RESUMEN
Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Encéfalo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritas/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión/fisiología , Encéfalo/crecimiento & desarrollo , Células COS , Chlorocebus aethiops , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Espectrometría de Masas , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/ultraestructura , Neuritas/ultraestructura , Células PC12 , Fosforilación , Unión Proteica/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ratas , Fracciones SubcelularesRESUMEN
In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.
Asunto(s)
División Celular/fisiología , Proteínas Fúngicas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Schizosaccharomyces/fisiología , Clonación Molecular , Colorantes Fluorescentes/metabolismo , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas de Unión a Poli(A) , Proteína Fosfatasa 2 , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Transducción de Señal/fisiología , TemperaturaRESUMEN
In Saccharomyces cerevisiae, the phosphoprotein phosphatase Cdc14p plays a central role in exit from mitosis, by promoting B-type cyclin degradation and allowing accumulation of the cyclin-dependent kinase inhibitor Sic1p. Cdc14p is sequestered in the nucleolus during interphase, from where it is released at the end of mitosis, dependent upon mitotic exit network function. The CDC14 gene is essential and loss-of-function mutants arrest at the end of mitosis. We have identified a fission yeast orthologue of CDC14 through database searches. A Schizosaccharomyces pombe flp1 (cdc fourteen-like-phosphatase) null mutant is viable, divides at a reduced size and shows defects in septation. flp1p is not the essential effector of the S. pombe septation initiation network, but may potentiate signalling of the onset of septation. In contrast to S. cerevisiae Cdc14p, flp1p is not required for the accumulation or destruction of the B-type cyclin cdc13p, the cyclin-dependent kinase inhibitor rum1p, or for dephosphorylation of the APC/C specificity factor ste9p in G1. Like its budding yeast counterpart, flp1p is restricted to the nucleolus until mitosis, when it is dispersed through the nucleus. In contrast to S. cerevisiae Cdc14p, flp1p is also present on the mitotic spindle and contractile ring. The potential roles of flp1p in cell cycle control are discussed.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclina B/metabolismo , Proteínas Fúngicas/metabolismo , Mitosis/fisiología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Nucléolo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Genes cdc/fisiología , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiologíaRESUMEN
We have found that the replicative helicase E1 of bovine papillomavirus type 1 (BPV-1) interacts with a key cell cycle regulator of S phase, the cyclin E-Cdk2 kinase. The E1 helicase, which interacts with cyclin E and not with Cdk2, presents the highest affinity for catalytically active kinase complexes. In addition, E1, cyclin E, and Cdk2 expressed in Xenopus egg extracts are quantitatively coimmunoprecipitated from crude extracts by either anti-Cdk2 or anti-E1 antibodies. E1 protein is also a substrate of the cyclin E-Cdk2 kinase in vitro. Using the viral components required for in vitro BPV-1 replication and free-membrane cytosol from Xenopus eggs, we show that efficient replication of BPV plasmids is dependent on the addition of E1-cyclin E-Cdk2 complexes. Thus, the BPV initiator of replication and cyclin E-Cdk2 are likely to function together as a protein complex which may be the key to the cell cycle regulation of papillomavirus replication.