RIG1 suppresses Ras activation and induces cellular apoptosis at the Golgi apparatus

Retinoid-inducible gene 1 encodes RIG1 is a growth regulator, which inhibits the pathways of the RAS/mitogen-activated protein kinases by suppressing the activation of RAS. Confocal microscopic analysis demonstrated that RIG1 is localized in the endoplasmic reticulum (ER) and Golgi apparatus in HtTA cervical cancer cells. Carboxyterminal-deleted RIG1 targeted to the Golgi or ER was constructed and validated. The activation of HRAS was inhibited by 25.1% or 81.4% in cells cotransfected with wild-type or Golgi-targeted RIG1, respectively. Expression of wild-type or Golgi-targeted RIG1 for 24 h induced cellular apoptosis in HtTA cells, as assessed by MTT assay, the release of lactate dehydrogenase, and chromatin condensation. In contrast, ER-targeted RIG1 and carboxyterminal-deleted RIG1 (RIG1DeltaC) exhibited no activity. Caspase-2, -3, and -9 were activated following the expression of wild-type and Golgi-targeted RIG1. Although the caspase-3 inhibitor Z-DEVD-FMK partially or completely reversed the cell death induced by wild-type or Golgi-targeted RIG1, it did not prevent the anti-RAS effect of RIG1. In conclusion, the proapoptotic and anti-RAS activities of RIG1 are primarily associated with the Golgi localization of the protein. The proapoptotic activities of RIG1 are mediated through the activation of caspase-2 and -3 and are independent of its effect on RAS.     

Exchange factors of the RasGRP family mediate Ras activation in the Golgi

H-Ras and N-Ras become activated both at the plasma membrane and in endomembrane structures such as the Golgi apparatus. This compartmentalized activation is relevant from a signaling standpoint, because effector molecules can become activated differently depending on the region of the cell where Ras proteins are activated. An unsolved question in this new regulatory mechanism is the understanding of how Ras proteins become activated in endomembranes. To approach this problem, we have studied the subcellular distribution and activities of a number of Ras guanosine nucleotide exchange factors. Our results indicate that Ras activation at the plasma membrane and endoplasmic reticulum is an unspecific process that can be achieved by most Ras activators. In contrast, GTP loading of Ras at the Golgi is only induced by members of the Ras guanosine nucleotide releasing protein family. In agreement with these observations, Ras guanosine nucleotide releasing proteins are the only Ras activators showing localization in the Golgi. These results indicate that the compartmentalized activation of effector pathways by Ras proteins depends not only on the specific localization of the GTPases but also in the availability of GDP/GTP exchange factors capable of activating Ras proteins in specific subcellular compartments.     

Lipid-transfer proteins in membrane trafficking at the Golgi complex

The Golgi complex (GC) represents the central junction for membrane trafficking. Protein and lipid cargoes continuously move through the GC in both anterograde and retrograde directions, departing to and arriving from diverse destinations within the cell. Nevertheless, the GC is able to maintain its identity and strict compartmentalisation, having a different composition in terms of protein and lipid content compared to other organelles. The discovery of coat protein complexes and the elucidation of their role in sorting cargo proteins into specific transport carriers have provided a partial answer to this phenomenon. However, it is more difficult to understand how relatively small and diffusible molecules like lipids can be concentrated in or excluded from specific subcellular compartments. The discovery of lipid-transfer proteins operating in the secretory pathway and specifically at the GC has shed light on one possible way in which this lipid compartmentalisation can be accomplished. The correct lipid distribution along the secretory pathway is of crucial importance for cargo protein sorting and secretion. This review focuses on what is now known about the putative and effective lipid-transfer proteins at the GC, and on how they affect the function and structure of the GC itself.     

Protein-lipid interactions in membrane trafficking at the Golgi complex

The integrated interplay between proteins and lipids drives many key cellular processes, such as signal transduction, cytoskeleton remodelling and membrane trafficking. The last of these, membrane trafficking, has the Golgi complex as its central station. Not only does this organelle orchestrates the biosynthesis, transport and intracellular distribution of many proteins and lipids, but also its own function and structure is dictated by intimate functional and physical relationships between protein-based and lipid-based machineries. These machineries are involved in the control of the fundamental events that govern membrane traffic, such as in the budding, fission and fusion of transport intermediates, in the regulation of the shape and geometry of the Golgi membranes themselves, and, finally, in the generation of "signals" that can have local actions in the secretory system, or that may affect other cellular systems. Lipid-protein interactions rely on the abilities of certain protein domains to recognize specific lipids. These interactions are mediated, in particular, through the headgroups of the phospholipids, although a few of these protein domains are able to specifically interact with the phospholipid acyl chains. Recent evidence also indicates that some proteins and/or protein domains are more sensitive to the physical environment of the membrane bilayer (such as its curvature) than to its chemical composition.     

A feedback loop in the polo-like kinase activation pathway

The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.     

Functional interactions of Raf and MEK with Jun-N-terminal kinase (JNK) result in a positive feedback loop on the oncogenic Ras signaling pathway

In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.     

The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation

Ras plays an important role in a variety of cellular functions, including growth, differentiation, and oncogenic transformation. For instance, Ras participates in the activation of Raf, which phosphorylates and activates mitogen-activated protein kinase kinase (MEK), which then phosphorylates and activates extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase. Activation of MAP kinase appears to be essential for propagating a wide variety of extracellular signals from the plasma membrane to the nucleus. N17Ras, a GDP-bound dominant negative mutant, is used widely as an interfering mutant to assess Ras function in vivo. Surprisingly, we observed that expression of N17Ras inhibited the activity and phosphorylation of Elk-1, a physiological substrate of MAP kinases, in response to phorbol myristate acetate. The activity and phosphorylation of the MAP kinase hemagglutinin epitope (HA)-ERK1 were not affected by N17Ras in response to the same stimulus. Additionally, expression of N17Ras, but not L61S186Ras, a GTP-bound interfering mutant, inhibited MEK-induced Elk-1 phosphorylation, suggesting that inhibition of Elk-1 may be unique to GDP-bound Ras mutants. Finally, we observed that V12Ras-induced focus formation in NIH3T3 cells is inhibited by coexpression of GDP-bound Ras mutants, such as N17, A15, and N17N69. Therefore, N17Ras and V12 Ras may be codominant with respect to Elk-1 activation and cellular transformation. These results indicate that N17Ras appears to have at least two distinguishable functions: interference with endogenous Ras activation and inhibition of Elk-1 and transfomation. Furthermore, our data imply the possibility that GDP-bound Ras, like N17Ras, may have a direct role in signal transduction.     

ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.     

Ras triggers ataxia-telangiectasia-mutated and Rad-3-related activation and apoptosis through sustained mitogenic signaling

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.     

Ras signalling on the endoplasmic reticulum and the Golgi

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.     

Non-hypoxic activation of the negative regulatory feedback loop of prolyl-hydroxylase oxygen sensors

Hypoxia inducible factors (HIF) coordinate cellular responses towards hypoxia. HIFs are mainly regulated by a group of prolyl-hydroxylases (PHDs) that in the presence of oxygen, target the HIFα subunit for degradation. Herein, we studied the role of nitric oxide (NO) in regulating PHD activities under normoxic conditions. In the present study we show that different NO-donors initially inhibited endogenous PHD2 activity which led to accumulation of HIF-1α subsequently to enhance HIF-1 dependent increased PHD2 promoter activity. Consequently PHD2 abundance and activity were strongly induced which caused downregulation of HIF-1α. Interestingly, upregulation of endogenous PHD2 activity by NO was not found in cells that lack an intact pVHL dependent degradation pathway. Recovery of PHD activity required intact cells and was not observed in cell extracts or recombinant PHD2. In conclusion induction of endogenous PHD2 activity by NO is dependent on a feedback loop initiated despite normoxic conditions.     

The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors

Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.     

Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT

Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of cellular responses including proliferation, differentiation, and apoptosis. We have developed an expression screening method to detect in vivo substrates of MAPKs in mammalian cells, and identified a membrane protein, linker for activation of T cells (LAT), as an MAPK target. LAT, an adapter protein essential for T-cell signaling, is phosphorylated at its Thr 155 by ERK in response to T-cell receptor stimulation. Thr 155 phosphorylation reduces the ability of LAT to recruit PLCgamma1 and SLP76, leading to attenuation of subsequent downstream events such as [Ca2+]i mobilization and activation of the ERK pathway. Our data reveal a new role for MAPKs in a negative feedback loop in T-cell activation via threonine phosphorylation of LAT.     

Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.     

The activation of RalGDS can be achieved independently of its Ras binding domain. Implications of an activation mechanism in Ras effector specificity and signal distribution.

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras x RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.     

Different ubiquitin signals act at the Golgi and plasma membrane to direct GAP1 trafficking

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.     

Regulation of Golgi signaling and trafficking by the KDEL receptor

Intracellular membrane transport involves the well-coordinated engagement of a series of organelles and molecular machineries that ensure that proteins are delivered to their correct cellular locations according to their function. To maintain the homeostasis of the secretory system, the fluxes of membranes and protein across the transport compartments must be precisely balanced. This control should rely on a mechanism that senses the movement of the traffic and generates the required homeostatic response. Due to its central position in the secretory pathway and to the large amounts of signaling molecules associated with it, the Golgi complex represents the ideal candidate for this regulation. The generation of autonomous signaling by the Golgi complex in response to the arrival of cargo from the endoplasmic reticulum (ER) has been experimentally addressed only in recent years. These studies have revealed that cargo moving from the ER to the Golgi activates a series of signaling pathways, the functional significance of which appears to be to maintain the homeostasis of the Golgi complex and to activate Golgi trafficking according to internal demand. We have termed this regulatory mechanism the Golgi control system. A key player in this Golgi control system is the KDEL receptor, which has previously been shown to retrieve chaperones back to the endoplasmic reticulum and more recently to behave as a signaling receptor. Here, we discuss the particular role of KDEL receptor signaling in the regulation of important pathways involved in the maintenance of the homeostasis of the transport apparatus, and in particular, of the Golgi complex.     

The Rsu-1-PINCH1-ILK complex is regulated by Ras activation in tumor cells

The link between Ras transformation and enhanced cell migration due to altered integrin signaling is well established in tumorigenesis, however there remain gaps in our understanding of its mechanism. The Ras suppressor, Rsu-1, has recently been linked to the IPP (integrin-linked kinase {ILK}, PINCH-1/LIMS1, parvin) focal adhesion complex based on its interaction with the LIM 5 domain of PINCH1. Defining the role of the Rsu1-PINCH1-ILK-parvin complex in tumorigenesis is important because both ILK and PINCH1 are elevated in certain tumors while ectopic expression of Rsu-1 blocks tumorigenesis. Our studies previously identified an alternatively spliced isoform of Rsu-1 in high-grade gliomas. We report here the detection of a truncated (p29) Rsu-1 protein, which correlates with the presence of the alternatively spliced Rsu-1 RNA. This RNA and the respective protein were detected in human tumor cell lines that contain high levels of activated Ras, and inhibitor studies demonstrate that the Mek-ERK pathway regulates expression of this truncated Rsu-1 product. We also show that Rsu-1 co-localizes with ILK at focal contacts and co-immunoprecipitates with the ILK-PINCH1 complex in non-transformed cells, but following Ras transformation the association of Rsu-1 with the PINCH1-ILK complex is greatly reduced. Using a human breast cancer cell line, our in vitro studies demonstrate that the depletion of Rsu-1 full-length protein enhances cell migration coincident with an increase in Rac-GTP while the depletion of the p29 Rsu-1 truncated protein inhibits migration. These findings indicate that Rsu-1 may inhibit cell migration by stabilizing the IPP adhesion complex and that Ras activation perturbs this inhibitory function by modulating both Rsu-1 splicing and association of full-length Rsu-1 with IPP. Hence, our findings demonstrate that Rsu-1 links the Ras pathway with the IPP complex and the perturbations of cell attachment-dependent signaling that occur in the malignant process.