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RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling.

ABSTRACT: The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

SUBMITTER: Nelson AC

PROVIDER: S-EPMC7363148 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Ca2+-dependent monomer and dimer formation switches CAPRI Protein between Ras GTPase-activating protein (GAP) and RapGAP activities.

Project description:CAPRI is a member of the GAP1 family of GTPase-activating proteins (GAPs) for small G proteins. It is known to function as an amplitude sensor for intracellular Ca(2+) levels stimulated by extracellular signals and has a catalytic domain with dual RasGAP and RapGAP activities. Here, we have investigated the mechanism that switches CAPRI between its two GAP activities. We demonstrate that CAPRI forms homodimers in vitro and in vivo in a Ca(2+)-dependent manner. The site required for dimerization was pinpointed by deletion and point mutations to a helix motif that forms a hydrophobic face in the extreme C-terminal tail of the CAPRI protein. Deletion of this helix motif abolished dimer formation but did not affect translocation of CAPRI to the plasma membrane upon cell stimulation with histamine. We found that dimeric and monomeric CAPRI coexist in cells and that the ratio of dimeric to monomeric CAPRI increases upon cell stimulation with histamine. Free Ca(2+) at physiologically relevant concentrations was both necessary and sufficient for dimer formation. Importantly, the monomeric and dimeric forms of CAPRI exhibited differential GAP activities in vivo; the wild-type form of CAPRI had stronger RapGAP activity than RasGAP activity, whereas a monomeric CAPRI mutant showed stronger RasGAP than RapGAP activity. These results demonstrate that CAPRI switches between its dual GAP roles by forming monomers or homodimers through a process regulated by Ca(2+). We propose that Ca(2+)-dependent dimerization of CAPRI may serve to coordinate Ras and Rap1 signaling pathways.

| S-EPMC3103366 | biostudies-literature

Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations.

Project description:Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the ?- relative to the ?-phosphates and the further rotation of the nonbridging oxygen atoms of ?- relative to the ?- and ?-phosphates by GAP. Further, the ?-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

| S-EPMC3458370 | biostudies-literature

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes.

Project description:Rasal, belonging to the GAP1 subfamily of Ras GTPase-activating proteins (RasGAPs) with dual RasGAP/RapGAP specificity, is epigenetically silenced in several tumor types. Surprisingly, the isolated protein has GAP activity on Rap but not on Ras. Its membrane recruitment is regulated by interaction with calcium and lipids, which simultaneously induces its RasGAP activity through a yet unknown mechanism. Here we show that the interaction of Rasal with membranes induces Rasal RasGAP activity by spatial and conformational regulation, although it does not have any effect on its RapGAP activity. Not only is colocalization of Rasal and Ras in the membrane essential for RasGAP activation, but direct and Ca-dependent interaction between the tandem C2 domains of Rasal and lipids of the membrane is also required. Whereas the C2A domain binds specifically phosphatidylserine, the C2B domain interacts with several phosphoinositol lipids. Finally we show, that similar to the C2 domains of synaptotagmins, the Rasal tandem C2 domains are able to sense and induce membrane curvature by the insertion of hydrophobic loops into the membrane.

| S-EPMC3538246 | biostudies-literature

Nonredundant functions for Ras GTPase-activating proteins in tissue homeostasis.

Project description:Inactivation of the small guanosine triphosphate-binding protein Ras during receptor signal transduction is mediated by Ras guanosine triphosphatase (GTPase)-activating proteins (RasGAPs). Ten different RasGAPs have been identified and have overlapping patterns of tissue distribution. However, genetic analyses are revealing critical nonredundant functions for each RasGAP in tissue homeostasis and as regulators of disease processes in mouse and man. Here, we discuss advances in understanding the role of RasGAPs in the maintenance of tissue integrity.

| S-EPMC5483993 | biostudies-literature

Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

Project description:High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

| S-EPMC4496354 | biostudies-literature

A Ras-GTPase-activating protein SH3-domain-binding protein.

Project description:We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

| S-EPMC231246 | biostudies-other

Tumor Suppression of Ras GTPase-Activating Protein RASA5 through Antagonizing Ras Signaling Perturbation in Carcinomas.

Project description:Aberrant RAS signaling activation is common in cancers with even few Ras mutations, indicating alternative dysregulation other than genetic mutations. We identified a Ras GTPase-activating gene RASA5/SYNGAP1, at the common 6p21.3 deletion, methylated/downregulated in multiple carcinomas and different from other RASA family members (RASA1-RASA4), indicating its special functions in tumorigenesis. RASA5 mutations are rare, unlike other RASA members, whereas its promoter CpG methylation is frequent in multiple cancer cell lines and primary carcinomas and associated with patient's poor survival. RASA5 expression inhibited tumor cell migration/invasion and growth in mouse model, functioning as a tumor suppressor. RASA5 suppressed RAS signaling, depending on its Ras GTPase-activating protein catalytic activity, which could be counteracted by oncogenic HRas Q61L mutant. RASA5 knockdown enhanced Ras signaling to promote tumor cell growth. RASA5 also inhibited epithelial-mesenchymal transition (EMT) through regulating actin reorganization. Thus, epigenetic inactivation of RASA5 contributing to hyperactive RAS signaling is involved in Ras-driven human oncogenesis.

| S-EPMC6820368 | biostudies-literature

ScanITD: Detecting internal tandem duplication with robust variant allele frequency estimation.

Project description:BACKGROUND:Internal tandem duplications (ITDs) are tandem duplications within coding exons and are important prognostic markers and drug targets for acute myeloid leukemia (AML). Next-generation sequencing has enabled the discovery of ITD at single-nucleotide resolution. ITD allele frequency is used in the risk stratification of patients with AML; higher ITD allele frequency is associated with poorer clinical outcomes. However, the ITD allele frequency data are often unavailable to treating physicians and the detection of ITDs with accurate variant allele frequency (VAF) estimation remains challenging for short-read sequencing. RESULTS:Here we present the ScanITD approach, which performs a stepwise seed-and-realignment procedure for ITD detection with accurate VAF prediction. The evaluations on simulated and real data demonstrate that ScanITD outperforms 3 state-of-the-art ITD detectors, especially for VAF estimation. Importantly, ScanITD yields better accuracy than general-purpose structural variation callers for predicting ITD size range duplications. CONCLUSIONS:ScanITD enables the accurate identification of ITDs with robust VAF estimation. ScanITD is written in Python and is open-source software that is freely accessible at https://github.com/ylab-hi/ScanITD.

| S-EPMC7450668 | biostudies-literature

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

Project description:KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by which mUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodoacetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to Ub(G76C) (native linker), Ub(X77C) (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Ras modified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues.

| S-EPMC3976977 | biostudies-literature

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.

Project description:Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

| S-EPMC3537887 | biostudies-literature

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