-1 domain of SLP-76, which associates with the SH3 domain of PLC-1. Notably, the P-1 mutant still binds inducibly to PLC-1, indicating that a different domain of SLP-76 mediates the inducible association. One might think that the inducible component reflects binding of the SH2 domains of PLC-1 to the tyrosine phosphorylation sites of SLP-76. However, we found no evidence to support this possibility. We observed no in vitro binding of the SH2 domains of PLC-1 to tyrosine phosphorylated SLP-76 from TCR-stimulated cells. Furthermore, the inducible component was not affected by mutation of the SLP-76 tyrosine phosphorylation sites. Rather, the inducible association depended on the Gads-binding domain of SLP-76, which mediates recruitment of SLP-76 to LAT, and followed a time course that paralleled the time course of LAT tyrosine phosphorylation. We conclude that the inducible association of SLP-76 with PLC-1 is indirect and is due to recruitment of both proteins to tyrosine-phosphorylated LAT. Thus, a web of interactions connects PLC-1 to the SLP-76, LAT, and Gads adapters (Fig. 8). Both PLC-1 and Gads bind inducibly to LAT via their SH2 domains (1, 29, 50). Concomitantly, the SH3 domain of Gads binds constitutively to SLP-76 (1, 26, 29), which, in turn, binds constitutively to the SH3 domain of PLC-1 (this study).

View larger version (47K): [in this window] [in a new window]   FIG. 8.   A web of interactions connects PLC-1 to the SLP-76, LAT, and Gads adapter proteins. The SH2-mediated interactions are inducible upon TCR stimulation, whereas the SH3-mediated interactions are constitutive. As predicted by this model, the constitutive component of the interaction of SLP-76 with PLC-1 depends on the P-I domain of SLP-76, whereas the inducible component depends on the Gads-binding domain of SLP-76.

How does this web of interactions bring about the activation of PLC-1? First, the recruitment of PLC-1 to LAT appears to be essential for its TCR-induced tyrosine phosphorylation and activation. Indeed, mutation of the single PLC-1 SH2 domain consensus binding site in LAT is sufficient to abrogate TCR-induced tyrosine phosphorylation of PLC-1 (68). SLP-76 is not required for the TCR-induced association of PLC-1 with LAT (64); furthermore, LAT is an integral membrane protein, suggesting that the PLC-1-LAT interaction is probably sufficient to localize PLC-1 to the membrane. Nonetheless, SLP-76 is required to fully activate the membrane-localized PLC-1 (64). We have found that the P-I domain of SLP-76 associates with the SH3 domain of PLC-1 and is required to mediate activation of PLC-1. Based on this finding, we suggest that this interaction is likely to play a role in the regulation of PLC-1 by SLP-76.

Examination of the interactions depicted in Fig. 8 suggests a number of mechanisms by which SLP-76 could contribute to the activation of PLC-1. One possibility is that SLP-76 stabilizes the recruitment of PLC-1 to LAT. SH2- and SH3-mediated interactions are generally of relatively low affinity, such that a single interaction between the N-terminal SH2 domain of PLC-1 and LAT might not be stable enough to keep PLC-1 at the membrane. However, the direct interaction of each of the proteins in the complex with two other proteins could serve to stabilize the complex as a whole, thereby stabilizing the recruitment of PLC-1 to the membrane. In support of this hypothesis, Zhang et al. have found that mutation of the Gads-binding sites on LAT significantly reduces the binding of PLC-1 to LAT (68). Nonetheless, we do not believe that the role of SLP-76 is limited to stabilizing the recruitment of PLC-1 to the membrane. Experiments in B cells show that membrane-targeted PLC- is not efficiently activated by BCR stimulation in the absence of other interactions mediated by the PLC- SH domains (9, 23). Thus, we believe that the interaction of SLP-76 with the SH3 domain of PLC-1 exerts an important regulatory influence on PLC-1 once it is at the membrane. In particular, the direct binding of the N-terminal SH2 and SH3 domains of PLC-1 to LAT and SLP-76, respectively, may conformationally constrain the SH region of PLC-1. The result of this conformational change may be a relief of the autoinhibitory function exerted by the SH domains of PLC-1 (19, 20) and/or a stretching of the intervening region between the SH2 and SH3 domains. Since two of the tyrosine phosphorylation sites of PLC-1 are located within this intervening region, a SLP-76-induced conformational change in the SH region of PLC-1 may result in better accessibility of these tyrosines to phosphorylation.

The above, speculative model accounts for the role of the P-I and Gads-binding domains of SLP-76 in mediating optimal activation of PLC-1. In addition, our data demonstrate that the N-terminal tyrosine phosphorylation sites of SLP-76 are required for PLC-1 activation. Recent studies suggest that these phosphorylation sites may bind to Tec family tyrosine kinases Itk and/or Rlk (3, 47, 52), which are required for TCR-mediated activation of PLC-1 (28, 45). It is possible that SLP-76 may not only affect the conformation of PLC-1 and its accessibility to tyrosine kinases but also recruit the appropriate tyrosine kinase required for phosphorylation of a subset of the tyrosine phosphorylation sites of PLC-1, leading to its activation.

This study identifies SLP-76 as a physiologic ligand for the SH3 domain of PLC-1 and provides evidence that this interaction plays an essential role in SLP-76-mediated activation of PLC-1. BCR-mediated activation of PLC-1 and -2 occurs by an analogous but not identical mechanism, due to the different adapter proteins expressed in the two cell types. TCR-mediated activation of PLC-1 requires SLP-76 and LAT (15, 64), whereas BCR-mediated PLC-1 activation requires BLNK (22), an adapter which bears overall structural similarity to SLP-76 but only 33% homology (16, 58). No analog of LAT has been identified in B cells, where BLNK is thought to fulfill the function of both adapters (16, 59). In keeping with this dual role of BLNK, the SH2 domains of PLC-1, which associate with tyrosine-phosphorylated LAT in T cells, bind to tyrosine phosphorylated BLNK in B cells (9, 23). Nonetheless, this interaction does not rule out an additional interaction between the SH3 domain of PLC-1 and the proline-rich domain of BLNK, which may be required for activation of PLC-1 in this system. This possibility awaits further investigation.

It is important to note that our analysis is limited to an investigation of the SLP-76 domains required for NFAT activation. SLP-76 is likely to participate in additional signaling pathways, such as those that lead to TCR-induced actin polymerization (55) or the activation of HPK1 (27). The requirements for SLP-76 domains and effector proteins for signaling through these pathways may differ and will require further analysis.