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.

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.

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.

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.

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.

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.

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.

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.

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. Orthologs of Tbf1 bind TTAGGG telomeric repeats in most eukaryotes, and TTAGGG cis-elements are present upstream of RP genes in plants and fungi, suggesting that Tbf1 was involved in both functions in ancestral eukaryotes. In all Hemiascomycetes, Rap1 substituted Tbf1 at telomeres and, in the S. cerevisiae lineage, this substitution also occurred independently at RP genes, illustrating the extreme adaptability and flexibility of transcriptional regulatory networks.

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 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.

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.

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. Deletion of HAP4 and CYT1, both of which are essential for respiration, revert the sch9Delta mutant respiratory rate back to a lower-than-wild-type level. These alterations of the electron transport chain almost completely blocked CLS extension by the sch9Delta mutation but had a minor impact on the RLS. SCH9 thus negatively regulates the CLS and RLS through inhibition of respiratory genes, but a large part of its action on life span seems to be respiration independent and might involve increased resistance to stress. Considering that TOR1 deletion also increases respiration and that Sch9p is a direct target of TOR signaling, we propose that SCH9 is one of the major effectors of TOR repression of respiratory activity in glucose grown cells.

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. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. CONCLUSION: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans.

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. The first set corresponded to mixtures consisting of three gases with no overlapping spectral bands (C(2)H(2), C(2)H(4), and R14). The second set corresponded to mixtures of three gases having significant spectral overlap (C(2)H(4), R114, and R134a). For each mixture the flow rates of individual constituents were adjusted to yield specific constituent optical-density (CL) ratios. These ratios were compared to the optical-density ratios retrieved from the measured infrared radiance spectra. Results of this study indicated that for both sets of multi-gas mixtures the optical-density ratios retrieved by the passive remote monitoring technique were in good agreement with those derived from the release flow rates, provided that a simple correction scheme was introduced to compensate for the limited accuracy of the fast radiance model implemented in the monitoring algorithm.

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.

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.

Passive standoff detection of SF6 at a distance of 5.7 km by differential Fourier transform infrared radiometry.

Recent results are presented on the passive detection, identification, and quantification of a vapor cloud of SF6 measured at a horizontal standoff distance of 5.7 km using a dual-beam interferometer optimized for background signal suppression. The measurements were performed at Defense Research and Development Canada (DRDC)-Valcartier during a number of recent open-air experiments. The measurement approach is based on the differential passive standoff detection method that has been developed by DRDC Valcartier during the past few years. This work represents the first such measurement reported in the open literature for a standoff distance as large as 5.7 km. These results clearly demonstrate the capability of the differential radiometry approach to the detection, identification, and quantification of chemical vapor clouds located at long distances from the sensor.