RESUMO
Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Clonagem Molecular , Feminino , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Proteínas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Treonina/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva , Células COS , Chlorocebus aethiops , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteína de Replicação C , TransfecçãoRESUMO
The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G2/M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RFCp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase.