RESUMEN
The actin cytoskeleton plays critical roles in numerous cellular events and functions, and its spatiotemporal dynamics are maintained and regulated by several actin cofactor proteins. MISP/Caprice is a recently reported actin-bundling protein that is also involved in the progression of mitosis. In this study, we investigated how the actin-regulatory function of MISP is modulated by phosphorylation. A series of mutation studies demonstrated that phosphorylation of S394, S395, and S400 induced stress fiber formation in interphase cells. In vitro studies revealed that these phosphorylation events increased the actin-bundling activity but not the actin-binding activity of MISP. Moreover, actin-binding activity was suppressed by mitotic phosphorylation, including that at S376, S471, and S541. These results indicate that phosphorylation during interphase and mitosis differentially regulates the actin-binding and -bundling activities of MISP, in turn regulating the higher-order architecture of the actin cytoskeleton during cell cycle.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Huso Acromático/metabolismo , Ciclo Celular/fisiología , Células Cultivadas , Humanos , Mitosis/fisiología , Fosforilación , Unión Proteica , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).