RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Publisher Correction: Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport.

In the original version of this Article, the fifth sentence of the abstract incorrectly read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.', and should have read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane tubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.'. This has been corrected in both the PDF and HTML versions of the Article.

Investigation of F-BAR domain PACSIN proteins uncovers membrane tubulation function in cilia assembly and transport.

The intracellular ciliogenesis pathway requires membrane trafficking, fusion, and reorganization. Here, we demonstrate in human cells and zebrafish that the F-BAR domain containing proteins PACSIN1 and -2 play an essential role in ciliogenesis, similar to their binding partner and membrane reorganizer EHD1. In mature cilia, PACSINs and EHDs are dynamically localized to the ciliary pocket membrane (CPM) and transported away from this structure on membrane tubules along with proteins that exit the cilium. PACSINs function early in ciliogenesis at the ciliary vesicle (CV) stage to promote mother centriole to basal body transition. Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell. Together, our work uncovers a function for F-BAR proteins and membrane tubulation in ciliogenesis and explains how the intracellular cilium emerges from the cell.

Oncogenic driver genes and the inflammatory microenvironment dictate liver tumor phenotype.

UNLABELLED: The majority of hepatocellular carcinoma develops in the background of chronic liver inflammation caused by viral hepatitis and alcoholic or nonalcoholic steatohepatitis. However, the impact of different types of chronic inflammatory microenvironments on the phenotypes of tumors generated by distinct oncogenes is largely unresolved. To address this issue, we generated murine liver tumors by constitutively active AKT-1 (AKT) and ß-catenin (CAT), followed by induction of chronic liver inflammation by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and carbon tetrachloride. Also, the impact of DDC-induced chronic liver inflammation was compared between two liver tumor models using a combination of AKT-CAT or AKT-NRAS(G12V) . Treatment with DDC and carbon tetrachloride significantly facilitated the adenoma-to-carcinoma conversion and accelerated the growth of AKT-CAT tumors. Furthermore, DDC treatment altered the morphology of AKT-CAT tumors and caused loss of lipid droplets. Transcriptome analysis of AKT-CAT tumors revealed that cellular growth and proliferation were mainly affected by chronic inflammation and caused up-regulation of Cxcl16, Galectin-3, and Nedd9, among others. Integration with transcriptome profiles from human hepatocellular carcinomas further demonstrated that AKT-CAT tumors generated in the context of chronic liver inflammation showed enrichment of poor prognosis gene sets or decrease of good prognosis gene sets. In contrast, DDC had a more subtle effect on AKT-NRAS(G12V) tumors and primarily enhanced already existent tumor characteristics as supported by transcriptome analysis. However, it also reduced lipid droplets in AKT-NRAS(G12V) tumors. CONCLUSION: Our study suggests that liver tumor phenotype is defined by a combination of driving oncogenes but also the nature of chronic liver inflammation. (Hepatology 2016;63:1888-1899).