RESUMO
Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.
Assuntos
Segregação de Cromossomos/genética , Proteínas de Ligação a DNA/genética , Cinesinas/genética , Meiose/genética , Proteínas Nucleares/genética , Oócitos/crescimento & desenvolvimento , Proteínas de Xenopus , Xenopus laevis/genética , Animais , Apoptose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Segregação de Cromossomos/efeitos dos fármacos , Ciclina B/genética , Ciclina B/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Feminino , Cinesinas/biossíntese , Cinesinas/deficiência , Meiose/efeitos dos fármacos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/deficiência , Oligorribonucleotídeos Antissenso , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Xenopus laevis/embriologiaRESUMO
Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteína SUMO-1/metabolismo , Vaccinia virus/metabolismo , Vaccinia virus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Western Blotting , DNA Viral/genética , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisina/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Peso Molecular , Mutagênese Sítio-Dirigida , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Proteína SUMO-1/química , Vaccinia virus/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/ultraestrutura , Montagem de Vírus , Replicação ViralRESUMO
Progression through the cell cycle is regulated by CDKs (cyclin-dependent kinases), which associate with activating partners, named cyclins, to efficiently phosphorylate substrates. We previously reported the identification of RINGO, a Xenopus protein that can activate CDK1 and CDK2 despite lack of sequence similarity to cyclins, which plays a role in the regulation of the meiotic cell cycle in oocytes. In the present study we report the characterization of four mammalian RINGO proteins, which are 53-68% identical with Xenopus RINGO in a central core of about 75 residues. We show that all RINGO family members can bind to and activate CDK1 and CDK2, albeit with different efficiencies, but they do not bind to CDK4 or CDK6. The core RINGO sequences are critical for CDK activation. We also identified key residues in CDK2 that are required for RINGO binding. All RINGO proteins can also bind the CDK inhibitor p27Kip1, but with an inverse efficiency of their ability to bind to CDK1. Our results identify a new family of mammalian proteins that can activate CDKs and therefore potentially function as cell cycle regulators. The ability of RINGO proteins to activate CDK1 and CDK2 suggest also cyclin-independent roles for these kinases.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ativadores de Enzimas/química , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , Quinase 2 Dependente de Ciclina , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/química , Oócitos/enzimologia , Oócitos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas/métodos , Alinhamento de Sequência/métodos , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/química , Proteínas de Xenopus/fisiologiaRESUMO
The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly nuclear during interphase and is actively exported before nuclear envelope breakdown to initiate prospindle assembly. It localizes to the spindle microtubules but not to the interdigitating polar microtubules during anaphase or to the phragmoplast as it is rapidly degraded during telophase. We characterized the Arabidopsis thaliana TPX2-targeting domains and show that the protein is able to rescue microtubule assembly in TPX2-depleted Xenopus laevis egg extracts. Injection of antibodies to TPX2 into living plant cells inhibits the onset of mitosis. These results demonstrate that plant TPX2 already functions before nuclear envelope breakdown. Thus, plants have adapted nuclear-cytoplasmic shuttling of TPX2 to maintain proper spindle assembly without centrosomes.