A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK

Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.
Nature (Impact Factor: 41.46). 03/2011; 472(7343):366-9. DOI: 10.1038/nature09860
Source: PubMed


In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.

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    • "If the NtA is phosphorylated on KSR1 or C-Raf, both can function as activator kinases of their partners C-Raf or B-Raf. Moreover, Brennan and co-workers showed that B-Raf dimerization with KSR2 could allosterically stimulate the kinase activity of KSR2 towards MEK [48], however it is not known yet whether this transactivation event is mediated by the NtA of B-Raf. Studies on B-Raf inhibitor drugs led to the observation that unphosphorylated KSR1, however, is able to block B-Raf activation by constitutively dimerizing with B-Raf [49]. "
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    ABSTRACT: NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR's function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR's discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs' effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.
    Full-text · Article · May 2014 · Journal of Molecular Signaling
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    • "SAHA-PIP 13 activated GPRC5B that got recently identified to contribute to neurogenesis5 (Figure 2i). In the case of second generation SAHA-PIPs, 17 activated PDLIM3, a gene belonging to the network shown in Figure S4c and 18 activated LEFTY1 and KSR2, the factors known to be associated with lung development32 (Figure 2j–l). Likewise, 19 activated TSTD1 and SMOC2, a factor known to be associated with hearing impairment33 (Figure 2m and n). "
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    ABSTRACT: The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.
    Full-text · Article · Jan 2014 · Scientific Reports
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    • "One unforeseen outcome of the drive to develop drugs that inhibit kinases (Davis et al., 2011) is the realization that protein–protein interactions can profoundly influence drug action (Prince and Ahn, 2010). For example, the association of a kinase with endogenous binding partners can confer resistance to ATP-analog inhibitors, which provides an explanation for why kinases, such as Akt (PKB) and B-Raf, can become refractory to certain anticancer drugs (Brennan et al., 2011; Knight et al., 2010; Okuzumi et al., 2010; Okuzumi et al., 2009; Poulikakos et al., 2010). The same might be true for the PKC inhibitor ruboxistaurin, which is being developed to manage diabetic microcirculatory complications and neuropathies (Short and Tuttle, 2005; Tuttle et al., 2005), as structural analogs of this compound have been found to be ineffective against PKC that is associated with AKAP150 (Hoshi et al., 2010; Prince and Ahn, 2010). "
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    ABSTRACT: The second messenger cyclic AMP (cAMP) operates in discrete subcellular regions within which proteins that synthesize, break down or respond to the second messenger are precisely organized. A burgeoning knowledge of compartmentalized cAMP signaling is revealing how the local control of signaling enzyme activity impacts upon disease. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight how misregulation of local cyclic AMP signaling can have pathophysiological consequences. We first introduce the core molecular machinery for cAMP signaling, which includes the cAMP-dependent protein kinase (PKA), and then consider the role of A-kinase anchoring proteins (AKAPs) in coordinating different cAMP-responsive proteins. The latter sections illustrate the emerging role of local cAMP signaling in four disease areas: cataracts, cancer, diabetes and cardiovascular diseases.
    Full-text · Article · Oct 2013 · Journal of Cell Science
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