A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.

RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS-binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB-domain-containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB-domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 µM) and very low (500 µM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain.

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a 'scaffolding domain' by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.

Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

Selectivity Determinants of RHO GTPase Binding to IQGAPs.

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

Structural snapshots of RAF kinase interactions.

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein-protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.

Deciphering the Molecular and Functional Basis of RHOGAP Family Proteins: A SYSTEMATIC APPROACH TOWARD SELECTIVE INACTIVATION OF RHO FAMILY PROTEINS.

RHO GTPase-activating proteins (RHOGAPs) are one of the major classes of regulators of the RHO-related protein family that are crucial in many cellular processes, motility, contractility, growth, differentiation, and development. Using database searches, we extracted 66 distinct human RHOGAPs, from which 57 have a common catalytic domain capable of terminating RHO protein signaling by stimulating the slow intrinsic GTP hydrolysis (GTPase) reaction. The specificity of the majority of the members of RHOGAP family is largely uncharacterized. Here, we comprehensively investigated the sequence-structure-function relationship between RHOGAPs and RHO proteins by combining our in vitro data with in silico data. The activity of 14 representatives of the RHOGAP family toward 12 RHO family proteins was determined in real time. We identified and structurally verified hot spots in the interface between RHOGAPs and RHO proteins as critical determinants for binding and catalysis. We have found that the RHOGAP domain itself is nonselective and in some cases rather inefficient under cell-free conditions. Thus, we propose that other domains of RHOGAPs confer substrate specificity and fine-tune their catalytic efficiency in cells.

IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.

IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features.

Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis.

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2(S2G) mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.

Functional cross-talk between ras and rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger.

The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

Diverging gain-of-function mechanisms of two novel KRAS mutations associated with Noonan and cardio-facio-cutaneous syndromes.

Activating somatic and germline mutations of closely related RAS genes (H, K, N) have been found in various types of cancer and in patients with developmental disorders, respectively. The involvement of the RAS signalling pathways in developmental disorders has recently emerged as one of the most important drivers in RAS research. In the present study, we investigated the biochemical and cell biological properties of two novel missense KRAS mutations (Y71H and K147E). Both mutations affect residues that are highly conserved within the RAS family. KRAS(Y71H) showed no clear differences to KRAS(wt), except for an increased binding affinity for its major effector, the RAF1 kinase. Consistent with this finding, even though we detected similar levels of active KRAS(Y71H) when compared with wild-type protein, we observed an increased activation of MEK1/2, irrespective of the stimulation conditions. In contrast, KRAS(K147E) exhibited a tremendous increase in nucleotide dissociation generating a self-activating RAS protein that can act independently of upstream signals. As a consequence, levels of active KRAS(K147E) were strongly increased regardless of serum stimulation and similar to the oncogenic KRAS(G12V). In spite of this, KRAS(K147E) downstream signalling did not reach the level triggered by oncogenic KRAS(G12V), especially because KRAS(K147E) was downregulated by RASGAP and moreover exhibited a 2-fold lower affinity for RAF kinase. Here, our findings clearly emphasize that individual RAS mutations, despite being associated with comparable phenotypes of developmental disorders in patients, can cause remarkably diverse biochemical effects with a common outcome, namely a rather moderate gain-of-function.

Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

Functional Classification and Interaction Selectivity Landscape of the Human SH3 Domain Superfamily.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein-protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain-PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain-PRM interactions and be utilized as a general approach for other domain-peptide interactions.

SIRT4 as a novel interactor and candidate suppressor of C-RAF kinase in MAPK signaling.

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.

Interaction characteristics of Plexin-B1 with Rho family proteins.

Plexin-B1 regulates various cellular processes interacting directly with several Rho proteins. Molecular details of these interactions are, however, not well understood. In this study, we examined in vitro and in silico the interaction of the Rho binding domain (B1RBD) of human Plexin-B1 with 11 different Rho proteins. We show that B1RBD binds in a GTP-dependent manner to Rac1, Rac2, Rac3, Rnd1, Rnd2, Rnd3, and RhoD, but not to RhoA, Cdc42, RhoG, or Rif. Interestingly, Rnd1 competitively displaces the Rac1 from B1RBD but not vice versa. Structure-function analysis revealed a negatively charged loop region, called B1L(31), which may facilitate a selective B1RBD interaction with Rho proteins.

New insight into the molecular switch mechanism of human Rho family proteins: shifting a paradigm.

Major advances have been made in understanding the structure, function and regulation of the small GTP-binding proteins of the Rho family and their involvement in multiple cellular process and disorders. However, intrinsic nucleotide exchange and hydrolysis reactions, which are known to be fundamental to Rho family proteins, have been partially investigated in the case of RhoA, Rac1 and Cdc42, but for others not at all. Here we present a comprehensive and quantitative analysis of the molecular switch functions of 15 members of the Rho family that enabled us to propose an active GTP-bound state for the rather uncharacterized isoforms RhoD and Rif under equilibrium and quiescent conditions.