Membranes prime the RapGEF EPAC1 to transduce cAMP signaling.

EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling 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 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery.

Publisher Correction: PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.

PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.

Post-transcriptional gene silencing activity of human GIGYF2.

In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression.

EWS-FLI1 inhibits TNFalpha-induced NFkappaB-dependent transcription in Ewing sarcoma cells.

Ewing sarcoma is primarily caused by a t(11;22) chromosomal translocation encoding the EWS-FLI1 fusion protein. To exert its oncogenic function, EWS-FLI1 acts as an aberrant transcription factor, broadly altering the gene expression profile of tumor cells. Nuclear factor-kappaB (NFkappaB) is a tightly regulated transcription factor controlling cell survival, proliferation and differentiation, as well as tumorigenesis. NFkappaB activity is very low in unstimulated Ewing sarcoma cells, but can be induced in response to tumor necrosis factor (TNF). We wondered whether NFkappaB activity could be modulated by EWS-FLI1 in Ewing sarcoma. Using a knockdown approach in Ewing sarcoma cells, we demonstrated that EWS-FLI1 has no influence on NFkappaB basal activity, but impairs TNF-induced NFkappaB-driven transcription, at least in part through inhibition of NFkappaB binding to DNA. We detected an in vivo physical interaction between the fusion protein and NFkappaB p65, which could mediate these effects. Our findings suggest that, besides directly controlling the activity of its primary target promoters, EWS-FLI1 can also indirectly influence gene expression in tumor cells by modulating the activity of key transcription factors such as NFkappaB.

The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells.

Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.