Src-like Adaptor Protein Research Articles

A Novel Src Homology 2 Domain-containing Molecule, Src-like Adapter Protein-2 (SLAP-2), Which Negatively Regulates T Cell Receptor Signaling

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.

Positive selection with SLAP

Regulation of the positive selection of thymocytes is the key to T-cell development, with double-positive CD4+CD8+ subsets playing a crucial role. The Src-like adaptor protein (SLAP) is a recently identified protein that negatively regulates mitogenesis. It is thought to regulate signaling events downstream of the T-cell receptor (TCR). Whether it plays a positive or negative regulatory role is still controversial. A recent report by Sosinowski and colleagues shows that the expression of SLAP is regulated developmentally. They found low-level expression in CD4−CD8− thymocytes, which increased to a peak in the CD4+CD8+ subset and decreased again to lower levels in more mature cells. Disruption of the gene encoding SLAP enhanced expression of TCR and CD5 at the double-positive thymocyte stage and rescued the development of ZAP-70-deficient thymocytes. This points the way to a role for SLAP in the down-regulation of TCR expression at the double-positive stage and, hence, as a regulator of positive selection in the thymus. Immunity (2001) 15, 457–466 AL

Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

The Src-like Adaptor Protein Downregulates the T Cell Receptor on CD4 +CD8 + Thymocytes and Regulates Positive Selection

In this report, we show that the Src-like adaptor protein (SLAP) plays an important role in thymocyte development. SLAP expression is developmentally regulated; it is low in CD4 −CD8 − thymocytes, it peaks in the CD4 +CD8 + subset, and it decreases to low levels in more mature cells. Disruption of the SLAP gene leads to a marked upregulation of TCR and CD5 expression at the CD4 +CD8 + stage. The absence of SLAP was also developmentally significant because it enhanced positive selection in mice expressing the DO11.10 transgenic T cell receptor. Moreover, SLAP deletion at least partially rescued the development of ZAP-70-deficient thymocytes. These results demonstrate that SLAP participates in a novel mechanism of TCR downregulation at the CD4 +CD8 + stage and regulates positive selection.

Characterization of promoter region and genomic structure of the murine and human genes encoding Src like adapter protein

Src-like adapter protein (SLAP) was identified as a signaling molecule in a yeast two-hybrid system using the cytoplasmic domain of EphA2, a receptor protein tyrosine kinase (Pandey et al., 1995. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201–19204). It is very similar to members of the Src family of cytoplasmic tyrosine kinases in that it contains very homologous SH3 and SH2 domains (Abram and Courtneidge, 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell. Res. 254, 1–13.). However, instead of a kinase domain at the C-terminus, it contains a unique C-terminal region. In order to exclude the possibility that an alternative form exists, we have isolated genomic clones containing the murine Slap gene as well as the human SLA gene. The coding regions of murine Slap and human SLA genes contain seven exons and six introns. Absence of any kinase domain in the genomic region confirm its designation as an adapter protein. Additionally, we have cloned and sequenced approximately 2.6 kb of the region 5′ to the initiator methionine of the murine Slap gene. When subcloned upstream of a luciferase gene, this fragment increased the transcriptional activity about 6-fold in a human Jurkat T cell line and approximately 52-fold in a murine T cell line indicating that this region contains promoter elements that dictate SLAP expression. We have also cloned the promoter region of the human SLA gene. Since SLAP is transcriptionally regulated by retinoic acid and by activation of B cells, the cloning of its promoter region will permit a detailed analysis of the elements required for its transcriptional regulation.

Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

Slap Negatively Regulates Src Mitogenic Function but Does Not Revert Src-Induced Cell Morphology Changes

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975–978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G1/S block induced by both SrcK− and a Slap mutant with a deletion of its C terminus (SlapΔC), but not the block induced by Slap or SlapΔSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcΔK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85α, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapΔC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling.

Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.

Src-like adaptor protein (Slap) is a negative regulator of mitogenesis.

The Src-like adaptor protein (Slap) is a recently identified adaptor protein containing Src homology 3 (SH3) and SH2 domains. Slap is found in a wide range of cell types and was shown to interact with the Eck receptor tyrosine kinase in a yeast two-hybrid interaction screen [1]. Here, we found that Slap is expressed in NIH3T3 cells and could associate with the activated platelet-derived growth factor (PDGF) receptor. Using mutated versions of the PDGF receptor and phosphopeptide competition experiments, we determined that Slap has the highest affinity for the Src-binding site of the PDGF receptor. Our inability to produce cell lines that stably expressed Slap suggested that Slap inhibited cell growth. We further investigated this issue by transiently expressing Slap by microinjection. Overexpression of Slap by this method inhibited DNA synthesis induced by PDGF and serum, whereas overexpression of the adaptor proteins Grb2 and Shc did not. Finally, microinjection of a Slap antibody into NIH3T3 cells that had been stimulated with suboptimal doses of growth factors potentiated the effects of the growth factors. These data suggest that, unlike other adaptor proteins, Slap is a negative regulator of signalling initiated by growth factors.

Expression of Src-like Adapter Protein mRNA Is Induced by All-trans Retinoic Acid

By using a differential display method, specific bands were selected from ladder PCR products derived from ATRA-dependent differentiated U937 cells, in comparison with those of untreated U937. By screening the cDNA library of ATRA-dependent differentiated U937 cells with one of the PCR products, we cloned the src-like adapter protein (SLAP). Northern blot analysis of U937 cells with or without ATRA treatment indicated that the SLAP mRNA was clearly induced by ATRA. The induction was inhibited by the addition of cycloheximide, indicating that ATRA acted indirectly through synthesis of other proteins. The SLAP mRNA was induced in HL60 and NB-4 but not in K562 or THP-1. Interestingly, these cells in which SLAP mRNA was induced by ATRA all showed ATRA-dependent cell differentiation. The relationship between SLAP and cell differentiation is unclear, but SLAP may transduce a signal for cell differentiation.

Chromosomal Localization of the Mouse Src-like Adapter Protein ( Slap) Gene and Its Putative Human Homolog SLA

Molecules containing Src-homology 2 (SH2) and Src-homology 3 (SH3) domains are critical components of signal transduction pathways that serve to relay signals originating from the cell surface to the interior of the cell. Src-like adapter protein (SLAP) is a recently described adapter protein that binds activated the Eck receptor protein-tyrosine kinase. Although SLAP bears a striking homology to the SH3 and SH2 domains of the Src family of nonreceptor tyrosine kinases, it does not contain a tyrosine kinase catalytic domain. In this report, the Slap gene was mapped by linkage analysis to mouse chromosome 15, while its putative human homolog (SLA) was identified and mapped to human 8q22.3-qter using a panel of somatic cell hybrids.

Receptor tyrosine kinase expression, a central component of rat hepatic stellate cell activation - characterization by homology PCR

SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

Characterization of a Novel Src-like Adapter Protein That Associates with the Eck Receptor Tyrosine Kinase

The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D.F., Saltiel, A. R., and Dixit, V.M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule.