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The CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction.

ABSTRACT: 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.

SUBMITTER: Maisonneuve P

PROVIDER: S-EPMC11257983 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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The CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction.

Maisonneuve Pierre P Sahmi Malha M Bergeron-Labrecque Fanny F Ma Xianjie Iris XI Queguiner Juliette J Arseneault Geneviève G Lefrançois Martin M Kurinov Igor I Fronzes Rémi R Sicheri Frank F Therrien Marc M

Nature structural & molecular biology 20240222 7

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 ...[more]

PMID: 38388830

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Project description:BackgroundHeart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3β acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown.MethodsTo define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis.ResultsFibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFβ1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition.ConclusionsGSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-β1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

Dataset Information

The CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction.

Publications

The CNK-HYP scaffolding complex promotes RAF activation by enhancing KSR-MEK interaction.

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