Functional Cloning of Src-like Adapter Protein-2 (SLAP-2), a Novel Inhibitor of Antigen Receptor Signaling

Abstract

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.

Cloning and characterization of human Src-like adaptor protein 2 and a novel splice isoform, SLAP-2-v

“…While the consequences of fluctuations in the relative levels of expression of p28 and p25 SLAP-2 remain to be more fully investigated, the p25 isoform could serve to target c-Cbl to a different subcellular location or antagonize the function of p28. The p28 and p25 murine isoforms have been shown to be differentially localized to the membrane and cytoplasm, respectively, and mutation of the amino-terminal myristoylation site of p28 was shown to reduce its capacity to negatively regulate TCR-mediated NFAT activation (Holland et al, 2001;Loreto et al, 2002;Pandey et al, 2002).…”

“…The numbering refers to the coding exons. The corresponding amino acid sequences are indicated by letters above the nucleotide sequences, which start and complete each of the exons mediated NFAT activation, suggesting that SLAP-2 is a negative regulator of TCR signal transduction (Holland et al, 2001;Loreto et al, 2002;Pandey et al, 2002). Importantly, the inhibition of TCR-mediated NFAT activation by SLAP-2 is dependent on its interaction with c-Cbl (Loreto et al, 2002).…”

“…Recently, the cloning and characterization of a novel adaptor protein, Src-like adaptor protein 2 (SLAP-2), bearing significant sequence and structural similarity to SLAP, has been described (Holland et al, 2001;Loreto et al, 2002;Pandey et al, 2002). The murine SLAP-2 protein consists of an amino-terminal myristoylation sequence that is responsible for targeting SLAP-2 to cellular membranes, a Src homology 3 (SH3) domain and a Src homology 2 (SH2) domain, as well as a unique carboxy-terminal region.…”

A Novel E3 Ubiquitin Ligase TRAC-1 Positively Regulates T Cell Activation

“…Phoenix A cells were grown in DMEM supplemented with 10% FCS, penicillin, and streptomycin (39). The tTA-BJAB or tTA-Jurkat cell lines are described in the study by Holland et al (40).…”

“…Cell sorts were performed on a MoFlo (DakoCytomation). Calcium mobilization assays were performed as described elsewhere (40).…”