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Mutation-deletion analysis of a Ca(2+)-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras.

ABSTRACT: p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.

SUBMITTER: Gawler DJ

PROVIDER: S-EPMC1136674 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila.

Project description:Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region was capable of binding RasGAP only when the tyrosine residue was phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP was a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevented tyrosine phosphorylation of RasGAP. RasGAP attenuated FGFR signalling in vivo and this ability was dependent on both its SH2 domains and its GAP activity. On the basis of these results, we propose that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway.

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Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein.

Project description:Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.

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Project description: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.

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Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers.

Project description:Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPase-activating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a Ca(2+)-regulated Ras GAP that decodes the frequency of Ca(2+) oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of Ca(2+) in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis.

| S-EPMC1941473 | biostudies-literature

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.

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A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

Project description:We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

| S-EPMC359218 | biostudies-other

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.

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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

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

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

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