A Method to Conditionally Measure Target Engagement at Intracellular RAS and RAF Complexes.

Dysfunction of the RAS/mitogen-activated protein kinase (MAPK) pathway is a common driver of human cancers. As such, both the master regulator of the pathway, RAS, and its proximal kinase effectors, RAFs, have been of interest as drug targets for decades. Importantly, signaling within the RAS/MAPK pathway is highly coordinated due to the formation of a higher-order complex called the RAS/RAF signalosome, which may minimally contain dimers of both RAS and RAF protomers. In the disease state, RAS and RAF assemble in homo- and/or heterodimeric forms. Traditionally, drug development campaigns for both RAS and RAF have utilized biochemical assays of purified recombinant protein. As these assays do not query the RAS or RAF proteins in their full-length and complexed forms in cells, potency results collected using these assays have often failed to correlate with inhibition of the MAPK pathway. To more accurately quantify engagement at this signaling components, we present a bioluminescence resonance energy transfer (BRET)-based method to conditionally measure target engagement at individual protomers within the RAS/RAF signalosome in live cells.

Inhibition of the RAF/MEK/ERK Signaling Cascade in Pancreatic Cancer: Recent Advances and Future Perspectives.

Pancreatic cancer represents a formidable challenge in oncology, primarily due to its aggressive nature and limited therapeutic options. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC), the main form of pancreatic cancer, remains disappointingly poor with a 5-year overall survival of only 5%. Almost 95% of PDAC patients harbor Kirsten rat sarcoma virus (KRAS) oncogenic mutations. KRAS activates downstream intracellular pathways, most notably the rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling axis. Dysregulation of the RAF/MEK/ERK pathway is a crucial feature of pancreatic cancer and therefore its main components, RAF, MEK and ERK kinases, have been targeted pharmacologically, largely by small-molecule inhibitors. The recent advances in the development of inhibitors not only directly targeting the RAF/MEK/ERK pathway but also indirectly through inhibition of its regulators, such as Src homology-containing protein tyrosine phosphatase 2 (SHP2) and Son of sevenless homolog 1 (SOS1), provide new therapeutic opportunities. Moreover, the discovery of allele-specific small-molecule inhibitors against mutant KRAS variants has brought excitement for successful innovations in the battle against pancreatic cancer. Herein, we review the recent advances in targeted therapy and combinatorial strategies with focus on the current preclinical and clinical approaches, providing critical insight, underscoring the potential of these efforts and supporting their promise to improve the lives of patients with PDAC.

Targeting CRAF kinase in anti-cancer therapy: progress and opportunities.

The RAS/mitogen-activated protein kinase (MAPK) signaling cascade is commonly dysregulated in human malignancies by processes driven by RAS or RAF oncogenes. Among the members of the RAF kinase family, CRAF plays an important role in the RAS-MAPK signaling pathway, as well as in the progression of cancer. Recent research has provided evidence implicating the role of CRAF in the physiological regulation and the resistance to BRAF inhibitors through MAPK-dependent and MAPK-independent mechanisms. Nevertheless, the effectiveness of solely targeting CRAF kinase activity remains controversial. Moreover, the kinase-independent function of CRAF may be essential for lung cancers with KRAS mutations. It is imperative to develop strategies to enhance efficacy and minimize toxicity in tumors driven by RAS or RAF oncogenes. The review investigates CRAF alterations observed in cancers and unravels the distinct roles of CRAF in cancers propelled by diverse oncogenes. This review also seeks to summarize CRAF-interacting proteins and delineate CRAF's regulation across various cancer hallmarks. Additionally, we discuss recent advances in pan-RAF inhibitors and their combination with other therapeutic approaches to improve treatment outcomes and minimize adverse effects in patients with RAF/RAS-mutant tumors. By providing a comprehensive understanding of the multifaceted role of CRAF in cancers and highlighting the latest developments in RAF inhibitor therapies, we endeavor to identify synergistic targets and elucidate resistance pathways, setting the stage for more robust and safer combination strategies for cancer treatment.

ITPR1-AS1 promotes small cell lung cancer metastasis by facilitating P21HRAS splicing and stabilizing DDX3X to activate the cRaf-MEK-ERK cascade.

The mechanisms underlying the involvement of long non-coding RNAs (lncRNAs) in the metastasis of small cell lung cancer (SCLC) remain largely unknown. Here, we identified that the lncRNA ITPR1-AS1 was upregulated in SCLC and lymph node metastasis tissues and positively correlated with SCLC malignant features. The overexpression of ITPR1-AS1 in SCLC was an independent risk factor for the overall survival of patients with SCLC. Our data confirmed that ITPR1-AS1 induces SCLC cell metastasis both in vitro and in vivo. Mechanistically, ITPR1-AS1 acts as a scaffold to enhance the interaction between SRC-associated in mitosis 68 kDa and heterogeneous nuclear ribonucleoprotein A1, which facilitates the alternative splicing of the H-Ras proto-oncogene (HRAS) pre-mRNA (P21HRAS). Moreover, we observed that ITPR1-AS1 could associate in a complex with and maintain the stability of DEAD-box polypeptide 3 (DDX3X), which inhibited the latter's ubiquitination and degradation. Our data provide evidence that ITPR1-AS1 activates the cRaf-MEK-ERK cascade by upregulating P21HRAS production and stabilizing DDX3X, to promote SCLC metastasis.

Sex disparities in non-small cell lung cancer: mechanistic insights from a cRaf transgenic disease model.

BACKGROUND: Women are at greater risk of developing non-small cell lung cancer (NSCLC), yet the underlying causes remain unclear. METHODS: We performed whole genome scans in lung tumours of cRaf transgenic mice and identified miRNA, transcription factor and hormone receptor dependent gene regulations. We confirmed hormone receptors by immunohistochemistry and constructed regulatory gene networks by considering experimentally validated miRNA-gene and transcription factor-miRNA/gene targets. Bioinformatics, genomic foot-printing and gene enrichment analysis established sex-specific circuits of lung tumour growth. Translational research involved a large cohort of NSCLC patients. We evaluated commonalities in sex-specific NSCLC gene regulations between mice and humans and determined their prognostic value in Kaplan-Meier survival statistics and COX proportional hazard regression analysis. FINDINGS: Overexpression of the cRaf kinase elicited an extraordinary 8-fold increase in tumour growth among females, and nearly 70% of the 112 differentially expressed genes (DEGs) were female specific. We identified oncogenes, oncomirs, tumour suppressors, cell cycle regulators and MAPK/EGFR signalling molecules, which prompted sex-based differences in NSCLC, and we deciphered a regulatory gene-network, which protected males from accelerated tumour growth. Strikingly, 41% of DEGs are targets of hormone receptors, and the majority (85%) are oestrogen receptor (ER) dependent. We confirmed the role of ER in a large cohort of NSCLC patients and validated 40% of DEGs induced by cRaf in clinical tumour samples. INTERPRETATION: We report the molecular wiring that prompted sex disparities in tumour growth. This allowed us to propose the development of molecular targeted therapies by jointly blocking ER, CDK1 and arginase 2 in NSCLC. FUNDING: We gratefully acknowledge the financial support of the Lower Saxony Ministry of Culture and Sciences and Volkswagen Foundation, Germany to JB (25A.5-7251-99-3/00) and of the Chinese Scholarship Council to SZ (202008080022). This publication is funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the "Open Access Publikationskosten" program.

RAS-independent ERK activation by constitutively active KSR3 in non-chordate metazoa.

During early development of the sea urchin embryo, activation of ERK signalling in mesodermal precursors is not triggered by extracellular RTK ligands but by a cell-autonomous, RAS-independent mechanism that was not understood. We discovered that in these cells, ERK signalling is activated through the transcriptional activation of a gene encoding a protein related to Kinase Suppressor of Ras, that we named KSR3. KSR3 belongs to a family of catalytically inactive allosteric activators of RAF. Phylogenetic analysis revealed that genes encoding kinase defective KSR3 proteins are present in most non-chordate metazoa but have been lost in flies and nematodes. We show that the structure of KSR3 factors resembles that of several oncogenic human RAF mutants and that KSR3 from echinoderms, cnidarians and hemichordates activate ERK signalling independently of RAS when overexpressed in cultured cells. Finally, we used the sequence of KSR3 factors to identify activating mutations of human B-RAF. These findings reveal key functions for this family of factors as activators of RAF in RAS-independent ERK signalling in invertebrates. They have implications on the evolution of the ERK signalling pathway and suggest a mechanism for its co-option in the course of evolution.

SH3 domain-binding kinase 1 promotes proliferation and inhibits apoptosis of cervical cancer via activating the Wnt/ß-catenin and Raf/ERK1/2 signaling pathways.

SH3 domain-binding kinase 1 (SBK1), is a member of the serine/threonine protein kinases family, and was confirmed to be upregulated in cervical cancer in our previous study. Nonetheless, the role of SBK1 in regulating cancer occurrence and development is unclear. In this study, the stable SBK1-knockdown and -overexpressed cell models were constructed by plasmid transfection technology. Cell viability and growth were assessed through CCK-8, colony formation, and BrdU methods. Cell cycle and apoptosis were analyzed by flow cytometry. The JC-1 staining assay was used to explore mitochondrial membrane potential. The scratch and Transwell assays were used to evaluate the cell metastatic ability. The nude mice models were utilized to explore the SBK1 expression affecting tumor growth in vivo. Our research indicated a high expression of SBK1 both in tissues and cells of cervical cancer. The proliferative, migratory, as well as invasive capacities of cervical cancer cells, were suppressed, and apoptosis was enhanced after SBK1 silence, whereas SBK1 upregulation led to opposite results. In addition, Wnt/ß-catenin and Raf/ERK1/2 pathways were activated by SBK1 upregulation. Furthermore, downregulation of c-Raf or ß-catenin, reversed the proliferation promotion and apoptosis inhibition effects in SBK1-overexpressed cells. The same results were observed with the use of the specific Raf inhibitor. SBK1 overexpression also contributed to tumor growth in vivo. Overall, SBK1 played a vital role in cervical tumorigenesis via activating the Wnt/ß-catenin and Raf/ERK1/2 pathways.

ZnT1 induces a crosstalk between T-type and L-type calcium channels through interactions with Raf-1 kinase and the calcium channel ß2 subunit.

ZnT1 is a major zinc transporter that regulates cellular zinc homeostasis. We have previously shown that ZnT1 has additional functions that are independent of its activity as a Zn2+ extruder. These include inhibition of the L-type calcium channel (LTCC) through interaction with the auxiliary ß-subunit of the LTCC and activation of the Raf-ERK signaling leading to augmented activity of the T-type calcium channel (TTCC). Our findings indicate that ZnT1 increases TTCC activity by enhancing the trafficking of the channel to the plasma membrane. LTCC and TTCC are co-expressed in many tissues and have different functions in a variety of tissues. In the current work, we investigated the effect of the voltage-gated calcium channel (VGCC) ß-subunit and ZnT1 on the crosstalk between LTCC and TTCC and their functions. Our results indicate that the ß-subunit inhibits the ZnT1-induced augmentation of TTCC function. This inhibition correlates with the VGCC ß-subunit-dependent reduction in ZnT1-induced activation of Ras-ERK signaling. The effect of ZnT1 is specific, as the presence of the ß-subunit did not change the effect of endothelin-1 (ET-1) on TTCC surface expression. These findings document a novel regulatory function of ZnT1 serving as a mediator in the crosstalk between TTCC and LTCC. Overall, we demonstrate that ZnT1 binds and regulates the activity of the ß-subunit of VGCC and Raf-1 kinase and modulates surface expression of the LTCC and TTCC catalytic subunits, consequently modulating the activity of these channels.

RAF1 deficiency causes a lethal syndrome that underscores RTK signaling during embryogenesis.

Somatic and germline gain-of-function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor-prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss-of-function of the proto-oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress-induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Consensus on the RAS dimerization hypothesis: Strong evidence for lipid-mediated clustering but not for G-domain-mediated interactions.

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.

Semilicoisoflavone B Induces Apoptosis of Oral Cancer Cells by Inducing ROS Production and Downregulating MAPK and Ras/Raf/MEK Signaling.

Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer worldwide. Despite advancement in treatment, advanced-stage OSCC is associated with poor prognosis and high mortality. The present study aimed to investigate the anticancer activities of semilicoisoflavone B (SFB), which is a natural phenolic compound isolated from Glycyrrhiza species. The results revealed that SFB reduces OSCC cell viability by targeting cell cycle and apoptosis. The compound caused cell cycle arrest at the G2/M phase and downregulated the expressions of cell cycle regulators including cyclin A and cyclin-dependent kinase (CDK) 2, 6, and 4. Moreover, SFB induced apoptosis by activating poly-ADP-ribose polymerase (PARP) and caspases 3, 8, and 9. It increased the expressions of pro-apoptotic proteins Bax and Bak, reduced the expressions of anti-apoptotic proteins Bcl-2 and Bcl-xL, and increased the expressions of the death receptor pathway protein Fas cell surface death receptor (FAS), Fas-associated death domain protein (FADD), and TNFR1-associated death domain protein (TRADD). SFB was found to mediate oral cancer cell apoptosis by increasing reactive oxygen species (ROS) production. The treatment of the cells with N-acetyl cysteine (NAC) caused a reduction in pro-apoptotic potential of SFB. Regarding upstream signaling, SFB reduced the phosphorylation of AKT, ERK1/2, p38, and JNK1/2 and suppressed the activation of Ras, Raf, and MEK. The human apoptosis array conducted in the study identified that SFB downregulated survivin expression to induce oral cancer cell apoptosis. Taken together, the study identifies SFB as a potent anticancer agent that might be used clinically to manage human OSCC.

HERC2 deficiency activates C-RAF/MKK3/p38 signalling pathway altering the cellular response to oxidative stress.

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases.

Development of fluorizoline analogues as prohibitin ligands that modulate C-RAF signaling, p21 expression and melanogenesis.

Fluorizoline is a cytotoxic trifluorothiazoline that targets the scaffold proteins prohibitins-1 and -2 (PHB1/2) to inhibit the kinase C-RAF and promote the expression of the cyclin-dependent kinase inhibitor p21 to induce cancer cell death. In melanocytes, fluorizoline also induces the synthesis of melanin. Herein we report the first structural requirement of fluorizoline analogues for these activities. We identified in particular some compounds that display enhanced anti-C-RAF and anti-MEK activities, and a higher cytotoxicity in HeLa cells compared to fluorizoline. These results provide a foundation for further optimization of PHB ligands for the treatment of cancers. We also discovered an analogue of fluorizoline that displays pharmacological effects opposed to those of fluorizoline and that can be used as a chemical tool to explore PHB signaling in cancers and other diseases.

Structure of SHOC2-PP1C-RAS Reveals a Mechanism of RAF Activation.

Determination of the SHOC2-PP1C-RAS structure uncovers the mechanism of substrate specificity for RAF.

Exploring CRD mobility during RAS/RAF engagement at the membrane.

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells.

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

Dioscorea nipponica Makino Relieves Ovalbumin-Induced Asthma in Mice through Regulating RKIP-Mediated Raf-1/MEK/MAPK/ERK Signaling Pathway.

Purpose: Dioscorea nipponica Makino (DNM) is a traditional herb with multiple medicinal functions. This study is aimed at exploring the therapeutic effects of DNM on asthma and the underlying mechanisms involving RKIP-mediated MAPK signaling pathway. Methods: An ovalbumin-induced asthma model was established in mice, which was further administrated with DNM and/or locostatin (RKIP inhibitor). ELISA was performed to detect the serum titers of OVA-IgE and OVA-IgG1, bronchoalveolar lavage fluid (BALF) levels of inflammation-related biomarkers, and tissue levels of oxidative stress-related biomarkers. The expression of RKIP was measured by quantitative real-time PCR, Western blot, immunohistochemistry, and immunofluorescence. HE staining was used to observe the pathological morphology of lung tissues. The protein expression of MAPK pathway-related proteins was detected by Western blot. Results: Compared with the controls, the model mice exhibited significantly higher serum titers of OVA-IgE and OVA-IgG1, BALF levels of IL-6, IL-8, IL-13, TGF-ß1, and MCP-1, tissue levels of MDA and ROS, lower BALF levels of IL-10 and IFN-γ, and tissue level of GSH. DNM relieved the allergic inflammatory response and oxidative stress in the model mice. DNM also recovered the downregulation of RKIP and the pathological injury of lung tissues in asthma mice. In addition, the Raf-1/MEK/MAPK/ERK pathway in the model mice was blocked by DNM. Silencing of RKIP by locostatin weakened the relieving effects of DNM on asthma through activating the Raf-1/MEK/MAPK/ERK pathway. Conclusion: DNM relieves asthma via blocking the Raf-1/MEK/MAPK/ERK pathway that mediated by RKIP upregulation.

The mechanism of activation of MEK1 by B-Raf and KSR1.

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1's role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.

Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.

Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers.

1α, 25-Dihydroxyvitamin D3 protects gastric mucosa epithelial cells against Helicobacter pylori-infected apoptosis through a vitamin D receptor-dependent c-Raf/MEK/ERK pathway.

CONTEXT: Due to the resistance of Helicobacter pylori to antibiotics, it is difficult to eradicate this pathogenic bacterium from the host. The role of 1α, 25-dihydroxyvitamin D3 (1,25-D3) in H. pylori-infected gastric mucosa epithelial cells remains unknown. OBJECTIVE: This study investigates the protective property of 1,25-D3 against H. pylori-infected apoptosis in gastric mucosa epithelial cells and its potential molecular mechanisms. MATERIALS AND METHODS: GES-1 cells were infected with H. pylori SS1 strain (MOI: 100) and treated with 1,25-D3 at 100, 200, and 300 nM for 24 h. Mice were orally gavaged with 108 CFUs of H. pylori and 25 µg/kg 1,25-D3 every other day for 1 month. CCK-8, LDH assay, TUNEL assay and western blot were used to determine the effect of 1,25-D3 on H. pylori-induced apoptosis. RESULTS: H. pylori infection decreased cell viability to 59.2%, while 100-300 nM 1,25-D3 increased cell viability to 62.2%, 78.4% and 87.1%, respectively. Compared with positive control (4.53-fold), 1,25-D3 reduced caspase-3 activity to 4.49-, 2.88- and 1.49-fold, reduced caspase-6 activity to 2.36-, 1.88- and 1.50-fold, reduced caspase-9 activity to 4.55-, 2.91- and 2.01-fold. 1,25-D3 alters Bcl-2 family, caspase protein expression and c-Raf/MEK/ERK phosphorylation levels in vivo and in vitro. Suppression of 1,25-D3 in apoptosis was reliant on binding to vitamin D receptor. The pharmacological inhibition of c-Raf/MEK/ERK phosphorylation blocked the anti-apoptotic effect of 1,25-D3. DISCUSSION AND CONCLUSION: 1,25-D3 protected gastric mucosa epithelial cells against H. pylori-infected apoptosis through a VDR-dependent c-Raf/MEK/ERK pathway.