RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Nucleares , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Domínios de Homologia de src , Sequência de Aminoácidos , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/genética , Humanos , Células Jurkat , Lectinas Tipo C , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Fatores de Transcrição NFATC , Fosforilação , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/imunologia , Homologia de Sequência de Aminoácidos , Tetraciclina/farmacologia , Transativadores , Fatores de Transcrição/genética , Ativação Transcricional , Tirosina/metabolismoRESUMO
To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.
