RESUMO
Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2-Stat5 pathway.
Assuntos
Mielopoese , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/classificação , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/metabolismoRESUMO
SOCS3 is essential for regulating the extent, duration, and specificity of cellular responses to cytokines such as G-CSF and IL-6. Here we describe the solution structure of SOCS3, the first structure determined for any SOCS protein, in complex with a phosphotyrosine-containing peptide from the IL-6 receptor signaling subunit gp130. The structure of the complex shows that seven peptide residues form a predominantly hydrophobic binding motif. Regions outside the SOCS3 SH2 domain are important for ligand binding, in particular, a single 15 residue alpha helix immediately N-terminal to the SH2 domain makes direct contacts with the phosphotyrosine binding loop and, in part, determines its geometry. The SH2 domain itself is remarkable in that it contains a 35 residue unstructured PEST motif insertion that is not required for STAT inhibition. The PEST motif increases SOCS3 turnover and affects its degradation pathway, implying that it has an important regulatory role inside the cell.
Assuntos
Mutagênese Insercional , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genética , Domínios de Homologia de src/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Genes Reporter , Meia-Vida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luciferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Análise Espectral Raman , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , TransfecçãoRESUMO
The suppressors of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing intracellular inhibitors of cytokine signal transduction that act by several different mechanisms. Recent evidence suggests that the action of the SOCS proteins may extend beyond the cytokine receptors to signaling initiated by members of the tyrosine kinase receptor family. In this study, the ability of SOCS-5 to negatively regulate signaling cascades downstream of the epidermal growth factor receptor (EGF-R) has been examined by using an EGF-responsive cell line engineered to constitutively express the EGF-R and SOCS-5 or SOCS-5 mutants. SOCS-5 associated with the EGF-R complex in an EGF-independent manner, and the mitogenic response to EGF of all SOCS-5-expressing cell lines was dramatically inhibited when compared with control cell lines. Furthermore, this effect was abrogated after deletion of the SOCS-5 SOCS box. This result suggests that the inhibition of signaling occurs through enhanced proteasomal degradation of the EGF-R through SOCS box recruitment of E3 ubiquitin ligase activity.