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The RAS-MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1-BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR-MEK interaction, which in turn enhances RAF-KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR-MEK-CNK-HYP complex reveals a ring-like arrangement of the CNK-HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR-MEK interaction., (© 2024. The Author(s).)

Cell motility is a critical feature of invasive tumour cells that is governed by complex signal transduction events. Particularly, the underlying mechanisms that bridge extracellular stimuli to the molecular machinery driving motility remain partially understood. Here, we show that the scaffold protein CNK2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to downstream activation of ARF6 GTPase. Mechanistically, AXL signalling induces PI3K-dependent recruitment of CNK2 to the plasma membrane. In turn, CNK2 stimulates ARF6 by associating with cytohesin ARF GEFs and with a novel adaptor protein called SAMD12. ARF6-GTP then controls motile forces by coordinating the respective activation and inhibition of RAC1 and RHOA GTPases. Significantly, genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model. Together, this work identifies CNK2 and its partner SAMD12 as key components of a novel pro-motility pathway in cancer cells, which could be targeted in metastasis., (© 2023. The Author(s).)

Objective: Conducting participatory research (PR) aimed at improving health implies considering inequitable power relations, including those related to sex/gender (S/G). This necessitates specific skills and methods and may be challenging especially since guidelines are scarce. Our objective was to perform a scoping review to provide a typology of existing guidelines for researchers on how to take account of S/G in the context of PR in public health, with a focus on occupational and environmental health., Methods: All steps of the research were conducted with the collaboration of an advisory committee, following PR principles. Nineteen documents were retained from 513 references identified in nine scientific databases and grey literature between 2000 and 2020. Data on recommendations were extracted and coded qualitatively. Cluster analysis based on similarities in recommendations proposed in the documents identified four types: (1) empowerment-centered; (2) concrete action-centered; (3) macrosystem-centered; and (4) stakeholder-centered., Synthesis: Many sources gave pointers on how to include S/G during data collection and analysis or during the dissemination of findings, but there was a dearth of suggestions for building partnerships with stakeholders and producing sustainable S/G sociopolitical transformations. Occupational health PR showed less similarities with other public health subfields including environmental health PR. Power relationships with workplace stakeholders generated specific obstacles related to S/G integration that require further attention. Intersectionality and reflexive practices emerged as overarching themes., Conclusion: This review provides helpful guidelines to researchers at different stages of planning PR, ranging from familiarizing themselves with S/G approaches to anticipating difficulties in their ongoing S/G-transformative PR., (© 2023. The Author(s) under exclusive license to The Canadian Public Health Association.)

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology., (© 2021 by The American Society of Hematology.)

For parents working in the transportation industry, atypical schedules are often a daily puzzle. Schedule planning systems allowing workers to choose shifts may affect job strenuousness and work-family balance (WFB) for both female and male workers. How could ergonomic interventions related to the implementation of those systems better consider gender dynamics regarding WFB strategies, and minimize inequities among workers? This article presents a joint analysis of two independent case studies related to ergonomic interventions in transport companies in Canada and Switzerland. Direct observation and semi-structured interviews shed light on the characteristics of schedule planning systems and their interaction with men's and women's WFB strategies. Issues related to each step of the planning process (shift construction, schedule choice, day-to-day schedule management) are discussed to inform interventions aimed at facilitating WFB, and ultimately gender equity, in atypical schedule contexts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this article is to examine the impacts of incorporating sex and gender (s/g) analysis in integrated knowledge translation (iKT) initiatives in the field of ergonomics and occupational health. The article presents findings based on a retrospective analysis of twelve intervention-research (IR) studies, including a thematic content analysis of in-depth interviews conducted with 15 researchers involved in these IRs. The findings offer an overview of various categories of impacts, such as changes in partners' views, in workplace settings and conditions, in practices and policies, and in economic outcomes. In these types of IR, health effects measurement is not the main objective, and direct health outcomes are difficult to assess. Explicitly talking about sex/gender led more often to system-level changes but less often to workplace-level changes, compared to interventions where sex/gender was not identified as a specific object of the intervention., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

To date, very little is known about the normal development trajectory of visual texture segregation, or how it is affected by preterm birth. The goal of this study was to characterize the development of visual texture segregation using texture segregation visual evoked potentials (tsVEPs) in children born full-term and children born preterm without major neurological impairment. Forty-five full-term and 43 preterm children were tested at either 12, 24 or 36 months of age (corrected age for prematurity at 12 and 24 months old). VEPs were obtained using two lower-level stimuli defined by orientation (oriVEP) and two higher-level stimuli defined by texture (texVEP). TsVEP was obtained by dividing by two the subtraction of oriVEP from texVEP. Results show a clear maturation of the processes underlying visual texture segregation in the full-term group, with a significant N2 latency reduction between 12 and 36 months of age for all conditions. Significant N2 amplitude reduction was observed for oriVEP between 12 and 24 months, as well as for texVEP between 12 and 24 months, and 12 and 36 months. Comparison between full-term and preterm children indicated significantly lower N2 amplitude for the preterm group at 12 months for oriVEP and texVEP. These differences were no longer apparent at 24 months of age, suggesting that children born preterm catch up with their full-term counterparts somewhere between 12 and 24 months of age. Our results appear to reflect a maturational delay in preterm children in both lower-level and higher-level visual processing during, at least, early childhood., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity., (Copyright © 2014 by the Genetics Society of America.)

The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing components can also specifically impact alternative splicing., Competing Interests: The authors have declared that no competing interests exist.

Functional near-infrared spectroscopy (fNIRS) has become increasingly established as a promising technique for monitoring functional brain activity. To our knowledge, no study has yet used fNIRS to investigate overt reading of irregular words and nonwords with a full coverage of the cerebral regions involved in reading processes. The aim of our study was to design and validate a protocol using fNIRS for the assessment of overt reading. Twelve healthy French-speaking adults underwent one session of fNIRS recording while performing an overt reading of 13 blocks of irregular words and nonwords. Reading blocks were separated by baseline periods during which participants were instructed to fixate a cross. Sources (n = 55) and detectors (n = 16) were placed bilaterally over frontal, temporal, parietal, and occipital regions. Two wavelengths were used: 690 nm, more sensitive to deoxyhemoglobin (HbR) concentration changes, and 830 nm, more sensitive to oxyhemoglobin (HbO) concentration changes. For all participants, total hemoglobin (HbT) concentrations (HbO + HbR) were significantly higher than baseline for both irregular word and nonword reading in the inferior frontal gyri, the middle and superior temporal gyri, and the occipital cortices bilaterally. In the temporal gyri, although the difference was not significant, [HbT] values were higher in the left hemisphere. In the bilateral inferior frontal gyri, higher [HbT] values were found in nonword than in irregular word reading. This activation could be related to the grapheme-to-phoneme conversion characterizing the phonological pathway of reading. Our findings confirm that fNIRS is an appropriate technique to assess the neural correlates of overt reading.

The development of agonists for the chemokine receptor CXCR4 could provide promising therapeutic candidates. On the basis of previously forwarded two site model of chemokine-receptor interactions, we hypothesized that linking the agonistic N-terminus of SDF-1 to the T140 backbone would yield new high-affinity agonists of CXCR4. We developed chimeras with the agonistic SDF-1 N-terminus grafted to a T140 side chain and tested their binding affinity and chemotactic agonist activity. While chimeras with the peptide grafted onto position 12 of T140 remained high-affinity antagonists, those bearing the peptide on position 14 were in part agonists. One chimera was a full CXCR4 agonist with 25 nM affinity, and several chimeras showed low nanomolar affinities with partial agonist activity. Our results confirmed that we have developed high-affinity agonists of CXCR4.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The ERK (extracellular signal-regulated kinase) pathway is an evolutionarily conserved signal transduction module that controls cellular growth, differentiation and survival. Activation of receptor tyrosine kinases (RTKs) by the binding of growth factors initiates GTP loading of RAS, which triggers the initial steps in the activation of the ERK pathway by modulating RAF family kinase function. Once activated, RAF participates in a sequential cascade of phosphorylation events that activate MEK, and in turn ERK. Unbridled signalling through the ERK pathway caused by activating mutations in RTKs, RAS or RAF has been linked to several human cancers. Of note, one member of the RAF family, BRAF, is the most frequently mutated oncogene in the kinase superfamily. Not surprisingly, there has been a colossal effort to understand the underlying regulation of this family of kinases. In particular, the process by which the RAF kinase domain becomes activated towards its substrate MEK remains of topical interest. Here, using Drosophila Schneider S2 cells, we demonstrate that RAF catalytic function is regulated in response to a specific mode of dimerization of its kinase domain, which we term the side-to-side dimer. Moreover, we find that the RAF-related pseudo-kinase KSR (kinase suppressor of Ras) also participates in forming side-to-side heterodimers with RAF and can thereby trigger RAF activation. This mechanism provides an elegant explanation for the longstanding conundrum about RAF catalytic activation, and also provides an explanation for the capacity of KSR, despite lacking catalytic function, to directly mediate RAF activation. We also show that RAF side-to-side dimer formation is essential for aberrant signalling by oncogenic BRAF mutants, and identify an oncogenic mutation that acts specifically by promoting side-to-side dimerization. Together, our data identify the side-to-side dimer interface of RAF as a potential therapeutic target for intervention in BRAF-dependent tumorigenesis.

Connector enhancer of KSR (CNK) is a multidomain-containing protein previously identified as a positive regulator of the RAS/MAPK pathway in Drosophila. Using transfection experiments and an RNAi-based rescue assay in Drosophila S2 cells, we demonstrate that CNK has antagonistic properties with respect to RAF activity. We show that CNK's N-terminal region contains two domains (SAM and CRIC) that are essential for RAF function. Unexpectedly, we also report that the C-terminal region of CNK contains a short bipartite element that strongly inhibits RAF catalytic function. Interestingly, CNK's opposite properties appear to prevent signaling leakage from RAF to MEK in the absence of upstream signals, but then transforms into a potent RAF activator upon signal activation. Together, these findings suggest that CNK not only participates in the elusive RAF activation process, but might also contribute to the switch-like behavior of the MAPK module.