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 tra nsplant rejection, we developed and performed a large-scale retroviral-base d functional screen to select for proteins that inhibit antigen receptor-me diated activation of lymphocytes. In addition to known regulators of antige n receptor signaling, we identified a novel adaptor protein, SLAP-2 which s hares 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 anti gen receptor-mediated signaling events, including CD69 surface marker upreg ulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and i onomycin was not significantly reduced, suggesting SLAP-2 functions proxima lly in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homol ogy domains, but lacks a tyrosine kinase domain. In antigen receptor-stimul ated 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 identificat ion of the negative regulator SLAP-2 demonstrates that a retroviral-based s creening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.