Raf-1 Protein Kinase Research Articles

Peptide V3 Inhibits the Growth of Human Hepatocellular Carcinoma by Inhibiting the Ras/Raf/MEK/ERK Signaling Pathway.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. Peptide V3 has shown anti-angiogenic and anti-tumor effects on S180 and H22 xenografts in nude mice. However, the detailed mechanism of action of peptide V3 has not yet been fully elucidated. In the present study, the effects of peptide V3 on the growth of human HCC cells were examined both in vitro and in vivo. Our results showed that peptide V3 inhibited the proliferation, viability, migration, and invasion of human HCC cells. However, no obvious effect was observed in HL-7702 cells. Peptide V3 increased the apoptosis and decreased the protein levels of H-RAS, phospho (p)-RAF, p-MEK, and p-extracellular signal-regulated protein kinase (ERK) in human HCC cells. Peptide V3 suppressed the growth of human HCC xenografts by down-regulating angiogenesis and up-regulating apoptosis. In conclusion, peptide V3 could inhibit the growth of human HCC by inhibiting the Ras/Raf/MEK/ERK signaling pathway. Novel peptides and modification strategies could be designed and applied for the treatment of different types of cancer.

USP10 promotes proliferation and migration and inhibits apoptosis of endometrial stromal cells in endometriosis through activating the Raf-1/MEK/ERK pathway.

Endometriosis has been initially described as endometrial-like tissue outside of the uterine cavity. The mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway playing an important role in the regulation of cell proliferation, apoptosis, and migration has been found to be activated in endometriosis. However, regulation of the MEK/ERK signaling pathway in endometriosis has not been fully understood. In this study, primary-cultured endometrial stromal cells were collected from patients with endometriosis and healthy controls, and the proliferation, apoptosis, and migration of ectopic endometrial stromal cells transfected with ubiquitin-specific protease 10 (USP10)-small-interfering RNA (siRNA) or pLVX-Puro-USP10 with or without MEK inhibitor PD-98059 or exogenous signaling stimulation such as epidermal growth factor (EGF) were measured by CCK-8, flow cytometry, and Transwell, respectively. The gene and protein expressions were measured by real-time PCR or Western blot. USP10 overexpression promoted ectopic endometrial stromal cell migration and proliferation, suppressed cell apoptosis, and activated MEK/ERK signaling that is a critical downstream target of the serine/threonine protein kinase Raf-1, which was significantly blocked by PD-98059. USP10 silencing demonstrated the inverse effects, and these effects induced by USP10 silencing were significantly blocked by EGF. USP10 overexpression promoted Raf-1 protein expression, but not mRNA expression, through deubiquitination. In conclusion, these results suggest that USP10 promotes proliferation and migration and inhibits apoptosis of endometrial stromal cells in endometriosis through activating the Raf-1/MEK/ERK pathway.

Ras-Mediated Activation of the Raf Family Kinases.

The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade components, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.

Usefulness of serum 5-S-cysteinyl-dopa as a biomarker for predicting prognosis and detecting relapse in patients with advanced stage malignant melanoma.

With the recent development of novel molecular targeted drugs for advanced stage malignant melanoma (MM), including RAF and mitogen-activated protein kinase kinase inhibitors and immune checkpoint blockers, the early detection of relapse is important for managing patients with MM. In this study, we retrospectively analyzed two conventional serum biomarkers, 5-S-cysteinyl-dopa and lactate dehydrogenase, in patients with MM (n = 140) who were treated at a single Japanese institute from June 2007 to June 2015. At the initial hospital visit, serum 5-S-cysteinyl-dopa levels were significantly increased in patients with stages III (n = 38) and IV (n = 20) MM compared with patients with stages 0-II (n = 62) MM. In addition, in patients with stages III and IV MM, serum 5-S-cysteinyl-dopa levels of more than 15.0 nmol/L at initial hospital visit correlated with a poor prognosis. In 11 of 14 patients whose disease progressed during follow up (mostly from stages III-IV), serum 5-S-cysteinyl-dopa levels exceeded the normal limit of 10.0 nmol/L during the clinical detection of distant metastases. These results indicate the usefulness of measuring serum 5-S-cysteinyl-dopa levels at initial hospital visit and during follow up for early and effective therapeutic interventions using newly developed molecular targeted drugs.

Advanced Glycation End Products (AGE) Potently Induce Autophagy through Activation of RAF Protein Kinase and Nuclear Factor κB (NF-κB)

Advanced glycation end products (AGE) accumulate in diabetic patients and aging people because of high amounts of three- or four-carbon sugars derived from glucose, thereby causing multiple consequences, including inflammation, apoptosis, obesity, and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to the activation of autophagy (self-eating) that might result in obesity. We detected AGE as one of the potent inducers of autophagy compared with doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3K and potentiated by the autophagosome maturation blocker bafilomycin. It increases autophagy in different cell types, and that correlates with the expression of its receptor, receptor for AGE. LC3B, the marker for autophagosomes, is shown to increase upon AGE stimulation. AGE-mediated autophagy is partially suppressed by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases sterol regulatory element binding protein activity, which leads to an increase in lipogenesis. Although AGE-mediated lipogenesis is affected by autophagy inhibitors, AGE-mediated autophagy is not influenced by lipogenesis inhibitors, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we provide data showing that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, that are involved in autophagy and simultaneously help with the accumulation of lipid droplets that are not cleared effectively by autophagy, therefore causing obesity.

The many-faced KSR1: a tumor suppressor in breast cancer.

Emerging evidence supports the dual function of kinase suppressor of Ras 1 (KSR1) as an active kinase and a scaffold, although it has been extensively referred as a pseudokinase, due to the absence of key residues in its catalytic domain [1, 2]. As a scaffolding protein, KSR1 orchestrates the assembly of the protein kinases RAF, mitogen activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK) in the canonical Ras-RAF-MAPKs pathway, in a Ras-dependent manner or upon growth factor treatment [1, 3]. Conversely, structural and biochemical studies reveal that KSR1 is capable of phosphorylating MEK and more importantly, the catalytic activity of KSR is markedly increased when BRAF or inhibitor-bound CRAF is introduced in the complexes [1, 4, 5]. Such findings add complexity to the ERK spatio-temporal pathway control and identify KSR1 as a modulator of these pathways. In light of its regulatory role in oncogenic Ras-RAF-MAPKs signaling, extensive efforts have attempted to establish KSR1 as an oncogene in Ras-dependent cancers. Indeed, KSR1 has been shown to contribute to oncogenesis in various forms of Ras-activated cancer, including skin, pancreatic and lung carcinomas [1]. First, absence of KSR1 inhibits tumor formation in different Ras-mediated mouse models (KSR1−/−), suggesting that KSR1 is required for Ras-transduced MAPK activated tumorigenesis. Moreover, depletion of KSR1 reduces tumor growth of K-Ras-dependent pancreatic and lung cancer xenografts in nude mice, further supporting KSR1 as an oncogene as well as a potential therapeutic target. However, our own studies reveal an interesting, and yet unexpected role of KSR1 in breast cancer, where Ras mutations are rare. Using tumor tissue microarrays in a large patient cohort with a long term follow-up, we observe that breast cancer patients with high KSR1 had better disease free- and overall survival, results also supported by Oncomine analyses, microarray data and genomic data from paired tumor and cell-free DNA (cfDNA) samples revealing loss of heterozygosity [6]. KSR1 expression is positively associated with breast cancer 1, early onset (BRCA1), BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels in breast cancer specimens. Intriguingly, phospho-profiling of major components of the canonical Ras-RAF-MAPKs pathway, including RAF, MEK and ERK, show no significant changes upon KSR1 overexpression or depletion. These results underline its role beyond coordinating MAPKs signaling and challenge its oncogenic function in breast cancer. Further experiments support a tumor suppressive role of KSR1. Of note, breast cancer cells overexpressing KSR1 form fewer and smaller size colonies compared to the parental ones, while an in vivo mouse model also demonstrates that the growth of xenograft tumors overexpressing KSR1 is inhibited. It appears that the tumor inhibitory effect of KSR1 is BRCA1-dependent as shown by in vitro 3D-matrigel and soft-agar assays. Consistent with the correlation between KSR1 and BRCA1 in clinical samples, in vitro assays demonstrate KSR1 overexpression increases BRCA1 protein levels by decreasing BRCA1 ubiquitination partly through elevating BARD1 protein abundance and the BRCA1-BARD1 interaction. These findings integrate KSR1 into a complicated signaling network involving BRCA1-BARD1 complex. Unquestionably, alternative mechanisms underlying BRCA1's up-regulation by KSR1 other than through BARD1 remain to be examined. Our current understanding of KSR1's function in breast cancer is far from complete (Figure ​(Figure1).1). Puzzlingly, in addition to executing its anti-tumor effects through BRCA1, KSR1 negatively regulates transcriptional activity of p53 by reducing p53 acetylation via modulation of deleted in breast cancer 1 (DBC1) phosphorylation [7]. How can KSR1 up-regulates BRCA1 and inhibits p53 activity at the same time? What determines its preferable binding partners and thus contributes to its inhibitory outcome? An interesting and simple theory might be the following: since DBC1 is a positive regulator of p53 activity and a repressor for BRCA1, it potentially positions KSR1 into a dynamic state connecting p53 and BRCA1. Ultimately, while BRCA1 compensates for the loss of p53 activity, KSR1 tilts the balance scale towards tumor suppression in cancer cells. Further investigation is much needed to address the underlying mechanisms. In addition, evidence regarding other aspects of its action are evolving: a very recent study characterizing the proteomic profile of KSR1-regulated signaling in response to genotoxic agents in breast cancer illustrates a broad functional network conferred by KSR1, highlighting its importance in the chemotherapy response [8].. Figure 1 KSR1-regulated signaling in cancer A better understanding of the molecular determinants of the distinctive behaviour of KSR1 in different types of cancer is essential to the optimal targeting of this dual functional kinase-scaffold protein. Our studies suggest the tumor suppressive action of KSR1 and its clinical relevance in patient stratification, placing KSR1 in the major oncoprotein pathways.

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.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1.

Discovery of an obesity susceptibility gene, KSR2, provides new insight into energy homeostasis pathways.

Obesity is a heterogeneous condition with considerable heritability. Multiple adiposity genes have now been identified through genome-wide association studies and studies of rare forms obesity caused by single-gene mutations. Several of these monogenic forms of obesity seem to replicate mouse obesity phenotypes, with notable examples including obesity caused by inactivating mutations in leptin, the leptin receptor, and the melanocortin-4 receptor. A large-scale screen for additional obese phenotypes in mice found that targeted deletion of Ksr2 leads to obesity,1 suggesting a role for Ksr2 in energy homeostasis. Ksr2 encodes the scaffolding protein kinase suppressor of ras 2 (KSR2), which may regulate multiple signaling pathways by binding kinases, such as Raf and AMP-activated protein kinase (AMPK). In this article, Pearce et al2 hypothesize that genetic variants in KSR2 may contribute to the development of severe, early onset obesity in humans. Using a combination of Sanger and whole-exome sequencing, the authors sequenced the coding region and intron/exon boundaries of KSR2 to identify rare variants (minor allele frequency, <0.5%) in 2101 individuals of mixed European ancestry who developed severe obesity before 10 years and in 1536 controls from a UK population-based study. They also performed a small replication study (238 cases and 1117 controls) and sequenced KSR2 in family members of severely obese probands to determine whether KSR2 variants cosegregated with overweight/obesity …

Expression of the Transcription Factor Egr-1 in Pancreatic Acinar Cells Following Stimulation of Cholecystokinin or Gαq-Coupled Designer Receptors

Backgound/Aims: The injection of cerulein, an analogue of the pancreatic secretagogue cholecystokinin (CCK), induces acute pancreatitis in mice that is accompanied by the synthesis of the transcription factor Egr-1. The signaling cascade that connects cerulein stimulation with enhanced Egr-1 biosynthesis was analyzed. Methods: AR42J rat pancreatic acinar cells were used as a model system to measure cerulein-induced Egr-1 biosynthesis. For comparison, the signaling cascade induced by activation of Gαq-coupled designer receptors with the designer drug clozapine-N-oxide (CNO) was investigated. Results: Stimulation of AR42J cells with cerulein induced a robust and transient biosynthesis of Egr-1. The signaling cascade connecting cerulein stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca²⁺ and the activation of the protein kinases PKC, Raf and ERK, while expression of MKP-1 prevented Egr-1 biosynthesis in cerulein-stimulated AR42J cells. In addition, ternary complex factors are required to connect cerulein stimulation with enhanced transcription of the Egr-1 gene. Egr-1 biosynthesis induced in CNO-stimulated AR42J pancreatic acinar cells expressing Gαq-coupled designer receptors required identical signaling molecules, although subtle differences were observed in comparison to cerulein/CCK receptor signaling. Conclusion: We propose that overstimulation of the canonical Gαq-induced signaling pathway may be crucial for inducing acute pancreatitis.

Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS.

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.

Different Raf Protein Kinases Mediate Different Signaling Pathways to Stimulate E3 Ligase RFFL Gene Expression in Cell Migration Regulation

We previously characterized a Gα12-specific signaling pathway that stimulates the transcription of the E3 ligase RFFL via the protein kinase ARAF and ERK. This pathway leads to persistent PKC activation and is important for sustaining fibroblast migration. However, questions remain regarding how Gα12 specifically activates ARAF, which transcription factor is involved in Gα12-mediated RFFL expression, and whether RFFL is important for cell migration stimulated by other signaling mechanisms that can activate ERK. In this study, we show that replacement of the Gα12 residue Arg-264 with Gln, which is the corresponding Gα13 residue, abrogates the ability of Gα12 to interact with or activate ARAF. We also show that Gα12 can no longer interact with and activate an ARAF mutant with its C-terminal sequence downstream of the kinase domain being replaced with the corresponding CRAF sequence. These results explain why Gα12, but not Gα13, specifically activates ARAF but not CRAF. Together with our finding that recombinant Gα12 is sufficient for stimulating the kinase activity of ARAF, this study reveals an ARAF activation mechanism that is different from that of CRAF. In addition, we show that this Gα12-ARAF-ERK pathway stimulates RFFL transcription through the transcription factor c-Myc. We further demonstrate that EGF, which signals through CRAF, and an activated BRAF mutant also activate PKC and stimulate cell migration through up-regulating RFFL expression. Thus, RFFL-mediated PKC activation has a broad significance in cell migration regulation.

Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.

ERK and RSK regulate distinct steps of a cellular program that induces transition from multicellular epithelium to single cell phenotype

The ERK (extracellular signal-regulated kinases) cascade has an evolutionarily conserved three tier architecture consisting of protein kinases Raf, MEK (MAPK/ERK kinase) and ERK. Following activation, ERK phosphorylates various cellular elements leading to diverse cellular responses. Downstream of ERK the family of p90 ribosomal S6 kinases (RSKs) has been proven to be an important conveyor of ERK signaling, however, little is known if ERK and RSK coordinate their functions to generate a specific biological response. Here we show that in epithelial cells conditional activation of the ERK pathway causes phenotypic conversion of epithelial cells to autonomously migrating cells. This process involves two sequential steps characterized by loss of apical–basal polarity followed by cell scattering. The activation of ERK, but not RSK, is sufficient for the execution of the first step and it requires calpain mediated remodeling of actin cytoskeleton. Conversely, RSK regulates the successive stage characterized by cell–cell contact weakening and increased cellular migration. Thus, ERK and RSK regulate different cellular subprograms and coordinated execution of these subprograms in time generates a relevant biological response. Our data also suggest that the mechanism by which the ERK pathway controls a cellular response may be distributed between ERK and RSK, rather than being elicited by a single effector kinase.

Role of MKP-1 (DUSP1) in clozapine-induced effects on the ERK1/2 signaling pathway in the rat frontal cortex

Clozapine affects the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the brain, which plays an important role in its antipsychotic action. However, previous findings are inconsistent, and related molecular mechanisms require further clarification. Time- and dose-dependent effects of clozapine on the ERK1/2 pathway and its regulatory mechanism were investigated in rat frontal cortex. At 15, 30, 60, and 120min after intraperitoneal injection of clozapine (5, 10, and 20mg/kg), changes in ERK1/2, its upstream canonical kinases (Raf1 and mitogen-activated protein kinase kinase 1/2 [MEK1/2]), and its downstream molecule (p90 ribosomal S6 kinase [p90RSK]) were investigated in rat frontal cortex. At 15min, p-Raf1, p-MEK1/2, p-ERK1/2, and p-p90RSK all increased dose-dependently. At 30min, p-ERK1/2 and p-p90RSK showed no significant changes, while dose-dependent increases in p-Raf1 and p-MEK1/2 were found. At 60 and 120min, although p-ERK1/2 and p-p90RSK decreased, increases in p-Raf1 and p-MEK1/2 were maintained. A clozapine-induced reduction in ERK1/2 phosphorylation was evident at both tyrosine and threonine residues, suggesting the involvement of dual specificity phosphatases (DUSPs; mitogen-activated protein kinase phosphatases [MKPs]). mRNA expression of seven Dusps that can dephosphorylate ERK1/2 were examined; Mkp-1 (Dusp1) mRNA increased following clozapine treatment. Moreover, MKP-1 protein and phosphatase activity increased, and binding of MKP-1 to ERK1/2 was also upregulated by clozapine administration. In rat frontal cortex, clozapine regulates ERK1/2 phosphorylation via MKP-1, which induces uncoupling between Raf1-MEK1/2 and ERK1/2-p90RSK activity. These findings suggest an important role of MKP-1 in the mechanism of action of clozapine.

Abstract 4295: PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity.

Abstract PHLPP belongs to a family of protein phosphatases that have previously been shown to inhibit Akt-mediated cell proliferation and survival signaling in cancers. Here, we demonstrate that PHLPP exerts its tumor suppressor function by negatively regulating the RAF/MEK/ERK pathway to inhibit cell migration and invasion. Specifically, we identify the oncogenic protein kinase RAF1 as an interacting protein and a novel substrate of PHLPP. PHLPP dephosphorylates the S338 site of RAF1 and inactivates the kinase both in vitro and in cells. Loss of PHLPP expression largely increases the amplitude and duration of RAF/MEK/ERK signaling in cells, whereas overexpression of PHLPP inhibits the pathway. Functionally, knockdown of PHLPP induces epithelial-mesenchymal transition (EMT)-like phenotype, which coincides with a significant increase in migration and invasion in colon cancer cells. Furthermore, we show that upregulation of transcription factors ZEB1 and ZEB2 is responsible for increased invasiveness observed in PHLPP knockdown cells. Taken together, we have identified a novel role of PHLPP in suppressing EMT and cell motility in colon cancer cells by directly antagonizing RAF1 function. Citation Format: Xin Li, Payton D. Stevens, Tianyan Gao. PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2013-4295

Signal transduction via TRPM3 channels in pancreatic β-cells

Transient receptor potential melastatin 3 (TRPM3) channels are non-selective cation channels that are expressed in insulinoma cells and pancreatic β-cells. Stimulation of TRPM3 with the neurosteroid pregnenolone sulfate induces an intracellular signaling cascade, involving a rise in intracellular Ca(2)(+) concentration, activation of the protein kinases Raf and ERK, and a change in the gene expression pattern of the cells. In particular, biosynthesis of insulin is altered following activation of TRPM3 by pregnenolone sulfate. Moreover, a direct effect of TRPM3 stimulation on insulin secretion has been reported. The fact that stimulation of TRPM3 induces a signaling cascade that is very similar to the signaling cascade induced by glucose in β-cells suggests that TRPM3 may influence main functions of pancreatic β-cells. The view that TRPM3 represents an ionotropic steroid receptor of pancreatic β-cells linking insulin release with steroid hormone signaling is discussed.

A Genome-Scale RNA Interference Screen Implicates NF1 Loss in Resistance to RAF Inhibition

RAF inhibitors such as vemurafenib and dabrafenib block BRAF-mediated cell proliferation and achieve meaningful clinical benefit in the vast majority of patients with BRAF(V600E)-mutant melanoma. However, some patients do not respond to this regimen, and nearly all progress to therapeutic resistance. We used a pooled RNA interference screen targeting more than 16,500 genes to discover loss-of-function events that could drive resistance to RAF inhibition. The highest ranking gene was NF1, which encodes neurofibromin, a tumor suppressor that inhibits RAS activity. NF1 loss mediates resistance to RAF and mitogen-activated protein kinase (MAPK) kinase kinase (MEK) inhibitors through sustained MAPK pathway activation. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor. NF1 mutations were observed in BRAF-mutant tumor cells that are intrinsically resistant to RAF inhibition and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus showing the clinical potential for NF1-driven resistance to RAF/MEK-targeted therapies.

Regulation of immediate‐early gene transcription following activation of Gαq‐coupled designer receptors

G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.

PKD controls mitotic Golgi complex fragmentation through a Raf–MEK1 pathway

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to-cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1-dependent manner, a process required for entry of the cells into mitosis.