RESUMO
To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas Culina , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/fisiologia , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor , Ubiquitinas/metabolismo , Proteínas de Xenopus , Animais , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclina E/genética , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Ligases/genética , Ligases/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Complexo de Reconhecimento de Origem , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Quinases Associadas a Fase S , Proteínas Ligases SKP Culina F-Box , Ubiquitina-Proteína Ligases , Ubiquitinas/genética , Xenopus laevisRESUMO
Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Cromatina/metabolismo , Ciclina E/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Oócitos/fisiologia , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Motivos de Aminoácidos , Animais , Western Blotting , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Humanos , Masculino , Modelos Biológicos , Oócitos/química , Complexo de Reconhecimento de Origem , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Espermatozoides/citologia , Espermatozoides/fisiologia , Proteínas de Xenopus , Xenopus laevisRESUMO
Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.
