Expression pattern and first functional characterization of riok-1 in Caenorhabditis elegans

Rio kinases are atypical serine/threonine kinases that emerge as potential cooperation partners in Ras-driven tumors. In the current study, we performed an RNAi screen in Caenorhabditis elegans to identify suppressors of oncogenic Ras signaling. Aberrant Ras/Raf signaling in C. elegans leads to the formation of a multi-vulva (Muv) phenotype. We found that depletion of riok-1, the C. elegans orthologue of the mammalian RioK1, suppressed the Muv phenotype. By using a promoter GFP construct, we could show that riok-1 is expressed in neuronal cells, the somatic gonad, the vulva, the uterus and the spermatheca. Furthermore, we observed developmental defects in the gonad upon riok-1 knockdown in a wildtype background. Our data suggest that riok-1 is a modulator of the Ras signaling pathway, suggesting implications for novel interventions in the context of Ras-driven tumors.     

Improved Binding of Raf to Ras��GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.     

The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf

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.     

Raf-1激酶与肿瘤治疗

自Raf激酶被证明为逆转录病毒致癌基因的产物以来,逐渐成为人们研究的热点。研究表明,Raf激酶既是Ras的效应物,又能作为ERK信号通路中的重要组分,成为活化的Ras和ERK之间的一个重要纽带。Ras-Raf-MEK-ERK信号通路参与了细胞增殖、分化和凋亡等生物学过程。作为这一信号通路上的节点蛋白,Raf激酶在肿瘤发生过程中起着关键作用。Raf家族成员Raf-1（cRaf）在调控细胞运动和凋亡过程中发挥关键作用,它既可以通过抑制促凋亡激酶ASK1和MST2活性来抑制细胞凋亡,也可以通过激活Rok-α的活性来促进细胞迁移。该文主要综述了Raf-1激酶的调控机制及其在肿瘤发生过程中的作用,同时也总结了以Raf-1为靶点的肿瘤治疗的最新进展。     

The CRAL/TRIO and GOLD domain protein TAP-1 regulates RAF-1 activation

The activation of the protein kinase Raf at the cell membrane is a critical step in cell signaling during development, but the mechanisms that regulate Raf activity remain incompletely defined. We previously demonstrated that the C. elegans cgr-1 gene encodes a CRAL/TRIO domain-containing protein that is a critical modulator of Ras-dependent cell fate specification during C. elegans development. Here we identify the mammalian α-tocopherol associated protein-1 (TAP-1) as a functional ortholog of cgr-1. TAP-1 mRNA was expressed in many tissues, and TAP-1 protein colocalized with Ras and Raf at the cell membrane. Reducing TAP-1 expression by RNA interference increased Ras/ERK signaling in multiple cell types. These functional studies demonstrate that CRAL/TRIO domain proteins play a conserved role in regulating Ras signaling. Biochemical analyses indicated that TAP-1 operates at the level of Raf, since TAP-1 function negatively regulated the amount of Raf-1 recruited to GTP-bound Ras at the cell membrane. TAP-1 plays a significant physiological role in controlling cell division, since reducing TAP-1 expression increased the oncogenic capacity of Ras transformed human cancer cell lines. These studies identify TAP-1 as a critical modulator of Ras-mediated cellular signaling.     

Erbin inhibits RAF activation by disrupting the sur-8-Ras-Raf complex

Erbin is a member of the LAP (leucine-rich repeat (LRR) and PDZ domain) family. It inhibits Ras-mediated activation of ERK in response to growth factors. In this study, we investigated the mechanisms by which Erbin regulates the Ras-Raf-MEK pathway. The N-terminal LRR domain was necessary and sufficient to inhibit neuregulin-activated expression of epsilon416-Luc, a reporter of ERK activation. On the other hand, Erbin had no effect on Ras activation, but it attenuated neuregulin-induced Raf activation, suggesting that Erbin may regulate Raf activation by Ras. Via the LRR domain, Erbin interacts with Sur-8, a scaffold protein necessary for the Ras-Raf complex. Expression of Erbin attenuated the interaction of Sur-8 with active Ras and Raf. Moreover, Erbin-shRNA, which suppressed Erbin expression at mRNA and protein levels, increased the interaction of Sur-8 with Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous acetylcholine receptor epsilon-subunit mRNA. These results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras/Raf interaction.     

Mammalian Raf-1 is activated by mutations that restore Raf signaling in Drosophila.

An interaction with the Ras proto-oncogene product is a requirement for Raf-1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras-Raf interaction severely impairs the function of both mammalian (Raf-1) and Drosophila (D-Raf) Raf proteins. In D-Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras-binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf-1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras-Raf-1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf-1, allowing Raf-1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf-1, it also abolishes the ability of wild-type Raf-1 to become activated by Ras. This mutation occurs in the cysteine-rich domain (CRD) of Raf-1 and demonstrates the importance of this region for a productive Ras-Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf-1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf-1 kinase domain.     

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

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.     

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras–Raf and PI3K–Akt signaling pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras–Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K–Akt signaling pathways.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

DA-Raf1, a competent intrinsic dominant-negative antagonist of the Ras-ERK pathway, is required for myogenic differentiation

Ras activates Raf, leading to the extracellular-regulated kinase (ERK)-mitogen-activated protein kinase pathway, which is involved in a variety of cellular, physiological, and pathological responses. Thus, regulators of this Ras-Raf interaction play crucial roles in these responses. In this study, we report a novel regulator of the Ras-Raf interaction named DA-Raf1. DA-Raf1 is a splicing isoform of A-Raf with a wider tissue distribution than A-Raf. It contains the Ras-binding domain but lacks the kinase domain, which is responsible for activation of the ERK pathway. As inferred from its structure, DA-Raf1 bound to activated Ras as well as M-Ras and interfered with the ERK pathway. The Ras-ERK pathway is essential for the negative regulation of myogenic differentiation induced by growth factors. DA-Raf1 served as a positive regulator of myogenic differentiation by inducing cell cycle arrest, the expression of myogenin and other muscle-specific proteins, and myotube formation. These results imply that DA-Raf1 is the first identified competent, intrinsic, dominant-negative antagonist of the Ras-ERK pathway.     

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.     

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 p21^Cip1. 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 p21^Cip1 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.     

Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

Background

Previously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.

Methods

Retroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.

Results

We have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.

Conclusions

Presented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.     

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.