Specificity in Ras and Rap Signaling

Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-activating proteins, determining one level of specificity. In addition, although the effector domains are highly similar, Rap and Ras interact with largely different sets of effectors, providing a second level of specificity. In this review, we discuss the regulatory proteins and effectors of Ras and Rap, with a focus on those of Rap.Ras-like small G-proteins are ubiquitously expressed, conserved molecular switches that couple extracellular signals to various cellular responses. Different signals can activate GEFs² that induce the small G-protein to switch from the inactive, GDP-bound state to the active, GTP-bound state. This induces a conformational change that allows downstream effector proteins to bind specifically to and be activated by the GTP-bound protein to mediate diverse biological responses. Small G-proteins are returned to the GDP-bound state by hydrolyzing GTP with the help of GAPs. Ras (Ha-Ras, Ki-Ras, and N-Ras) and Rap proteins (Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C) have similar effector-binding regions that interact predominantly with RA domains or the structurally similar RBDs present in a variety of different proteins. Both protein families operate in different signaling networks. For instance, Ras is central in a network controlling cell proliferation and cell survival, whereas Rap1 predominantly controls cell adhesion, cell junction formation, cell secretion, and cell polarity. These different functions are reflected in a largely different set of GEFs and GAPs. Also the downstream effector proteins operate in a selective manner in either one of the networks.     

Biochemical clustering of monomeric GTPases of the Ras superfamily

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.     

Differential Oncogenic Ras Signaling and Senescence in Tumor Cells

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.     

Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases

Signaling proteins from the same family can have markedly different roles in a given cellular context. Here, we show that expression of one hundred constitutively active human small GTPases induced cell morphologies that fell into nine distinct classes. We developed an algorithm for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues were mostly outside known interaction surfaces and were structurally far apart from each other. Our study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants.     

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.     

Ras/Erk MAPK Signaling in Epidermal Homeostasis and Neoplasia

Epidermis provides the cutaneous barrier to the external environment and undergoes a continual process of proliferative self-renewal, with human epidermis undergoing complete turnover approximately 1,000 times in a lifetime. Recent work suggests that this ongoing proliferative replenishment of epidermal cells depends, in part, on continual signals for cell division and survival transmitted by the Ras/Erk MAPK pathway. Such constant cell proliferation, however, requires tight regulation to avoid the uncontrolled tissue expansion characteristic of epidermal neoplasia. Recent studies provide new insight into Ras/Erk MAPK pathway function in the control of normal skin development and homeostasis as well as how its deregulation promotes epidermal tumorigenesis.     

Crosstalk Between Pten and Ras Signaling Pathways in Tumor Development

The Pten and Ras pathways are disrupted or activated, respectively, in a substantial proportion of cancers. Skin tumors induced by the classical two stage carcinogenesis protocols show consistent activating mutations of the H-ras gene, but in tumors from Pten heterozygous mice, the frequency of these mutations is markedly decreased, suggesting some redundancy between these pathways. Pten heterozygous mice develop more papillomas and have earlier onset of carcinomas than their control counterparts, but molecular analysis of these tumors indicated that complete loss of Pten and activation of H-ras are mutually exclusive. Pten loss is however not functionally equivalent to H-ras activation, as Pten(-/-) tumors occur earlier and are generally more aggressive. Tumors with Pten loss or H-ras activation have different biochemical properties, suggestive of alternative routes to malignancy. These findings in this mouse model have important implications for the rational design of new targeted therapies for human tumors.     

Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity

The members of the mechanistically diverse enolase superfamily catalyze different overall reactions. Each shares a partial reaction in which an active site base abstracts the alpha-proton of the carboxylate substrate to generate an enolate anion intermediate that is stabilized by coordination to the essential Mg(2+) ion; the intermediates are then directed to different products in the different active sites. In this minireview, our current understanding of structure/function relationships in the divergent members of the superfamily is reviewed, and the use of this knowledge for our future studies is proposed.     

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.     

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.     

Ras Signaling Is Required for Serum-Induced Hyphal Differentiation in Candida albicans

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the RAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 of Saccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the only RAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1 (ras1-2/ras1-3) are viable. The Candida ras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active RAS1(V13) allele manifest enhanced hyphal growth, whereas those expressing a dominant negative RAS1(A16) allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.     

Unusual interplay of two types of Ras activators, RasGRP and SOS, establishes sensitive and robust Ras activation in lymphocytes

Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition, RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here, we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1, initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)—hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation.     

The Ras protein superfamily: evolutionary tree and role of conserved amino acids

The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.     

Relating biochemistry and function in the myosin superfamily

All characterized myosins share a common ATPase mechanism. However, detailed kinetic analyses suggest that modulation of the rate and equilibrium constants that define the ATPase cycle confers specific properties to these motor proteins, suiting them to specific physiological tasks. Understanding the kinetic mechanisms allows potential cellular functions of the different myosin classes and isoforms to be better defined.     

Structure and function in the uracil-DNA glycosylase superfamily

Deamination of cytosine to uracil is one of the major pro-mutagenic events in DNA, causing G:C-->A:T transition mutations if not repaired before replication. Repair of uracil-DNA is achieved in a base-excision pathway initiated by a uracil-DNA glycosylase (UDG) enzyme of which four families have so far been identified. Family-1 enzymes are active against uracil in ssDNA and dsDNA, and recognise uracil explicitly in an extrahelical conformation via a combination of protein and bound-water interactions. Extrahelical recognition requires an efficient process of substrate location by 'base-sampling' probably by hopping or gliding along the DNA. Family-2 enzymes are mismatch specific and explicitly recognise the widowed guanine on the complementary strand rather than the extrahelical scissile pyrimidine. This allows a broader specificity so that some Family-2 enzymes can excise uracil and 3, N(4)-ethenocytosine from mismatches with guanine. Although structures are not yet available for Family-3 (SMUG) and Family-4 enzymes, sequence analysis suggests similar overall folds, and identifies common active site motifs but with a surprising lack of conservation of catalytic residues between members of the super-family.