Dataset Information

Membranes prime the RapGEF EPAC1 to transduce cAMP signaling

ABSTRACT: EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling in normal cells and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 in the absence of cAMP, that they increase its affinity for cAMP by 2 orders of magnitude, and that they synergize with cAMP to yield maximal GEF activity. Thus, EPAC1 has four states that differ in their affinity for cAMP and GEF efficiency. In cells, where cytosolic cAMP is low, this implies that EPAC1 must be primed by membranes to bind cAMP. Examination of the inhibitory mechanism of the cardiomyocyte-active chemical CE3F4 in this new framework further reveals that it targets only only membrane- and cAMP-activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling to include a hitherto overlooked role of membranes through EPAC proteins, with important implications for drug discovery.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Marie LEY

LAB HEAD: CIANFERANI Sarah

PROVIDER: PXD040227 | Pride | 2023-04-07

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

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.

Dataset Information

Membranes prime the RapGEF EPAC1 to transduce cAMP signaling

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure