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1.
Cancer Res ; 73(14): 4311-22, 2013 Jul 15.
Article in English | MEDLINE | ID: mdl-23704209

ABSTRACT

Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance.


Subject(s)
Autophagy/physiology , Cytokinesis/physiology , Genomic Instability , rhoA GTP-Binding Protein/metabolism , Animals , Autophagy/genetics , Carcinoma/genetics , Carcinoma/metabolism , Carcinoma/pathology , Cell Line , Cell Line, Tumor , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Membrane/physiology , Cytokinesis/genetics , Giant Cells/metabolism , Giant Cells/physiology , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lysosomes/genetics , Lysosomes/metabolism , Lysosomes/physiology , Mice , Phagosomes/genetics , Phagosomes/metabolism , Phagosomes/physiology , Proteolysis , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism , rhoA GTP-Binding Protein/genetics
2.
Mol Biol Cell ; 17(6): 2824-38, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16571678

ABSTRACT

The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP-GTP cycling is critical for efficient cell fusion.


Subject(s)
Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Saccharomyces cerevisiae/physiology , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae/physiology , Amino Acid Sequence , Cell Fusion , Cell Polarity , Genotype , Molecular Sequence Data , Mutagenesis , Plasmids , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/physiology
3.
Eukaryot Cell ; 3(4): 1049-61, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15302837

ABSTRACT

During Saccharomyces cerevisiae mating, chemotropic growth and cell fusion are critical for zygote formation. Cdc24p, the guanine nucleotide exchange factor for the Cdc42 G protein, is necessary for oriented growth along a pheromone gradient during mating. To understand the functions of this critical Cdc42p activator, we identified additional cdc24 mating mutants. Two mating-specific mutants, the cdc24-m5 and cdc24-m6 mutants, each were isolated with a mutated residue in the conserved catalytic domain. The cdc24-m6 mutant responds normally to pheromone and orients its growth towards a mating partner yet accumulates prezygotes during mating. cdc24-m6 prezygotes have two apposed intact cell walls and do not correctly localize proteins required for cell fusion, despite normal exocytosis. Our results indicate that the exchange factor Cdc24p is necessary for maintaining or restricting specific proteins required for cell fusion to the cell contact region during mating.


Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , cdc42 GTP-Binding Protein/metabolism , Amino Acid Sequence , Cell Wall/metabolism , Cell Wall/ultrastructure , Chemotaxis/physiology , Molecular Sequence Data , Mutation , Pheromones/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , cdc42 GTP-Binding Protein/genetics
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