The molecular adapter SLP-76 relays signals from platelet integrin alphaIIbbeta3 to the actin cytoskeleton

Platelet adhesion to fibrinogen through integrin alpha(IIb)beta(3) triggers actin rearrangements and cell spreading. Mice deficient in the SLP-76 adapter molecule bleed excessively, and their platelets spread poorly on fibrinogen. Here we used human platelets and a Chinese hamster ovary (CHO) cell expression system to better define the role of SLP-76 in alpha(IIb)beta(3) signaling. CHO cell adhesion to fibrinogen required alpha(IIb)beta(3) and stimulated tyrosine phosphorylation of SLP-76. SLP-76 phosphorylation required coexpression of Syk tyrosine kinase and stimulated association of SLP-76 with the adapter, Nck, and with the Rac exchange factor, Vav1. SLP-76 expression increased lamellipodia formation induced by Syk and Vav1 in adherent CHO cells (p < 0.001). Although lamellipodia formation requires Rac, SLP-76 functioned downstream of Rac by potentiating adhesion-dependent activation of PAK kinase (p < 0.001), a Rac effector that associates with Nck. In platelets, adhesion to fibrinogen stimulated the association of SLP-76 with the SLAP-130 adapter and with VASP, a SLAP-130 binding partner implicated in actin reorganization. Furthermore, SLAP-130 colocalized with VASP at the periphery of spread platelets. Thus, SLP-76 functions to relay signals from alpha(IIb)beta(3) to effectors of cytoskeletal reorganization. Therefore, deficient recruitment of specific adapters and effectors to sites of adhesion may explain the integrin phenotype of SLP-76(-/-) platelets.     

Functional association between SLAP-130 and SLP-76 in Jurkat T cells

T cell antigen receptor (TCR) engagement results in protein-tyrosine kinase activation which initiates signaling cascades leading to induction of the interleukin-2 gene. Previous studies identified two substrates of the TCR-induced protein-tyrosine kinases, SH2 domain-containing leukocyte specific protein of 76 kDa (SLP-76) and SLP-76-associated phosphoprotein of 130 kDa (SLAP-130). While SLP-76 appears to couple the TCR with downstream signals, SLAP-130 may play a negative regulatory role in T cell activation. In this study, we demonstrate that consistent with its ability to abrogate the SLP-76 augmentation of TCR-induced activation of the NFAT/AP1 region of the interleukin-2 promoter, overexpression of SLAP-130 also interferes with the rescue of signaling in SLP-76-deficient Jurkat cells in co-transfection experiments. The effect of SLAP-130 on SLP-76 function is specific for regulating TCR-induced ERK activation, but not phospholipase Cgamma 1 phosphorylation. By generating both deletion and point mutants of SLAP-130, we identified tyrosine 559 as critical for the interaction between SLP-76 and SLAP-130. We show that mutation of this residue in context of full-length SLAP-130 diminishes the ability of SLAP-130 to abrogate SLP-76 function. These data suggest that the SLAP-130/SLP-76 association is important for the negative regulatory role that SLAP-130 appears to play in T cell signaling.     

Cutting edge: SLP-76 cooperativity with FYB/FYN-T in the Up-regulation of TCR-driven IL-2 transcription requires SLP-76 binding to FYB at Tyr595 and Tyr651

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.     

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.     

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.     

Cas-L is required for beta 1 integrin-mediated costimulation in human Tcells.

Beta 1 integrins provide a costimulus for TCR/CD3-driven T cell activation and IL-2 production in human peripheral T cells. However, this beta 1 integrin-mediated costimulation is impaired in a human T lymphoblastic line, Jurkat. We studied the molecular basis of this impaired costimulation and found that Cas-L, a 105-kDa docking protein, is marginally expressed in Jurkat T cells, whereas Cas-L is well expressed in peripheral T cells. Cas-L is a binding protein and a substrate for focal adhesion kinase and is tyrosine phosphorylated by beta 1 integrin stimulation. We here show that the transfection of wild-type Cas-L in Jurkat T cells restores beta 1 integrin-mediated costimulation. However, Cas-L transfection had no effect on CD28-mediated costimulation, indicating that Cas-L is specifically involved in the beta 1 integrin-mediated signaling pathway. Furthermore, transfection of the Cas-L Delta SH3 mutant failed to restore beta 1 integrin-mediated costimulation in Jurkat cells. Cas-L Delta SH3 mutant lacks the binding site for focal adhesion kinase and is not tyrosine phosphorylated after beta 1 integrin stimulation. These findings strongly suggest that the tyrosine phosphorylation of Cas-L plays a key role in the signal transduction in the beta 1 integrin-mediated T cell costimulation.     

The Src homology 2 domain-containing leukocyte protein of 76-kDa adaptor links integrin ligation with p44/42 MAPK phosphorylation and podosome distribution in murine dendritic cells

The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c+ B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.     

Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes

B cell linker protein (BLNK) is a SLP-76-related adaptor protein essential for signal transduction from the BCR. To identify components of BLNK-associated signaling pathways, we performed a phosphorylation-dependent yeast two-hybrid analysis using BLNK probes. Here we report that the serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1), which is activated upon antigen-receptor stimulation and which has been implicated in the regulation of MAP kinase pathways, interacts physically and functionally with BLNK in B cells and with SLP-76 in T cells. This interaction requires Tyr(379) of HPK1 and the Src homology 2 (SH2) domain of BLNK/SLP-76. Via homology modeling, we defined a consensus binding site within ligands for SLP family SH2 domains. We further demonstrate that the SH2 domain of SLP-76 participates in the regulation of AP-1 and NFAT activation in response to T cell receptor (TCR) stimulation and that HPK1 inhibits AP-1 activation in a manner partially dependent on its interaction with SLP-76. Our data are consistent with a model in which full activation of HPK1 requires its own phosphorylation on tyrosine and subsequent interaction with adaptors of the SLP family, providing a mechanistic basis for the integration of this kinase into antigen receptor signaling cascades.     

Evidence for glycosylphosphatidylinositol (GPI)-anchored eosinophil-derived neurotoxin (EDN) on human granulocytes

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST–SLAP-130–SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.     

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.     

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.     

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.     

Distinct role of ZAP-70 and Src homology 2 domain-containing leukocyte protein of 76 kDa in the prolonged activation of extracellular signal-regulated protein kinase by the stromal cell-derived factor-1 alpha/CXCL12 chemokine

Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1alpha is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1alpha that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1alpha stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1alpha-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1alpha to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1alpha stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.     

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.     

Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.     

SLP-76 binding to p56lck: a role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

Nonreceptor protein tyrosine kinases and associated substrates play a pivotal role in Ag receptor stimulation of resting cells and in the initiation of activation-induced cell death (AICD) of preactivated T cells. CD4-associated p56lck has been implicated not only in the activation of primary T cells, but also in the inhibition of T cell responses. We have previously shown that CD4+ T cell clones can be rescued from AICD when surface CD4 is engaged before the TCR stimulus. In this study, we show that prevention of AICD is associated with a CD4-dependent inhibition of TCR-triggered tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and Vav. We provide evidence for a SLP-76 interaction with Src homology 3 domains of p56lck and identify amino acids 185-194 of SLP-76 as relevant docking site. In view of the multiple functions of p56lck and SLP-76/Vav in the initiation of TCR/CD3/CD4 signaling, we propose a model for the CD4-dependent inhibition of TCR signaling and AICD of preactivated T cells. Our data suggest that preformed activation complexes of adapter proteins and enzymes in the vicinity of the CD4/p56lck complex are no longer available for the TCR signal when CD4 receptors are engaged before TCR stimulation.     

T cell receptor-induced activation of phospholipase C-gamma1 depends on a sequence-independent function of the P-I region of SLP-76

SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-gamma1 (SH3(PLC)) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-gamma1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-gamma1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-gamma1 activation, we mapped the PLC-gamma1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3(PLC), but is fully functional, mediating TCR-induced phosphorylation of PLC-gamma1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3(PLC) to activate PLC-gamma1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.