RESUMO
The T-cell specific adapter protein (TSAd) encoded by the SH2D2A gene is up-regulated in activated human CD4+ T-cells in a cAMP-dependent manner. Expression of SH2D2A is important for proper activation of T-cells. Here, we show that SH2D2A expression is regulated both at the transcriptional and translational level. cAMP signaling alone induces TSAd-mRNA expression but fails to induce increased TSAd protein levels. By contrast, TCR engagement provides signals for both TSAd transcription and translation. We further show that cAMP signaling can prime T-cells for a more prompt expression of TSAd protein upon TCR stimulation. Our study thus points to a novel mechanism for how cAMP signaling may modulate T-cell activation through transcriptional priming of resting cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Transcrição Gênica , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Modelos Imunológicos , Biossíntese de Proteínas/efeitos dos fármacos , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239-256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239-334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239-334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239-334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/imunologia , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/enzimologia , Núcleo Celular/metabolismo , Éxons , Expressão Gênica , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Dados de Sequência Molecular , Fosforilação , Prolina/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.