Cation diffusion facilitator proteins modulate Raf-1 activity

The Ras-extracellular signal-regulated kinase (ERK) cascade is a critical intracellular signaling pathway that regulates growth, survival, and differentiation. Previous work established that Ras-GTP binds to, and facilitates the activation of, the protein kinase Raf-1. Recently, it was demonstrated that the cation diffusion facilitator (CDF) proteins are involved in Ras-ERK signaling by use of a Caenorhabditis elegans genetic screen that identified suppressors of activated Ras. In the current work, we demonstrate that CDF proteins may function downstream of Ras, but upstream of Raf-1 in Xenopus oocytes. We also show that the C. elegans protein CDF-1 and its mammalian homologue ZnT-1 bind to the amino-terminal regulatory portion of Raf-1 and promote the biological and enzymatic activity of Raf-1. Furthermore, we show that Zn(2+) inhibits Raf-1 binding to ZnT-1. We propose a model in which CDF protein binding facilitates Raf-1 activation.     

Fine-tuning the RAS signaling pathway: Zn(2+) makes the difference

In the May, 2002 issue of Developmental Cell, Bruinsma et al. report that the CDF-1 cation diffusion facilitator protein is required for efficient Ras-mediated signaling in C. elegans. CDF-1 reduces intracellular Zn(2+) levels, indicating an inhibitory effect of Zn(2+) on the Ras pathway.     

Modulation of KSR activity in Caenorhabditis elegans by Zn ions, PAR-1 kinase and PP2A phosphatase

Vulval differentiation in Caenorhabditis elegans is controlled by a conserved signal transduction pathway mediated by Ras and a kinase cascade that includes Raf, Mek and MAPK. Activation of this cascade is positively regulated by a number of proteins such as KSR (kinase suppressor of Ras), SUR-8/SOC-2, SUR-6/PP2A-B and CDF-1. We describe the functional characterization of sur-7 and several genes that regulate signaling downstream of ras. We identified sur-7 by isolating a mutation that suppresses an activated ras allele, and showed that SUR-7 is a divergent member of the cation diffusion facilitator family of heavy metal ion transporters that is probably localized to the endoplosmic recticulum membrane and regulates cellular Zn(2+) concentrations. Genetic double mutant analyses suggest that the SUR-7-mediated effect is not a general toxic response. Instead, Zn(2+) ions target a specific step of the pathway, probably regulation of the scaffolding protein KSR. Biochemical analysis in mammalian cells indicates that high Zn(2+) concentration causes a dramatic increase of KSR phosphorylation. Genetic analysis also indicates that PP2A phosphatase and PAR-1 kinase act downstream of Raf to positively and negatively regulate KSR activity, respectively.     

Rap1 signalling: adhering to new models

Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.     

C. elegans ksr-1 and ksr-2 have both unique and redundant functions and are required for MPK-1 ERK phosphorylation

Kinase Suppressor of Ras (KSR) is a conserved protein that positively regulates Ras signaling and may function as a scaffold for Raf, MEK, and ERK. However, the precise role of KSR is not well understood, and some observations have suggested that KSR might act in a parallel pathway. In C. elegans, ksr-1 is only required for a specific Ras-mediated process (sex myoblast migration) and is a nonessential positive regulator of other Ras-mediated developmental events. We report the existence of a second C. elegans ksr gene, ksr-2, which is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with ksr-1 during development of the excretory system, hermaphrodite vulva, and male spicules. Thus, while the ksr-1 and ksr-2 genes are individually required only for specific Ras-dependent processes, together these two genes appear necessary for most aspects of Ras-mediated signaling in C. elegans. The finding that ksr-2; ksr-1 double mutants have strong ras-like phenotypes and severely reduced or absent levels of diphosphorylated MPK-1 ERK strongly supports models where KSR acts to promote the activation or maintenance of the Raf/MEK/ERK kinase cascade.     

Spatial Segregation of Ras Signaling—New Evidence from Fission Yeast

The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question—how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras.     

Bcl-2, via its BH4 domain,blocks apoptotic signaling mediated by mitochondrial Ras

Bcl-2 protects cells against Ras-mediated apoptosis; this protection coincides with its binding to Ras. However, the protection mechanism has remained enigmatic. Here, we demonstrate that, upon apoptotic stimulation, newly synthesized Bcl-2 redistributes to mitochondria, interacts there with activated Ras, and blocks Ras-mediated apoptotic signaling. We also show, by employing bcl-2 mutants, that the BH4 domain of Bcl-2 binds to Ras and regulates its anti-apoptotic activity. Experiments with a C-terminal-truncated Ras or a farnesyltransferase inhibitor demonstrate that the CAAX motif of Ras is essential for apoptotic signaling and Bcl-2 association. The results indicate a potential mechanism by which Bcl-2 protects cells against Ras-mediated apoptotic signaling.     

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.     

C. elegans SUR-6/PR55 cooperates with LET-92/protein phosphatase 2A and promotes Raf activity independently of inhibitory Akt phosphorylation sites.

Protein phosphatase 2A (PP2A) can both positively and negatively influence the Ras/Raf/MEK/ERK signaling pathway, but its relevant substrates are largely unknown. In C. elegans, the PR55/B regulatory subunit of PP2A, which is encoded by sur-6, positively regulates Ras-mediated vulval induction and acts at a step between Ras and Raf. We show that the catalytic subunit (C) of PP2A, which is encoded by let-92, also positively regulates vulval induction. Therefore SUR-6/PR55 and LET-92/PP2A-C probably act together to dephosphorylate a Ras pathway substrate. PP2A has been proposed to activate the Raf kinase by removing inhibitory phosphates from Ser259 from Raf-1 or from equivalent Akt phosphorylation sites in other Raf family members. However, we find that mutant forms of C. elegans LIN-45 RAF that lack these sites still require sur-6. Therefore, SUR-6 must influence Raf activity via a different mechanism. SUR-6 and KSR (kinase suppressor of Ras) function at a similar step in Raf activation but our genetic analysis suggests that KSR activity is intact in sur-6 mutants. We identify the kinase PAR-1 as a negative regulator of vulval induction and show that it acts in opposition to SUR-6 and KSR-1. In addition to their roles in Ras signaling, SUR-6/PR55 and LET-92/PP2A-C cooperate to control mitotic progression during early embryogenesis.     

Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways

The small G-protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient-dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct-Rac1), a member of the Rho family of G proteins. Ct-Rac1 expression is downregulated by DARas under limiting nutrition. Co-expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild-type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac-mediated MAPK activation is responsible for reversion to the wild-type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH-DA fluorescence. Co-expression with DNRac decreases ROS generation to wild-type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild-type phenotype. These findings suggest that Ras-mediated ROS generation is dependent on a Rac-cPLA(2)-linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.     

Ras proteins control mitochondrial biogenesis and function in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The evolutionarily conserved Ras proteins function as a point of convergence for different signaling pathways in eukaryotes and have been implicated in both aging and cancer development. In Saccharomyces cerevisiae the plasma membrane proteins Ras1 and Ras2 are sensing the nutritional status of the environments, e.g., the abundance and quality of available carbon sources. The cAMP-protein kinase A pathway is the most explored signaling pathway controlled by Ras proteins; it affects a large number of genes, some of which are important to defend the cell against oxidative stress. In addition, recent analysis has shown that the Ras system of yeast is involved in the development of mitochondria and in regulating their activity. As a sensor of environmental status and an effector of mitochondrial activity, Ras serves as a Rosetta stone of cellular energy transduction. This review summarizes the physical and functional involvement of Ras proteins and Ras-dependent signaling pathways in mitochondrial function in S. cerevisiae. Since mitochondria produce harmful reactive oxygen species as an inevitable byproduct and are partly under control of Ras, illuminating these regulatory interactions may improve our understanding of both cancer and aging.     

An evolutionarily conserved Rit GTPase-p38 MAPK signaling pathway mediates oxidative stress resistance

Ras-related small GTP-binding proteins control a wide range of cellular processes by regulating a variety of effector pathways, including prominent roles in the control of mitogen-activated protein kinase (MAPK) cascades. Although the regulatory role(s) for many Ras family GTPases are well established, the physiological function for the Rit/Rin subfamily has been lacking. Here, using both knockout mice and Drosophila models, we demonstrate an evolutionarily conserved role for Rit subfamily GTPases (mammalian Rit and Rin, and the Drosophila RIC homologue) in governing survival in response to oxidative stress. Primary embryonic fibroblasts derived from Rit knockout mice display increased apoptosis and selective disruption of MAPK signaling following reactive oxygen species (ROS) exposure but not in response to endoplasmic reticulum stress or DNA damage. These deficits include a reduction in ROS-mediated stimulation of a p38-MK2-HSP27 signaling cascade that controls Akt activation, directing Bad phosphorylation to promote cell survival. Furthermore, D-RIC null flies display increased susceptibility to environmental stresses and reduced stress-dependent p38 signaling, extending the Rit-p38 survival pathway to Drosophila. Together, our studies establish the Rit GTPases as critical regulators of an evolutionarily conserved, p38 MAPK-dependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.     

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.     

Novel CIPK1-associated proteins in Arabidopsis contain an evolutionarily conserved C-terminal region that mediates nuclear localization

Environmental stimuli, including light, pathogens, hormones, and abiotic stresses, elicit changes in the cytosolic Ca(2+) signatures of plant cells. However, little is known about the molecular mechanisms by which plants sense and transmit the specific cytoplasmic Ca(2+) signal into the nucleus, where gene regulation occurs to respond appropriately to the stress. In this study, we have identified two novel Arabidopsis (Arabidopsis thaliana) proteins specifically associated with Calcineurin B-Like-Interacting Protein Kinase1 (CIPK1), a member of Ser/Thr protein kinases that interact with the calcineurin B-like Ca(2+)-binding proteins. These two proteins contain a very similar C-terminal region (180 amino acids in length, 81% similarity), which is required and sufficient for both interaction with CIPK1 and translocation to the nucleus. Interestingly, the conserved C-terminal region was also found in many proteins from various eukaryotic organisms, including humans. However, none of them have been characterized so far. Taken together, these findings suggest that the two proteins containing the evolutionarily conserved C-terminal region (ECT1 and ECT2) may play a critical role in relaying the cytosolic Ca(2+) signals to the nucleus, thereby regulating gene expression.     

N-type Ca2+ channels as scaffold proteins in the assembly of signaling molecules for GABAB receptor effects

An emerging concept in signal transduction is the organization of neuronal receptors and channels into microdomains in which signaling proteins are brought together to regulate functional responses. With the multiplicity of potential protein-protein interactions arises the need for the regulation and timing of these interactions. We have identified N-type Ca(2+) channel-signaling molecule complexes formed at different times upon activation of gamma-aminobutyric acid, type B, receptors. The first type of interaction involves pre-association of signaling proteins such as Src kinase with the Ca(2+) channel, because it is rapidly activated by the receptors and regulates the magnitude of the inhibition of the Ca(2+) channel. The second type of interaction involves signaling molecules that are recruited to the channel by receptor activation and control the rate of the channel response. Recruitment of members of the Ras pathway has two effects as follows: 1) modulation of the rate of onset of the gamma-aminobutyric acid-mediated inhibition of Ca(2+) current, and 2) activation of MAP kinase. Our results suggest that the Ca(2+) channel alpha(1) subunit functions as a dynamic scaffold allowing assembly of intracellular signaling components that alter channel activity and route signals to the MAP kinase pathway.     

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.     

Annexin A6-Linking Ca(2+) signaling with cholesterol transport

Annexin A6 (AnxA6) belongs to a conserved family of Ca(2+)-dependent membrane-binding proteins. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in cellular membranes in a dynamic and reversible fashion, in particular during the regulation of endocytic and exocytic pathways. High amounts of AnxA6 sequester cholesterol in late endosomes, thereby lowering the levels of cholesterol in the Golgi and the plasma membrane. These AnxA6-dependent redistributions of cellular cholesterol pools give rise to reduced cytoplasmic phospholipase A2 (cPLA(2)) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. In addition to regulating cholesterol and caveolin distribution, AnxA6 acts as a scaffold/targeting protein for several signaling proteins, the best characterized being the Ca(2+)-dependent membrane targeting of p120GAP to downregulate Ras activity. AnxA6 also stimulates the Ca(2+)-inducible involvement of PKC in the regulation of HRas and possibly EGFR signal transduction pathways. The ability of AnxA6 to recruit regulators of the EGFR/Ras pathway is likely potentiated by AnxA6-induced actin remodeling. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to modulate intracellular cholesterol homeostasis, (ii) to create a scaffold for the formation of multifactorial signaling complexes, and (iii) to regulate transient membrane-actin interactions during endocytic and exocytic transport. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.