RESUMO
p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Humanos , Imunofilinas , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação Puntual , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Alinhamento de Sequência , Proteínas de Ligação a Tacrolimo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
