Molecular mechanisms involved in Ras inactivation: the annexin A6-p120GAP complex

In mammalian cells, a complex network of signaling pathways tightly regulates a variety of cellular processes, such as proliferation and differentiation. New insights from one of the most-important signaling cascades involved in oncogenesis, the Ras-Raf-MAPK pathway, suggest that the subcellular localisation and assembly of signaling modules of this pathway is crucial to control the biological response. This commonly requires membrane targeting events that are mediated by adaptor/scaffold proteins. Of particular interest is the translocation and complex formation of GTPase-activating proteins (GAPs), such as p120GAP, at the plasma membrane to inactivate Ras. Recent studies indicate that one member of the annexin family, annexin A6 acts as a targeting protein for p120GAP. This review discusses how annexin A6 modulates the involvement of negative regulators of the Ras-Raf-MAPK pathway contributing to Ras inactivation.     

Germline Mutations in Components of the Ras Signaling Pathway in Noonan Syndrome and Related Disorders

Ras proteins control a variety of critical cellular processes, and somatic mutations in RAS genes (and other members of signaling networks regulated by Ras) are common in human malignancies. Ras proteins are guanosine triphosphate (GTP)-binding proteins that cycle between active GTP-bound and inactive guanosine diphosphate (GDP) bound conformations. Cancer-associated Ras mutations typically alter amino acids G12, G13 or Q61. These mutant Ras proteins display impaired GTPase activity and are resistant to GTPase activating proteins (GAPs). We and others recently discovered novel germline KRAS mutations in individuals diagnosed with Noonan or cardio-facio-cutanous (CFC) syndrome, two clinically overlapping disorders characterized by short stature, distinct facial anomalies, heart defects, and other developmental abnormalities. We found that the mutant K-Ras proteins encoded by NS-associated alleles have less pronounced biochemical defects than known Ras oncoproteins, which likely explains why these mutations are tolerated in the germline. Together with the recent findings of mutations in other members of the Ras signaling cascade in CFC syndrome and in Costello syndrome, another clinically related disorder, it is now clear that Noonan-like features are common phenotypic consequences of systemic deregulation of the Ras pathway. The discovery of germline mutations in this group of related genetic disorders underscores the pivotal role of the degree and duration of Ras activation in cell fate decisions during embryonic development and morphogenesis.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.     

Requirement for Plk2 in orchestrated ras and rap signaling, homeostatic structural plasticity, and memory

Ras and Rap small GTPases are important for synaptic plasticity and memory. However, their roles in homeostatic plasticity are unknown. Here, we report that polo-like kinase 2 (Plk2), a homeostatic suppressor of overexcitation, governs the activity of Ras and Rap via coordination of their regulatory proteins. Plk2 directs elimination of Ras activator RasGRF1 and Rap inhibitor SPAR via phosphorylation-dependent ubiquitin-proteasome degradation. Conversely, Plk2 phosphorylation stimulates Ras inhibitor SynGAP and Rap activator PDZGEF1. These Ras/Rap regulators perform complementary functions to downregulate dendritic spines and AMPA receptors following elevated activity, and their collective regulation by Plk2 profoundly stimulates Rap and suppresses Ras. Furthermore, perturbation of Plk2 disrupts Ras and Rap signaling, prevents homeostatic shrinkage and loss of dendritic spines, and impairs proper memory formation. Our study demonstrates a critical role of Plk2 in the synchronized tuning of Ras and Rap and underscores the functional importance of this regulation in homeostatic synaptic plasticity.     

Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.     

Choose your own path: specificity in Ras GTPase signaling

The Ras superfamily of small G proteins contributes importantly to numerous cellular and physiological processes (M. F. Olsen and R. Marais, Semin. Immunol., 2000, 12, 63). This family comprises a large class of proteins (more than 150) which all share a common enzymatic function: hydrolysis of the gamma-phosphate of guanosine triphosphate (GTP) to create the products guanosine diphosphate (GDP) and inorganic phosphate (Y. Takai, T. Sasaki and T. Matozaki, Physiol. Rev., 2001, 81, 153). For this reason Ras family proteins, which include the Ras, Rho, Arf/Sara, Ran and Rab subfamilies, are classified as GTPases (G. W. Reuther and C. J. Der, Curr. Opin. Cell Biol., 2000, 12, 157). Guanine nucleotide coupling is a key regulator of enzymatic function; thus, Ras family GTPases participate in signal transduction. Ras signaling depends on binding to effectors. Many of the known effectors can bind to multiple Ras isotypes, often leading to common cellular outcomes, but each Ras isotype also engages specific effector pathways to mediate unique functions. Further, each Ras isotype can propagate multiple signaling pathways, indicating the presence of cellular determinants which allow for promiscuity in Ras-effector interactions while also maintaining specificity. Small distinctions in sequence, structure, and/or cellular regulation contribute to these differences in Ras-effector binding and subsequent cellular effects. A major focus of investigation in the Ras signaling field is identifying the determinants of these individualized functions. This review will attempt to summarize the current state of understanding of this question (with a particular focus on the Ras subfamily) and the approaches being taken to address it, and will discuss prospective areas for future investigation.     

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.     

Divergent Functions Through Alternative Splicing: The Drosophila CRMP Gene in Pyrimidine Metabolism, Brain, and Behavior

DHP and CRMP proteins comprise a family of structurally similar proteins that perform divergent functions, DHP in pyrimidine catabolism in most organisms and CRMP in neuronal dynamics in animals. In vertebrates, one DHP and five CRMP proteins are products of six genes; however, Drosophila melanogaster has a single CRMP gene that encodes one DHP and one CRMP protein through tissue-specific, alternative splicing of a pair of paralogous exons. The proteins derived from the fly gene are identical over 90% of their lengths, suggesting that unique, novel functions of these proteins derive from the segment corresponding to the paralogous exons. Functional homologies of the Drosophila and mammalian CRMP proteins are revealed by several types of evidence. Loss-of-function CRMP mutation modifies both Ras and Rac misexpression phenotypes during fly eye development in a manner that is consistent with the roles of CRMP in Ras and Rac signaling pathways in mammalian neurons. In both mice and flies, CRMP mutation impairs learning and memory. CRMP mutant flies are defective in circadian activity rhythm. Thus, DHP and CRMP proteins are derived by different processes in flies (tissue-specific, alternative splicing of paralogous exons of a single gene) and vertebrates (tissue-specific expression of different genes), indicating that diverse genetic mechanisms have mediated the evolution of this protein family in animals.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

RAC/ROP GTPases and auxin signaling

Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin-RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch.     

Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling

The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.     

Isolation of a novel ras gene from Trichomonas vaginalis: a possible evolutionary ancestor of the Ras and Rap genes of higher eukaryotes.

The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by which such family members have evolved their specific functions. A cDNA clone of TvRsp was isolated and sequenced from a cDNA expression library of the primitive eukaryote Trichomonas vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified by PCR and sequenced. Sequence analysis suggested that TvRsp was an intronless gene. This gene encoded a protein of 181 amino acids and contained the 5 conserved G domains that designated it as a Ras or Rap subfamily member. However, the deduced amino acid sequence shared only 34%-37% overall identity with other Ras subfamily members of different species, and the presence of motifs characteristic of both the Ras and Rap families of GTPase confused the familial classification of this gene. Phylogenetic analysis showed its origins at the divergence point of the Ras/Rap families and suggested that TvRsp was a possible evolutionary ancestral gene of the ras/rap genes of higher eukaryotes. This information was of importance not only from the perspective of understanding the evolution and diversity of eukaryotic signal transduction pathways but also in providing a framework by which to understand protein processing in the growth and differentiation of single-celled microorganisms.     

Eph receptor-ephrin bidirectional signals that target Ras and Rho proteins

The ability of cells to respond to their surrounding environment and relay signals to the cell interior is essential for numerous processes during the development and maintenance of tissues. Eph receptors and their membrane-bound ligands, the ephrins, are unique in the receptor tyrosine kinase family in that their signaling is bidirectional, through both the receptor and the ligand. Eph receptors and ephrins are essential for a variety of biological processes, and play a particularly important role in regulating cell shape and cell movement. Recent data have linked Eph receptor-ephrin signaling complexes to the Ras and Rho families of small molecular weight GTPases and also to heterotrimeric G proteins. Understanding the signaling networks involved is an important step to understand the molecular basis for normal and defective cell-cell communication through Eph receptors and ephrins.     

Rap G protein signal in normal and disordered lymphohematopoiesis

Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy.     

Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications

Epac proteins respond to the second messenger cyclic AMP (cAMP) and are activated by Gs coupled receptors. They act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. A plethora of studies using 8-pCPT-2′-O-Me-cAMP, an Epac agonist, has revealed the importance of these multi-domain proteins in the control of key cellular functions such as cell division, migration, growth and secretion. Epac and protein kinase A (PKA) may act independently but are often associated with the same biological process, in which they fulfill either synergistic or opposite effects. In addition, compelling evidence is now accumulating about the formation of molecular complexes in distinct cellular compartments that influence Epac signaling and cellular function. Epac is spatially and temporally regulated by scaffold protein and its effectors are interconnected with other signaling pathways. Pathophysiological changes in Epac signaling may underlie certain diseases.     

Signaling interplay in Ras superfamily function

Ras proteins function as signaling hubs that are activated by convergent signaling pathways initiated by extracellular stimuli. Activated Ras in turn regulates a diversity of downstream cytoplasmic signaling cascades. Ras proteins are founding members of a large superfamily of small GTPases that have significant sequence and biochemical similarities. Recent observations have established a complex signaling interplay between Ras and other members of the family. A key biochemical mechanism facilitating this crosstalk involves guanine nucleotide exchange factors (GEFs), which serve as regulators and effectors, as well as signaling integrators, of Ras signaling.     

Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1.

Activation of growth factor receptors by ligand binding initiates a cascade of events leading to cell growth and division. Progression through the cell cycle is controlled by cyclin-dependent protein kinases (Cdks), but the mechanisms that link growth factor signaling to the cell cycle machinery have not been established. We report here that Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade.     

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.