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1.
Cell Death Differ ; 22(9): 1526-39, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26021298

ABSTRACT

Cellular stress defense mechanisms have evolved to maintain homeostasis in response to a broad variety of environmental challenges. Stress signaling pathways activate multiple cellular programs that range from the activation of survival pathways to the initiation of cell death when cells are damaged beyond repair. To identify novel players acting in stress response pathways, we conducted a cell culture RNA interference (RNAi) screen using caffeine as a xenobiotic stress-inducing agent, as this compound is a well-established inducer of detoxification response pathways. Specifically, we examined how caffeine affects cell survival when Drosophila kinases and phosphatases were depleted via RNAi. Using this approach, we identified and validated 10 kinases and 4 phosphatases that are essential for cell survival under caffeine-induced stress both in cell culture and living flies. Remarkably, our screen yielded an enrichment of Hippo pathway components, indicating that this pathway regulates cellular stress responses. Indeed, we show that the Hippo pathway acts as a potent repressor of stress-induced cell death. Further, we demonstrate that Hippo activation is necessary to inhibit a pro-apoptotic program triggered by the interaction of the transcriptional co-activator Yki with the transcription factor p53 in response to a range of stress stimuli. Our in vitro and in vivo loss-of-function data therefore implicate Hippo signaling in the transduction of cellular survival signals in response to chemical stress.


Subject(s)
Caffeine/toxicity , Drosophila Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological/physiology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Line , Cell Survival/drug effects , Cell Survival/physiology , Drosophila melanogaster , Signal Transduction , Stress, Physiological/drug effects
2.
Cell Death Differ ; 16(8): 1118-25, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19282869

ABSTRACT

High temperature requirement A2 (HtrA2/Omi) is a mitochondrial protease that exhibits proapoptotic and cell-protective properties and has been linked to Parkinson's disease (PD). Impaired mitochondrial function is a common trait in PD patients, and is likely to play a significant role in pathogenesis of parkinsonism, but the molecular mechanisms remain poorly understood. Genetic studies in Drosophila have provided valuable insight into the function of other PD-linked genes, in particular PINK1 and parkin, and their role in maintaining mitochondrial integrity. Recently, HtrA2 was shown to be phosphorylated in a PINK1-dependent manner, suggesting it might act in the PINK1 pathway. Here, we describe the characterization of mutations in Drosophila HtrA2, and genetic analysis of its function with PINK1 and parkin. Interestingly, we find HtrA2 appears to be dispensable for developmental or stress-induced apoptosis. In addition, we found HtrA2 mutants share some phenotypic similarities with parkin and PINK1 mutants, suggesting that it may function in maintaining mitochondrial integrity. Our genetic interaction studies, including analysis of double-mutant combinations and epistasis experiments, suggest HtrA2 acts downstream of PINK1 but in a pathway parallel to Parkin.


Subject(s)
Apoptosis , Drosophila Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Serine Endopeptidases/metabolism , Animals , Animals, Genetically Modified , Animals, Newborn , Drosophila/metabolism , Drosophila Proteins/genetics , Female , Fertility/genetics , High-Temperature Requirement A Serine Peptidase 2 , Male , Mitochondria/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Phosphorylation , Serine Endopeptidases/genetics , Ubiquitin-Protein Ligases
3.
Curr Opin Cell Biol ; 13(6): 731-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11698190

ABSTRACT

The development of a complex multicellular organism requires a coordination of growth and cell division under the control of patterning mechanisms. Studies in yeast have pioneered our understanding of the relationship between growth and cell division. In recent years, many of the pathways that regulate growth in multicellular eukaryotes have been identified. This work has revealed interesting and unexpected relationships between mechanisms that regulate growth and the cell cycle machinery.


Subject(s)
Cell Cycle , Cell Division , Drosophila/growth & development , Yeasts/growth & development , Animals , Cyclin-Dependent Kinases/physiology , Cyclins/physiology , Drosophila/cytology , Insulin/physiology , Models, Biological , Proto-Oncogene Proteins/physiology , Repressor Proteins/physiology , Signal Transduction , Yeasts/cytology
4.
Cell ; 105(3): 345-55, 2001 May 04.
Article in English | MEDLINE | ID: mdl-11348591

ABSTRACT

The inherited human disease tuberous sclerosis, characterized by hamartomatous tumors, results from mutations in either TSC1 or TSC2. We have characterized mutations in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions.


Subject(s)
Cell Cycle/physiology , Genes, Tumor Suppressor/genetics , Photoreceptor Cells, Invertebrate/cytology , Proteins/genetics , Repressor Proteins/genetics , Tuberous Sclerosis/genetics , Animals , Cell Size , Cyclin A/metabolism , Cyclin E/metabolism , DNA/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Female , Flow Cytometry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Humans , Immunohistochemistry , Insect Proteins/genetics , Insect Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Phenotype , Photoreceptor Cells, Invertebrate/physiology , Photoreceptor Cells, Invertebrate/ultrastructure , Ploidies , Proteins/metabolism , Repressor Proteins/metabolism , Tuberous Sclerosis/physiopathology , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins
5.
EMBO J ; 17(5): 1395-404, 1998 Mar 02.
Article in English | MEDLINE | ID: mdl-9482736

ABSTRACT

The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has previously shown that these two activities are mediated by distinct cellular targets, though their identity is not known. Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. We present evidence that neither is the Rac target mediating JNK activation in Cos-1 cells. We have used yeast two-hybrid selection and identified a new target of Rac, POSH. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. We propose that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes.


Subject(s)
Adaptor Proteins, Signal Transducing , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carrier Proteins/metabolism , Cytoskeletal Proteins/metabolism , GTP-Binding Proteins/metabolism , MAP Kinase Kinase Kinases , Mitogen-Activated Protein Kinases , NF-kappa B/metabolism , src Homology Domains , 3T3 Cells , Amino Acid Sequence , Animals , Apoptosis , Biological Transport , COS Cells , Carrier Proteins/analysis , Carrier Proteins/genetics , Cell Nucleus/metabolism , Cloning, Molecular , Cytoskeletal Proteins/analysis , Cytoskeletal Proteins/genetics , Enzyme Activation , JNK Mitogen-Activated Protein Kinases , Mice , Molecular Sequence Data , Protein Serine-Threonine Kinases/physiology , Sequence Analysis, DNA , Signal Transduction/physiology , p21-Activated Kinases , rac GTP-Binding Proteins
6.
Curr Opin Cell Biol ; 9(1): 86-92, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9013670

ABSTRACT

Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.


Subject(s)
Actins/metabolism , Cell Cycle Proteins/metabolism , Cytoskeleton/enzymology , GTP-Binding Proteins/metabolism , Fungal Proteins/metabolism , Plant Proteins/metabolism , cdc42 GTP-Binding Protein, Saccharomyces cerevisiae , rac GTP-Binding Proteins , rho GTP-Binding Proteins
7.
Cell ; 87(3): 519-29, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8898204

ABSTRACT

Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.


Subject(s)
Actins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Cycle Proteins/physiology , G1 Phase/physiology , GTP-Binding Proteins/physiology , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases , Protein Kinases/physiology , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/physiology , Proteins/physiology , Signal Transduction/physiology , 3T3 Cells , Animals , COS Cells , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , DNA Replication , Enzyme Activation , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins , Integrins/metabolism , Mice , Mutagenesis, Site-Directed , Phosphorylation , Polymers , Proteins/genetics , Pseudopodia/ultrastructure , Recombinant Fusion Proteins/metabolism , Transfection , cdc42 GTP-Binding Protein , p21-Activated Kinases , ras GTPase-Activating Proteins
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