Functional characterization of a PROTAC directed against BRAF mutant V600E.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated.

ERK signalling: a master regulator of cell behaviour, life and fate.

The proteins extracellular signal-regulated kinase 1 (ERK1) and ERK2 are the downstream components of a phosphorelay pathway that conveys growth and mitogenic signals largely channelled by the small RAS GTPases. By phosphorylating widely diverse substrates, ERK proteins govern a variety of evolutionarily conserved cellular processes in metazoans, the dysregulation of which contributes to the cause of distinct human diseases. The mechanisms underlying the regulation of ERK1 and ERK2, their mode of action and their impact on the development and homeostasis of various organisms have been the focus of much attention for nearly three decades.

MEK drives BRAF activation through allosteric control of KSR proteins.

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK).

Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry.

The Polo kinase is a master regulator of mitosis and cytokinesis conserved from yeasts to humans. Polo is composed of an N-term kinase domain (KD) and a C-term polo-box domain (PBD), which regulates its subcellular localizations. The PBD and KD can interact and inhibit each other, and this reciprocal inhibition is relieved when Polo is phosphorylated at its activation loop.

RAF inhibitors promote RAS-RAF interaction by allosterically disrupting RAF autoinhibition.

First-generation RAF inhibitors paradoxically induce ERK signaling in normal and tumor cells exhibiting RAS activity. Compound-induced RAF dimerization through stabilization of the RAF ON/active state by inhibitors has emerged as a critical contributing factor. RAF inhibitors also enhance RAS-RAF association.

Time-resolved Phosphoproteome Analysis of Paradoxical RAF Activation Reveals Novel Targets of ERK.

Small molecules targeting aberrant RAF activity, like vemurafenib (PLX4032), are highly effective against cancers harboring the V600E BRAF mutation and are now approved for clinical use against metastatic melanoma. However, in tissues showing elevated RAS activity and in RAS mutant tumors, these inhibitors stimulate RAF dimerization, resulting in inhibitor resistance and downstream "paradoxical" ERK activation. To understand the global signaling response of cancer cells to RAF inhibitors, we profiled the temporal changes of the phosphoproteome of two colon cancer cell lines (Colo205 and HCT116) that respond differently to vemurafenib.

Inhibition of RAS function through targeting an allosteric regulatory site.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition.

Regulation of RAF protein kinases in ERK signalling.

RAF family kinases were among the first oncoproteins to be described more than 30 years ago. They primarily act as signalling relays downstream of RAS, and their close ties to cancer have fuelled a large number of studies. However, we still lack a systems-level understanding of their regulation and mode of action.

Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation.

Reported RAF kinase domain structures adopt a side-to-side dimer configuration reflective of an 'on' state that underpins an allosteric mechanism of regulation. Atomic details of the monomer 'off' state have been elusive. Reinspection of the BRAF kinase domain structures revealed that sulfonamide inhibitors induce features of an off state, primarily a laterally displaced helix αC stabilized by the activation segment helix 1 (AS-H1).

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood.

Dimerization-induced allostery in protein kinase regulation.

The ability of protein kinases to switch between inactive and active states is critical to control the outputs of cellular signaling pathways. In several protein kinases, the conformation of helix αC is a key hub on which regulatory inputs converge to induce catalytic switching. An emerging mechanism involved in regulating helix αC orientation is the allosteric coupling with kinase domain surfaces involved in homo- or heterodimerization.

The role of verbal memory in regressions during reading is modulated by the target word's recency in memory.

During reading, a number of eye movements are made backward, on words that have already been read. Recent evidence suggests that such eye movements, called regressions, are guided by memory. Several studies point to the role of spatial memory, but evidence for the role of verbal memory is more limited.

Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization.

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth.

The exon junction complex controls the splicing of MAPK and other long intron-containing transcripts in Drosophila.

Signaling pathways are controlled by a vast array of posttranslational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a key element of this pathway.

Experimental annotation of the human pathogen Candida albicans coding and noncoding transcribed regions using high-resolution tiling arrays.

Background: Compared to other model organisms and despite the clinical relevance of the pathogenic yeast Candida albicans, no comprehensive analysis has been done to provide experimental support of its in silico-based genome annotation.

Results: We have undertaken a genome-wide experimental annotation to accurately uncover the transcriptional landscape of the pathogenic yeast C. albicans using strand-specific high-density tiling arrays.

Evolutionary tinkering with conserved components of a transcriptional regulatory network.

Gene expression variation between species is a major contributor to phenotypic diversity, yet the underlying flexibility of transcriptional regulatory networks remains largely unexplored. Transcription of the ribosomal regulon is a critical task for all cells; in S. cerevisiae the transcription factors Rap1, Fhl1, Ifh1, and Hmo1 form a multi-subunit complex that controls ribosomal gene expression, while in C.

Rearrangements of the transcriptional regulatory networks of metabolic pathways in fungi.

Growing evidence suggests that transcriptional regulatory networks in many organisms are highly flexible. Here, we discuss the evolution of transcriptional regulatory networks governing the metabolic machinery of sequenced ascomycetes. In particular, recent work has shown that transcriptional rewiring is common in regulons controlling processes such as production of ribosome components and metabolism of carbohydrates and lipids.

Genome-wide mapping of the coactivator Ada2p yields insight into the functional roles of SAGA/ADA complex in Candida albicans.

The SAGA/ADA coactivator complex, which regulates numerous cellular processes by coordinating histone acetylation, is widely conserved throughout eukaryotes, and analysis of the Candida albicans genome identifies the components of this complex in the fungal pathogen. We investigated the multiple functions of SAGA/ADA in C. albicans by determining the genome-wide occupancy of Ada2p using chromatin immunoprecipitation (ChIP).

A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans.

Background: Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent.

Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags.

Increased respiration in the sch9Delta mutant is required for increasing chronological life span but not replicative life span.

Loss of the protein kinase Sch9p increases both the chronological life span (CLS) and the replicative life span (RLS) of Saccharomyces cerevisiae by mimicking calorie restriction, but the physiological consequences of SCH9 deletion are poorly understood. By transcriptional profiling of an sch9Delta mutant, we show that mitochondrial electron transport chain genes are upregulated. Accordingly, protein levels of electron transport chain subunits are increased and the oxygen consumption rate is enhanced in the sch9Delta mutant.

Transcription factor substitution during the evolution of fungal ribosome regulation.

Coordinated ribosomal protein (RP) gene expression is crucial for cellular viability, but the transcriptional network controlling this regulon has only been well characterized in the yeast Saccharomyces cerevisiae. We have used whole-genome transcriptional and location profiling to establish that, in Candida albicans, the RP regulon is controlled by the Myb domain protein Tbf1 working in conjunction with Cbf1. These two factors bind both the promoters of RP genes and the rDNA locus; Tbf1 activates transcription at these loci and is essential.

Remote monitoring of multi-gas mixtures by passive standoff Fourier transform infrared radiometry.

The passive remote monitoring of multi-gas mixtures was experimentally investigated using Fourier transform infrared (FT-IR) radiometry. The spectral radiance data were collected using a dual-port radiometrically balanced interferometer for a variety of multi-gas plumes at a standoff distance of 60 m. Two basic sets of mixtures were studied.