GTPase-activating Protein Function Research Articles

Identification of an inhibitory domain in GTPase-activating protein p190RhoGAP responsible for masking its functional GAP domain

The GTPase-activating protein (GAP) p190RhoGAP (p190A) is encoded by ARHGAP35 which is found mutated in cancers. p190A is a negative regulator of the GTPase RhoA in cells and must be targeted to RhoA-dependent actin-based structures to fulfill its roles. We previously identified a functional region of p190A called the PLS (protrusion localization sequence) required for localization of p190A to lamellipodia but also for regulating the GAP activity of p190A. Additional effects of the PLS region on p190A localization and activity need further characterization. Here, we demonstrated that the PLS is required to target p190A to invadosomes. Cellular expression of a p190A construct devoid of the PLS (p190AΔPLS) favored RhoA inactivation in a stronger manner than WT p190A, suggesting that the PLS is an autoinhibitory domain of p190A GAP activity. To decipher this mechanism, we searched for PLS-interacting proteins using a two-hybrid screen. We found that the PLS can interact with p190A itself. Coimmunoprecipitation experiments demonstrated that the PLS interacts with a region in close proximity to the GAP domain. Furthermore, we demonstrated that this interaction is abolished if the PLS harbors cancer-associated mutations: the S866F point mutation and the Δ865-870 deletion. Our results are in favor of defining PLS as an inhibitory domain responsible for masking the p190A functional GAP domain. Thus, p190A could exist in cells under two forms: an inactive closed conformation with a masked GAP domain and an open conformation allowing p190A GAP function. Altogether, our data unveil a new mechanism of p190A regulation.

Abstract P5-13-04: NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics

Abstract Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (>4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination. Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-04.

Pathophysiological Mechanisms in Neurodevelopmental Disorders Caused by Rac GTPases Dysregulation: What's behind Neuro-RACopathies.

Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1–3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

BackgroundCirculating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium‐derived prostacyclin and nitric oxide stimulate cAMP‐ and cGMP‐dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. ObjectivesWe wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide‐dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. MethodsPhosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos‐tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull‐down assays were performed to analyze protein interaction and function. ResultsOur data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA‐specific GTPase‐activating protein Myo9b and the guanine nucleotide exchange factor GEF‐H1. We identify Myo9b S1354 and guanine nucleotide exchange factor‐H1 (GEF‐H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA‐GTP levels. GEF‐H1 S886 phosphorylation stimulates binding of 14‐3‐3β and has been shown to inhibit GEF function by facilitating binding of GEF‐H1 to microtubules. Microtubule disruption increases RhoA‐GTP levels confirming the importance of GEF‐H1 in platelets. ConclusionPhosphorylation of RhoA regulatory proteins Myo9b and GEF‐H1, but not RhoA itself, is involved in cyclic nucleotide‐mediated control of RhoA in human platelets.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Allosteric regulation of a prokaryotic small Ras-like GTPase contributes to cell polarity oscillations in bacterial motility.

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.

Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the Rag GTPases

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex.

Fidelity of HIS4 start codon selection influences 3-amino-1,2,4-triazole sensitivity in GTPase activating protein function defective eIF5

The eIF5 protein plays an important role in the fidelity of AUG start codon selection. However, the hyper GTPase eIF5G31R mutation in yeast causes preferential utilization of UUG as initiation codon and is termed as suppressor of initiation codon (Sui-) phenotype. The eIF5G31R mutant recognizes upUUG initiation codon from the 5' regulatory leader region of GCN4 transcript and dominantly represses GCN4 expression thereby conferring sensitivity to 3-amino-1,2,4-triazole (3AT)-induced starvation. The 3AT sensitivity was rescued by supplementing HIS4UUG allele. The eIF5G31R mutant has a better efficiency of UUG codon recognition from the HIS4UUG allele under starvation conditions. Moreover, the expression of HIS4UUG allele was significantly lower than the critical level causing additional derepression of GCN4 expression in eIF5G31R mutant to rescue its 3AT sensitivity. The overexpression of eIF1 improved expression of HIS4AUG allele and GCN4 transcript causing 3AT resistance, whereas overexpression of eIF1 resulted in diminished UUG codon recognition of HIS4UUG allele causing 3AT sensitivity, despite having higher GCN4 expression. This paper reports the critical role of HIS4 expression necessary in response to 3AT-induced starvation in the eIF5G31R mutant which is ostensibly not a direct target of 3AT inhibition.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui–) phenotype due to its hyper GTPase activity. The eIF5G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn– phenotype) in the eIF5G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5′ regulatory region present between uORF1 and the main GCN4 ORF.

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation

ABSTRACTIn humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.

Recessive Inactivating Mutations in TBCK, Encoding a Rab GTPase-Activating Protein, Cause Severe Infantile Syndromic Encephalopathy

Infantile encephalopathies are a group of clinically and biologically heterogeneous disorders for which the genetic basis remains largely unknown. Here, we report a syndromic neonatal encephalopathy characterized by profound developmental disability, severe hypotonia, seizures, diminished respiratory drive requiring mechanical ventilation, brain atrophy, dysgenesis of the corpus callosum, cerebellar vermis hypoplasia, and facial dysmorphism. Biallelic inactivating mutations in TBCK (TBC1-domain-containing kinase) were independently identified by whole-exome sequencing as the cause of this condition in four unrelated families. Matching these families was facilitated by the sharing of phenotypic profiles and WES data in a recently released web-based tool (Geno2MP) that links phenotypic information to rare variants in families with Mendelian traits. TBCK is a putative GTPase-activating protein (GAP) for small GTPases of the Rab family and has been shown to control cell growth and proliferation, actin-cytoskeleton dynamics, and mTOR signaling. Two of the three mutations (c.376C>T [p.Arg126(∗)] and c.1363A>T [p.Lys455(∗)]) are predicted to truncate the protein, and loss of the major TBCK isoform was confirmed in primary fibroblasts from one affected individual. The third mutation, c.1532G>A (p.Arg511His), alters a conserved residue within the TBC1 domain. Structural analysis implicated Arg511 as a required residue for Rab-GAP function, and insilico homology modeling predicted impaired GAP function in the corresponding mutant. These results suggest that loss of Rab-GAP activity is the underlying mechanism of disease. In contrast to other disorders caused by dysregulated mTOR signaling associated with focal or global brain overgrowth, impaired TBCK function results in progressive loss of brain volume.

Physiology of RGS10 in Neurons and Immune Cells.

Regulator of G protein signaling-10 (RGS10), a GTPase-activating protein (GAP) for Gαi3, Gαq, and Gαz, belongs to the D/R12 subfamily based on the homology within the RGS domain and is one of the smallest RGS proteins, outside the RGS box. Although RGS10 lacks the flanking domains or motifs, RGS10 can be targeted to membrane by palmitoylation which markedly increases its net GAP activity. The cAMP-dependent protein kinase A phosphorylates RGS10 on serine 168 (Ser(168)) which promotes translocation of RGS10 from the cytosol to nucleus. In addition to its GAP function, RGS10 modulates adenylyl cyclase (AC) activity as well as the G protein-gated inwardly rectifying potassium channels. Although the roles of various RGS proteins have been well addressed in previous reviews, the function and mechanism of RGS10 in particular has not been reviewed in-depth. Moreover, recent arrays of studies implicate a role for RGS10 in immune and neuronal cells. RGS10 has been localized to various cell compartments including the cytoplasm and nucleus. In this chapter, we will review its role in neurons and immune cells.

Regulating Rac in the nervous system: molecular function and disease implication of Rac GEFs and GAPs.

Rho family GTPases, including RhoA, Rac1, and Cdc42 as the most studied members, are master regulators of actin cytoskeletal organization. Rho GTPases control various aspects of the nervous system and are associated with a number of neuropsychiatric and neurodegenerative diseases. The activity of Rho GTPases is controlled by two families of regulators, guanine nucleotide exchange factors (GEFs) as the activators and GTPase-activating proteins (GAPs) as the inhibitors. Through coordinated regulation by GEFs and GAPs, Rho GTPases act as converging signaling molecules that convey different upstream signals in the nervous system. So far, more than 70 members of either GEFs or GAPs of Rho GTPases have been identified in mammals, but only a small subset of them have well-known functions. Thus, characterization of important GEFs and GAPs in the nervous system is crucial for the understanding of spatiotemporal dynamics of Rho GTPase activity in different neuronal functions. In this review, we summarize the current understanding of GEFs and GAPs for Rac1, with emphasis on the molecular function and disease implication of these regulators in the nervous system.

Abstract IA18: Activation of RAS by post-translational modification: Ubiquitination and thiol oxidation

Abstract Ras proteins are critical regulators of multiple pathways involved in cellular growth control. Activating mutations in Ras genes are found in 33% of human cancers, with Ras well recognized as the most commonly mutated oncogene in human cancer. Mutations in Ras associated with cancer promote cellular activation by populating the active state of Ras, most frequently by impairing the action of proteins that downregulate its activity. We have investigated novel mechanisms of Ras activation by posttranslational modification. One such modification is monoubiquitination. While polyubiquitination of proteins leads to protein degradation, reversible covalent modification by monoubiquitination can lead to alterations in trafficking and subcellular localization. It has recently been shown that K-Ras monoubiquitination, upregulates its activity by populating the active GTP-bound state, enhances interactions with certain downstream effectors and promotes Ras-mediated tumorigenesis [1]. These findings represent a paradigm shift in our understanding of the role monoubiquitination may play in protein regulation as well as new mechanisms for regulation of Ras activity. To determine the mechanism by which ubiquitination activates Ras, we conducted NMR, modeling, biochemical and biological studies of monoubiquitinated H-Ras and K-Ras. We find that monoubiquitination of K- and H-Ras occurs at distinct sites, both of which can upregulate Ras activity, but by different mechanisms [2-4]. We will also present our recent work on an oncogenic variant prevalent in lung cancer, K-Ras G12C. We find that this activating mutation introduces a redox active cysteine that renders Ras G12C sensitive to thiol oxidation. We will present recent findings on how thiol oxidation at this site modulates Ras activity. 1. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors. Sasaki AT, Carracedo A, Locasale JW, Anastasiou D, Takeuchi K, Kahoud ER, Haviv S, Asara JM, Pandolfi PP, Cantley LC. Sci Signal. 2011 4(163):ra13. 2. Site-Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function. Baker R, Lewis SM, Wilkerson EM, Sasaki AT, Cantley LC, Kuhlman B, Dohlman H and Campbell, SL. Nat Struct Mol Biol. 2013 Jan;20(1):46-52. 3. Differences in the regulation of k-ras and h-ras isoforms by monoubiquitination.Baker R, Wilkerson EM, Sumita K, Isom DG, Sasaki AT, Dohlman HG, Campbell SL.J Biol Chem. 2013 Dec 27;288(52):36856-62. 4.Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Hobbs GA, Gunawardena HP, Baker R, Campbell SL. Small GTPases. 2013 Sep 12;4(3). Citation Format: Rachael Baker, Aaron Hobbs, Minh Huynh, Atsuo Sasaki, Henrik Dohlman, Sharon L. Campbell. Activation of RAS by post-translational modification: Ubiquitination and thiol oxidation. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA18. doi: 10.1158/1557-3125.RASONC14-IA18

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

Regulation of phospholipase C-β1GTPase-activating protein (GAP) function and relationship to Gqefficacy

How cells regulate Gq efficacy (initiation and termination of Gq signaling) to effect response remains a central question in pharmacology and drug discovery. Phospholipase C-β1 (PLC-β1) is an effector and a GTPase activating protein (GAP) specific to Gαq. The physiological function of PLC-β1 GAP remains unclear and controversial. GAPs are generally thought to function in deactivation of Gq signaling. However, PLC-β1 GAP has also been shown to increase signaling efficiency through kinetic coupling with the ligand-activated GPCR. GPCRs function as guanine nucleotide exchange factors (GEF) on the G protein activation cycle. This article sets forth a new hypothesis that could unify these conflicting paradigms as it pertains to physiological signaling and native levels of protein. It is proposed that the physiological function of PLC-β1 GAP is context-dependent and regulated by phosphatidic acid (PA). PA stimulates PLC-β1 GAP activity. In the absence of ligand, PLC-β1 GAP does indeed deactivate Gq signaling, limiting leaky activation to set the threshold for stimulation to sharpen signal kinetics. However in the presence of activating ligand, the increase in levels of PA would stimulate PLC-β1 GAP to kinetically couple with GPCR GEF to increase signaling efficiency. We found that PA-increased Gq efficiency is dependent on signaling via the unique PLC-β1 PA binding domain.

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

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

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

SUMMARYCell 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 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 Ras GTP binding, 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.