Unknown,Transcriptomics,Genomics,Proteomics

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CAR1 rescues developmental gene expression defects of rasC/rasG double mutants

ABSTRACT: It has been shown previously that rasC- cells do not aggregate, predominantly as a result of a defective cAMP relay, although there are also subtle defects in cAMP chemotaxis. There is no appreciable effect on early gene expression in response to starvation. In contrast, rasG-/rasC- cells are totally blocked in aggregation, cAMP relay and cAMP chemotaxis and, in addition, there is no induction of carA and gpaB gene expression, in response to starvation. However, expression of carA from the actin15 promoter in vegetative rasG-/rasC- cells restores the induced expression of carA and gpaB in response to starvation, but has no effect on the defects in aggregation, cAMP chemotaxis and cAMP relay. In order to determine the generality of the effect on early gene expression, micro array analysis was undertaken. These results revealed 121 genes that were significantly (p<0.05) induced in expression 5 fold or more in wild type cells, in response to starvation. Of these genes, the expression of 100 was not induced significantly in rasG-/rasC- cells, but the expression of most of these was restored in rasG-/rasC-/[act15:carA] cells, in response to starvation. These results indicate that the inhibition of early gene expression in rasG-/rasC- cells and its restoration in response to the expression of carA in vegetative cells is a fairly general phenomenon. Thus, signaling through RasG or RasC is required for the induction of expression of many genes that occurs in response to starvation, but the constitutive expression of carA is able to bypass this requirement.

ORGANISM(S): Dictyostelium discoideum

SUBMITTER: Gareth Bloomfield

PROVIDER: E-TABM-1085 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The second messenger cAMP acts via protein kinase A (PKA) to induce apoptosis by mechanisms that are poorly understood. Here, we assessed a role for mitochondria and analyzed gene expression in cAMP/PKA-promoted apoptosis by comparing wild-type (WT) S49 lymphoma cells and the S49 variant, D- (cAMP-deathless), which lacks cAMP-promoted apoptosis but has wild-type levels of PKA activity and cAMP-promoted G1 growth arrest. Treatment of WT, but not D-, S49 cells with 8-CPT-cAMP for 24 h induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and Smac and increase in caspase-3 activity. Gene expression analysis (using Affymetrix 430 2.0 Arrays) revealed that WT and D- cells incubated with 8-CPT-cAMP have similar, but non-identical, extents of cAMP-regulated gene expression at 2h (~800 transcripts) and 6h (~1000 transcripts) (|Fold|>2, P<0.06); by contrast, at 24h ~2500 and ~1100 transcripts were changed in WT and D- cells, respectively. Using an approach that combined regression analysis, clustering and functional annotation to identify transcripts that showed differential expression between WT and D- cells, we found differences in cAMP-mediated regulation of mRNAs involved in transcriptional repression, apoptosis, the cell cycle, RNA splicing, Golgi and lysosomes. The 2 cell lines differed in CREB phosphorylation and expression of the transcriptional inhibitor Icer and in cAMP-regulated expression of genes in the Inhibitor of apoptosis (IAP) and Bcl families. The findings indicate that cAMP/PKA-promoted apoptosis of lymphoid cells occurs via mitochondrial-mediated events and imply that such apoptosis involves gene networks in multiple biochemical pathways. Experiment Overall Design: S49 D- cells were treated with 8-CPT-cAMP over the course of 24 hours. Cells were prepared for hybridization to microarrays at times 0-untreated, 2h, 6h, and 24h after treatment with 8-CPT-cAMP. Experimental replicates were as follows n=4 for time 0 and n=3 for 2, 6, and 24h post treatment.

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CAR1 rescues developmental gene expression defects of rasC/rasG double mutants

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