RESUMEN
Analysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein-driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55-kDa B-type subunit identified in the Chlamydomonas flagellar proteome. The B-subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B- and C- subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re-establish nearly wild-type motility. The revertants confirmed that the slow-swimming Pf4 phenotype is a result of the defective PP2A B-subunit. These results demonstrate that the axonemal B-subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility.
Asunto(s)
Axonema/metabolismo , Chlamydomonas/metabolismo , Proteína Fosfatasa 2/metabolismo , Axonema/ultraestructura , Chlamydomonas/ultraestructura , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Microscopía Electrónica de Transmisión , Proteína Fosfatasa 2/química , Subunidades de ProteínaRESUMEN
The purpose of this chapter is to review the methodology and advances that have revealed conserved signaling proteins that are localized in the 9+2 ciliary axoneme for regulating motility. Diverse experimental systems have revealed that ciliary and eukaryotic flagellar motility is regulated by second messengers including calcium, pH, and cyclic nucleotides. In addition, recent advances in in vitro functional studies, taking advantage of isolated axonemes, pharmacological approaches, and biochemical analysis of axonemes have demonstrated that otherwise ubiquitous, conserved protein kinases and phosphatases are transported to and anchored in the axoneme. Here, we focus on the functional/pharmacological, genetic, and biochemical approaches in the model genetic system Chlamydomonas that have revealed highly conserved kinases, anchoring proteins (e.g., A-kinase anchoring proteins), and phosphatases that are physically located in the axoneme where they play a direct role in control of motility.
Asunto(s)
Axonema/enzimología , Bioensayo/métodos , Chlamydomonas/enzimología , Dineínas/metabolismo , Flagelos/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Proteínas Algáceas/metabolismo , Axonema/química , Transporte Biológico , Modelos Biológicos , Mutación/genética , PerfusiónRESUMEN
Ras proteins are members of a superfamily of related small GTPases. Some members, such as Ras, are oncogenic. However, other members seem to serve as tumor suppressors, such as Rig and Noey2. We now identify and characterize a novel member of the Ras superfamily, RRP22. Like Ras, RRP22 can be posttranslationally modified by farnesyl. Unlike Ras, RRP22 inhibits cell growth and promotes caspase-independent cell death. Examination of human tumor cells shows that RRP22 is frequently down-regulated due to promoter methylation. Moreover, reexpression of RRP22 in an RRP22-negative neural tumor cell line impairs its growth in soft agar. Unusually for a Ras-related protein, RRP22 localizes to the nucleolus in a GTP-dependent manner, suggesting a novel mechanism of action. Thus, we identify a new member of the Ras superfamily that can serve as a potential tumor suppressor.
Asunto(s)
Genes Supresores de Tumor , Proteínas ras/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Muerte Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Secuencia Conservada , Metilación de ADN , Regulación hacia Abajo , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Regiones Promotoras Genéticas , Prenilación de Proteína , Transfección , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The high frequency with which the novel tumor suppressor RASSF1A is inactivated by promoter methylation suggests that it plays a key role in the development of many primary human tumors. Yet the mechanism of RASSF1A action remains unknown. We now show that RASSF1A associates with microtubules and that this association is essential for RASSF1A to mediate its growth inhibitory effects. Overexpression of RASSF1A promotes the formation of stable microtubules, whereas a dominant-negative fragment of RASSF1A destabilizes microtubule networks. The RASSF1 protein is expressed as two main isoforms, 1A and 1C. The smaller 1C isoform also associates with microtubules but is less effective at stabilizing them. Because RASSF1A and RASSF1C localize to the mitotic spindle, we examined their effects upon genomic instability. RASSF1A and RASSF1C block activated Ras-induced genomic instability. However, a point mutant of RASSF1C, identified in human tumors, was severely defective for stabilizing tubulin and was unable to block the genomic destabilizing effects of Ras. Thus, we identify a role for RASSF1A/C in the control of microtubule polymerization and potentially in the maintenance of genomic stability.
Asunto(s)
Inestabilidad Genómica/fisiología , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor/fisiología , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Transfección , Tubulina (Proteína)/genética , Proteínas Supresoras de Tumor/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.