RESUMO
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.
Assuntos
Antígenos CD4/análise , Antígenos CD8/análise , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Receptores de Antígenos de Linfócitos T/análise , Subpopulações de Linfócitos T/fisiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos CD5/análise , Regulação para Baixo , Feminino , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/fisiologia , Proteína-Tirosina Quinase ZAP-70RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Etiquetas de Sequências Expressas , Humanos , Células Jurkat , Luciferases/genética , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Linfócitos T/fisiologiaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Proto-Oncogênicas pp60(c-src)/farmacologia , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Células 3T3 , Animais , Cálcio/metabolismo , Linhagem Celular , Detergentes , Regulação para Baixo , Células HeLa , Humanos , Células Jurkat , Camundongos , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Domínios de Homologia de srcRESUMO
Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region. CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.5 h. Although a carboxyl-terminal HSF transcriptional activation domain is critical for the activation of CUP1 transcription in response to both heat shock stress and glucose starvation, this region is dispensable for transient heat shock activation of at least two genes encoding members of the S. cerevisiae hsp70 family. Furthermore, inactivation of the chromosomal SNF1 gene, encoding a serine-threonine protein kinase, or the SNF4 gene, encoding a SNF1 cofactor, abolishes CUP1 transcriptional activation in response to glucose starvation without altering heat shock-induced transcription. These studies demonstrate that the S. cerevisiae HSF responds to multiple, distinct stimuli to activate yeast metallothionein gene transcription and that these stimuli elicit responses through nonidentical, genetically separable signalling pathways.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Metalotioneína/genética , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Quinases Ativadas por AMP , Sequência de Bases , Primers do DNA/química , Fatores de Transcrição de Choque Térmico , Temperatura Alta , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , RNA Mensageiro/genética , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/fisiologia , Transcrição Gênica , Ativação TranscricionalRESUMO
The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.