Spatial regulation of Raf kinase signaling by RKTG.
ABSTRACT: Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.
Project description:Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.
Project description:The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.
Project description:Epidermal growth factor (EGF) receptor (EGFR/HER1) is overexpressed in human pancreatic cancers. However, anti-EGFR therapy does not exhibit significant therapeutic activity with oncogenic K-ras mutation. We sought to assess the signaling relationship between EGFR and mutant K-ras, which is commonly detected in pancreatic cancer.Pancreatic cancer cells harboring mutated K-ras were treated with EGF to assess signaling from EGFR to mitogen-activated protein kinase (MAPK) pathway. The role of Ras family of proteins in transducing EGFR signals was assessed using short interfering RNA. Other components of MAPK and PI3K (phosphoinositide 3-kinase) pathways were examined for their roles in EGFR signaling.First, EGF signaling in pancreatic cancer cells occurs selectively through HER1. Second, knockdown of all Ras isoforms failed to block EGF-mediated phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of Raf was observed to partially abrogate ERK phosphorylation, whereas MEK inhibition resulted in complete attenuation of EGF-mediated ERK phosphorylation. Finally, inhibition of phosphoinositide 3-kinase/AKT and CDC42/PAK pathways did not block EGFR signaling.Our study results demonstrate that EGFR-mediated signaling in mutant K-ras pancreatic cancer cells does not follow canonical MAPK signaling. Our novel findings suggest the existence of alternate signaling pathways to downstream MAPK in the presence of mutant K-ras.
Project description:The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.
Project description:IQGAP1 modulates several cellular functions, including cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds ERK and MAPK kinase (MEK) and regulates EGF-stimulated MEK and ERK activity. Here we characterize the interaction between IQGAP1 and B-Raf, the molecule immediately upstream of MEK in the Ras/MAPK signaling cascade. B-Raf binds directly to IQGAP1 in vitro and coimmunoprecipitates with IQGAP1 from cell lysates. Importantly, IQGAP1 modulates B-Raf function. EGF is unable to stimulate B-Raf activity in IQGAP1-null cells and in cells transfected with an IQGAP1 mutant construct that is unable to bind B-Raf. Interestingly, binding to IQGAP1 significantly enhances B-Raf activity in vitro. Our data identify a previously unrecognized interaction between IQGAP1 and B-Raf and suggest that IQGAP1 is a scaffold necessary for activation of B-Raf by EGF.
Project description:The expression of contractile proteins in vascular smooth muscle cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific alpha-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of ERK1/2. Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerization of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be critical intermediates. Analysis of the expression of protease-activated receptor 1, heparin-binding EGF (HB-EGF), and the EGF receptor revealed that pro-HB-EGF is significantly up-regulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Because inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The small interfering RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a yellow fluorescent protein-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signaling strength within the Ras/Raf/MEK/ERK cascade.
Project description:MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Gialpha1/3-GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Gialpha1, which binds and recruits RGS14 to the plasma membrane. Gialpha1 binding to RGS14 inhibits Raf binding, indicating that Gialpha1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.
Project description:Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAF(V600E) tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAF(V600E). In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAF(V600E) tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.
Project description:Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.
Project description:Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK. We conclude that Raf-1 phosphorylation on serine 338 (S338) is a critical step that is inhibited in suspended cells. Restoration of phosphorylation at S338, either by expression of highly active PAK or by expression of an S338 phospho-mimetic Raf-1 mutation, led to a partial rescue of ERK activation in suspended cells. Raf-1 inhibition in suspension was not due to excessive negative regulation on inhibitory sites S43 and S259, as these serines were largely dephosphorylated in suspended cells. Finally, strong phosphorylation of Raf-1 S338 provided resistance to PKA-mediated inhibition of ERK activation. Phosphorylation at Raf-1 S43 and S259 by PKA only weakly inhibited EGF activation of Raf-1 and ERK when cells maintained high Raf-1 S338 phosphorylation.