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. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.

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. In this Review, we discuss the current understanding of this important class of kinases. We begin with a brief overview of the structure, regulation, substrate recognition and subcellular localization of ERK1 and ERK2. We then systematically discuss how ERK signalling regulates six fundamental cellular processes in response to extracellular cues. These processes are cell proliferation, cell survival, cell growth, cell metabolism, cell migration and cell differentiation.

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). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

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. Although this event is thought to play a key role in priming RAF activation, the underlying mechanism is not known. Here we report that RAF inhibitors induce the disruption of intramolecular interactions between the kinase domain and its N-terminal regulatory region independently of RAS activity. This provides a molecular basis to explain the induction of RAS-RAF association by RAF inhibitors, as well as the co-operativity observed between RAS activity and RAF kinase inhibitors in driving RAF activation. Profiling of second-generation RAF inhibitors confirmed their improved mode of action, but also revealed liabilities that allowed us to discern two properties of an ideal RAF inhibitor: high-binding affinity to all RAF paralogs and maintenance of the OFF/autoinhibited state of the enzyme.

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. How Polo activation and localization are coupled during mitotic entry is unknown. Here we report that PBD binding to the KD masks a nuclear localization signal (NLS). Activating phosphorylation of the KD leads to exposure of the NLS and entry of Polo into the nucleus before nuclear envelope breakdown. Failures of this mechanism result in misregulation of the Cdk1-activating Cdc25 phosphatase and lead to mitotic and developmental defects in Drosophila. These results uncover spatiotemporal mechanisms linking master regulatory enzymes during mitotic entry.

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. Comprehensive data mining and filtering identified a total of 37,910 phosphorylation sites, 660 of which were dynamically modulated upon treatment with vemurafenib. We established that 83% of these dynamic phosphorylation sites were modulated in accordance with the phospho-ERK profile of the two cell lines. Accordingly, kinase substrate prediction algorithms linked most of these dynamic sites to direct ERK1/2-mediated phosphorylation, supporting a low off-target rate for vemurafenib. Functional classification of target proteins indicated the enrichment of known (nuclear pore, transcription factors, and RAS-RTK signaling) and novel (Rho GTPases signaling and actin cytoskeleton) ERK-controlled functions. Our phosphoproteomic data combined with experimental validation established novel dynamic connections between ERK signaling and the transcriptional regulators TEAD3 (Hippo pathway), MKL1, and MKL2 (Rho serum-response elements pathway). We also confirm that an ERK-docking site found in MKL1 is directly antagonized by overlapping actin binding, defining a novel mechanism of actin-modulated phosphorylation. Altogether, time-resolved phosphoproteomics further documented vemurafenib selectivity and identified novel ERK downstream substrates.

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. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

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. The recent discovery that the catalytic activity of RAF depends on an allosteric mechanism driven by kinase domain dimerization is providing a vital new piece of information towards a comprehensive model of RAF function. The fact that current RAF inhibitors unexpectedly induce ERK signalling by stimulating RAF dimerization also calls for a deeper structural characterization of this family of kinases.

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). These features correlated with the ability of sulfonamides to disrupt human BRAF homodimers in cells, in vitro and in crystals yielding a structure of BRAF in a monomer state. The crystal structure revealed exaggerated, nonproductive positions of helix αC and AS-H1, the latter of which is the target of potent BRAF oncogenic mutations. Together, this work provides formal proof of an allosteric link between the RAF dimer interface, the activation segment and the catalytic infrastructure.

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. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

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. In this review, we discuss dimerization-mediated regulation of the rapidly accelerated fibrosarcoma (RAF) and eIF2α kinase families and draw parallels with the analogous behavior of the epidermal growth factor receptor (EGFR) and serine/threonine-protein kinase endoribonuclease 1 (IRE1)/ribonuclease L (RNAse L) kinase families. Given that resistance to RAF-targeted therapeutics often stems from dimerization-dependent mechanisms, we suggest that a better understanding of dimerization-induced allostery may assist in developing alternate therapeutic strategies.

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. In the present study, we examined the factors that modulate the role of verbal memory in regressions. Participants were required to make regressions on target words located in sentences displayed on one or two lines. Verbal interference was shown to affect regressions, but only when participants executed a regression on a word located in the first part of the sentence, irrespective of the number of lines on which the sentence was displayed. Experiments 2 and 3 showed that the effect of verbal interference on words located in the first part of the sentence disappeared when participants initiated the regression from the middle of the sentence. Our results suggest that verbal memory is recruited to guide regressions, but only for words read a longer time ago.

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 mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.

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. The EJC binds the exon-exon junctions of mRNAs and thus far, has been linked exclusively to postsplicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC.

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. RNAs were purified from cells growing under conditions relevant to C. albicans pathogenicity, including biofilm, lab-grown yeast and serum-induced hyphae, as well as cells isolated from the mouse caecum. This work provides a genome-wide experimental validation for a large number of predicted ORFs for which transcription had not been detected by other approaches. Additionally, we identified more than 2,000 novel transcriptional segments, including new ORFs and exons, non-coding RNAs (ncRNAs) as well as convincing cases of antisense gene transcription. We also characterized the 5' and 3' UTRs of expressed ORFs, and established that genes with long 5' UTRs are significantly enriched in regulatory functions controlling filamentous growth. Furthermore, we found that genomic regions adjacent to telomeres harbor a cluster of expressed ncRNAs. To validate and confirm new ncRNA candidates, we adapted an iterative strategy combining both genome-wide occupancy of the different subunits of RNA polymerases I, II and III and expression data. This comprehensive approach allowed the identification of different families of ncRNAs. CONCLUSIONS: In summary, we provide a comprehensive expression atlas that covers relevant C. albicans pathogenic developmental stages in addition to the discovery of new ORF and non-coding genetic elements.

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. albicans this regulation is under the control of Tbf1 and Cbf1. Here, we analyzed, using full-genome transcription factor mapping, the roles, in both S. cerevisiae and C. albicans, of each orthologous component of this complete set of regulators. We observe dramatic changes in the binding profiles of the generalist regulators Cbf1, Hmo1, Rap1, and Tbf1, while the Fhl1-Ifh1 dimer is the only component involved in ribosomal regulation in both fungi: it activates ribosomal protein genes and rDNA expression in a Tbf1-dependent manner in C. albicans and a Rap1-dependent manner in S. cerevisiae. We show that the transcriptional regulatory network governing the ribosomal expression program of two related yeast species has been massively reshaped in cis and trans. Changes occurred in transcription factor wiring with cellular functions, movements in transcription factor hierarchies, DNA-binding specificity, and regulatory complexes assembly to promote global changes in the architecture of the fungal transcriptional regulatory network.

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. We note that dramatic rearrangements of the transcriptional regulatory components of metabolic functions have occurred among ascomycetes species.