GDP-bound Form Research Articles

Abstract 2589: Dysregulation of RalGAPA2 in hepatocellular carcinoma

Abstract RalGTPases are Ras-like G-proteins which cycle between the active GTP-bound and the inactive GDP-bound forms. Elevation of expression and activity of RalGTPase has been demonstrated to play an oncogenic role in human cancers. RalA activity could be positively regulated by RalGEF and negatively regulated by RalGAP. The functional RalGAP complex consists of either one of the catalytic subunits, RalGAPA1 or RalGAPA2, together with the regulatory subunit, RalGAPB. In this study, we aimed to investigate the potential role of RalGAPA2 dysregulation and its functional significance in hepatocellular carcinoma (HCC). By RNA-sequencing analysis in human HCC tissues and cell lines, we found that RalGAPA2 was the predominantly expressed RalGAP catalytic subunit in the liver. To examine the RalGAPA2 expression in HCC, we performed qPCR analysis in paired HCC tissues (n=90), and 37.8% of the HCC samples showed a 2-fold downregulation of RalGAPA2. Clinically, downregulation of RalGAPA2 was significantly correlated with a lower cellular differentiation (P=0.034) and a shorter overall survival rate (P=0.045). Interestingly, HCC tumors showing combined upregulation of RalA and downregulation of RalGAPA2 tended to be associated with more aggressive tumor behaviour, including higher occurrence of venous invasion (P=0.001) and more advanced TNM stage (P=0.007). On the other hand, by targeted-DNA sequencing, RalGAPA2 was identified to be recurrently mutated in 7.4% of the patients in a HBV-associated HCC cohort (n=95). Functionally, CRISPR/Cas9-mediated RalGAPA2 knockout HCC cells showed an enhanced migratory ability when compared to the control cells, underlying an upregulation of RalA activity. In HCC, expression of RalA isoform was found to be specifically upregulated and functionally supporting HCC metastasis. Also, inhibition of RalGAP has been demonstrated to promote RalA activity and enhance cell invasion in several cancer types, including oral squamous cell carcinoma, prostate and bladder cancer. Consistently, the inverse correlation between the RalA activity and RalGAPA2 expression was also observed in a panel of HCC cell lines. Furthermore, HCC cells possessing a higher RalA activity were demonstrated to be more prone to RBC8 treatment, a RalGTPase-specific inhibitor. Taken together, our results suggested that RalGAPA2 might play a potential tumor suppressive role in HCC by negatively regulating RalA activity. Citation Format: Lu Tian, Lo Kong Chan, Daniel Wai Hung HO, LuQing Zhao, Irene Oi Lin NG. Dysregulation of RalGAPA2 in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2589.

Fission Yeast Polarization: Modeling Cdc42 Oscillations, Symmetry Breaking, and Zones of Activation and Inhibition.

Cells polarize for growth, motion, or mating through regulation of membrane-bound small GTPases between active GTP-bound and inactive GDP-bound forms. Activators (GEFs, GTP exchange factors) and inhibitors (GAPs, GTPase activating proteins) provide positive and negative feedbacks. We show that a reaction–diffusion model on a curved surface accounts for key features of polarization of model organism fission yeast. The model implements Cdc42 membrane diffusion using measured values for diffusion coefficients and dissociation rates and assumes a limiting GEF pool (proteins Gef1 and Scd1), as in prior models for budding yeast. The model includes two types of GAPs, one representing tip-localized GAPs, such as Rga3; and one representing side-localized GAPs, such as Rga4 and Rga6, that we assume switch between fast and slow diffusing states. After adjustment of unknown rate constants, the model reproduces active Cdc42 zones at cell tips and the pattern of GEF and GAP localization at cell tips and sides. The model reproduces observed tip-to-tip oscillations with periods of the order of several minutes, as well as asymmetric to symmetric oscillations transitions (corresponding to NETO “new end take off”), assuming the limiting GEF amount increases with cell size.

Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium

Salmonella enterica serovar Typhimurium, an intracellular Gram-negative bacterial pathogen, employs two type III secretion systems to deliver virulence effector proteins to host cells. One such effector, SseK3, is a Golgi-targeting arginine GlcNAc transferase. Here, we show that SseK3 colocalizes with cis-Golgi via lipid binding. Arg-GlcNAc-omics profiling reveals that SseK3 modifies Rab1 and some phylogenetically related Rab GTPases. These modifications are dependent on C-termini of Rabs but independent of the GTP- or GDP-bound forms. Arginine GlcNAcylation occurs in the switch II region and the third α-helix and severely disturbs the function of Rab1. The arginine GlcNAc transferase activity of SseK3 is required for the replication of Salmonella in RAW264.7 macrophages and bacterial virulence in the mouse model of Salmonella infection. Therefore, this SseK3 mechanism of action represents a new understanding of the strategy adopted by Salmonella to target host trafficking systems.

Abstract IA03: KRAS Q61H preferentially signals through the MAPK pathway in non-small cell lung cancer

Abstract RAS proteins are guanosine triphosphatases (GTPases) that function as binary switches cycling between inactive (GDP-bound) and active (GTP-bound) states. Activated RAS proteins bind RAS effectors including RAF kinases, PI3K, RalGDS, and others. Conversion from GDP to GTP is stimulated by guanine nucleotide exchange factors (GEFs) while conversion to the inactive GDP-bound form is mediated by GTPase activating proteins (GAPs). Mutations can alter the signaling properties of RAS proteins through a number of means including modification of the GTPase activity, nucleotide exchange rate, or effector binding characteristics. Understanding these differences may enable new therapeutic approaches to specific RAS mutations associated with human disease. We will discuss how codon 61 mutations in KRAS can result in profoundly reduced intrinsic and GAP-stimulated GTPase activity, and a relatively high affinity for RAF-RBD compared to other oncogenic KRAS mutations, and how these features may enable therapeutic options for cancers bearing RAS mutations at codon 61. Citation Format: Zhiwei Zhou, Chiara Ambrogio, Asim Bera, Li Qing, Xu Cheng-Xiong, Pasi Janne, Ken Westover. KRAS Q61H preferentially signals through the MAPK pathway in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA03.

Rac1 activation in human breast carcinoma as a prognostic factor associated with therapeutic resistance.

RAS-related C3 botulinus toxin substrate 1 (Rac1) is a molecular switch fluctuating between GDP-bound inactive form (Rac1-GDP) and GTP-bound active form (Rac1-GTP) and involved in diverse function in both normal and malignant cells such as breast carcinoma cells. Although several studies have demonstrated immunolocalization of Rac1 protein in human breast carcinoma tissues, activation status of Rac1 still remains to be elucidated. We immunolocalized active form of Rac1 (Rac1-GTP) as well as total Rac1 using antibody specific for them in 115 invasive breast carcinoma tissues and correlated with clinicopathological parameters and clinical outcomes. Rac1-GTP was frequently immunolocalized in the cytoplasm or cell membrane of breast carcinoma cells and it was positively correlated with Ki-67 labeling index and total Rac1 while negatively correlated with progesterone receptor. On the other hand, immunohistochemical Rac1-GTP status was significantly correlated with increased risk of recurrence and breast cancer-specific mortality of breast cancer patients and multivariate analyses did demonstrate Rac1-GTP as an independent worse prognostic factor for both disease-free and breast cancer-specific survival. In addition, Rac1-GTP was still correlated with worse prognosis in the patients who had received adjuvant chemotherapy or endocrine therapy. These findings suggested Rac1 activation played pivotal roles in the progression and therapeutic resistance of breast cancers and Rac1 might be an important therapeutic target for improvement of the therapy for breast cancer patients.

IA03 Therapeutic Approaches in KRAS-Driven Non-Small Cell Lung Cancer

RAS mutations (KRAS, HRAS, and NRAS) are the most common oncogenic drivers in non-small cell lung cancer (NSCLC). In metastatic NSCLC, KRAS mutations are associated with worse overall survival compared with KRAS wild-type tumors. They are also unique among the targetable alterations in NSCLC in that they are often associated with a patient smoking history. Though targeted therapies have led to significant improvements in survival of NSCLC patients with activating alterations in EGFR, ALK, ROS1, and BRAF, effective therapies targeting the RAS pathway have been elusive. The challenge in targeting KRAS reflects the complex biology of the RAS signaling pathway. KRAS proteins are membrane-bound effector proteins that link cell surface receptors to downstream growth and proliferation pathways. KRAS proteins are cytosolic protein that are linked to the cell membrane. They cycle between an inactive GDP-bound form and an active GTP-bound form with high affinity. Cycling between active and inactive states is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). When constitutively active, such as in KRAS mutated NSCLC, overlapping downstream growth and proliferation pathways such as PI3K/AKT, RAF/MEK/ERK, and RALGOS/RAL/RLBP1 become activated. Part of the challenge in blocking the oncogenic signaling pathways that originate from KRAS mutations is the crosstalk and redundancy within the pathway. Additionally, the comutational landscape of KRAS mutated NSCLC impacts responses to treatment and can have independent oncogenic activity, further adding to the challenge of blocking oncogenic signaling. Numerous therapeutic tactics have attempted to target this signaling pathway; however, until recently there has been limited success. Therapeutic approaches for KRAS-positive tumors include 1) targeting of the membrane attachment of the KRAS protein, 2) direct targeting of KRAS and its coactivation partners, 3) targeting of downstream and parallel growth and activation pathways, 4) targeting of synthetic lethal interactions, and 5) utilization of immunotherapy. Within and between each of these categories there are also combination therapies being developed. Despite the inherent complexity in developing treatments for KRAS mutated NSCLC, there are now multiple promising strategies in development that may change the treatment landscape of this disease. In this session, we will explore the background and current landscape of the therapeutic approaches for KRAS mutated NSCLC.

NMR resonance assignments for the active and inactive conformations of the small G protein RalA

The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1H, 15 N and 13C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.

Abstract PR10: Tri-complex inhibitors of the oncogenic, GTP-bound form of KRASG12C overcome RTK-mediated escape mechanisms and drive tumor regressions in vivo

Abstract RAS proteins are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins are found in approximately one-third of human cancers, and exist predominantly in the GTP-bound state, leading to excessive downstream signaling via interaction with effectors such as RAF. A KRAS mutation in which glycine-12 is mutated to cysteine (KRASG12C) is found in 11-12% of non-small cell lung cancers. Recently, multiple potent, covalent inhibitors of KRASG12C have been reported that target the inactive, GDP-bound form of KRASG12C, and thus rely on the residual intrinsic hydrolysis of GTP to cycle KRASG12C proteins through the inactive, GDP-bound state. This mechanism is vulnerable to adaptive responses in cancer cells that can activate RAS by increasing upstream signaling and further increase the relative abundance of KRASG12C(GTP) over KRASG12C(GDP). An inhibitor that directly targets the active, GTP-bound form of KRASG12C would overcome this limitation. Drawing inspiration from natural products like cyclosporine and rapamycin, we have developed tri-complex inhibitors of KRASG12C(GTP) that promote a ternary complex between KRASG12C and the abundant immunophilin cyclophilin A (CypA). These sanglifehrin-inspired inhibitors exploit significant non-covalent interactions in the SWI/SWII region of KRAS combined with an electrophilic cysteine-targeted warhead to potently and irreversibly inhibit KRASG12C(GTP). The inhibitors selectively drive formation of KRASG12C-inhibitor-CypA ternary complexes that are sterically prevented from interacting with the RAS Binding Domain (RBD) of BRAF in biochemical studies. In cellular models, KRASG12C(GTP) inhibitors attenuate both RAS-MAPK signaling and cell viability in cancer cell lines bearing KRASG12C mutations, but not other mutations in RAS or other pathway oncoproteins. In vivo administration of a KRASG12C(GTP) inhibitor drives dose-dependent tumor regressions in the NCI-H358 KRASG12C NSCLC xenograft mouse model and is well-tolerated. Consistent with targeting the KRAS(GTP) state, inhibitory activity in vitro is unaffected by RTK activation, whereas the activity of first generation KRASG12C(GDP) inhibitors is significantly attenuated. In addition, proliferation of NCI-H358 and MIA PaCa-2 cells in vitro is suppressed for a significantly longer duration with KRASG12C(GTP) inhibitor treatment compared to KRASG12C(GDP) inhibitors. The combination of sub-maximal concentrations of a MEK inhibitor and a KRASG12C(GTP) inhibitor drove pronounced cell death. In contrast, the MEK and KRASG12C(GDP) inhibitor combination evoked a modest enhancement of the antiproliferative effects and does not cause cell death. Tri-complex inhibitors that target the active, GTP-bound form of KRAS thus represent a second generation of KRASG12C inhibitor. Chemical modulation of the non-covalent and covalent interactions of our tri-complex inhibitors provides an exciting opportunity to step beyond KRASG12C to target the GTP-bound state of additional RAS variants, and we demonstrate in vitro covalent inhibition of KRASG13C. By directly targeting active RAS-GTP, tri-complex inhibitors have the potential to overcome adaptive resistance mechanisms that emerge following inhibition of aberrant RAS-MAPK pathway activation. Citation Format: Christopher J Schulze, Alun Bermingham, Tiffany J Choy, James J Cregg, Gert Kiss, Abby Marquez, Denise Reyes, Mae Saldajeno-Concar, Caroline E Weller, Daniel M Whalen, Yu C Yang, Elena S Koltun, Robert J Nichols, Mallika Singh, David Wildes, Adrian L Gill, Richard L Hansen, Steve Kelsey, Mark A Goldsmith, Jacqueline A.M. Smith. Tri-complex inhibitors of the oncogenic, GTP-bound form of KRASG12C overcome RTK-mediated escape mechanisms and drive tumor regressions in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR10. doi:10.1158/1535-7163.TARG-19-PR10

FolVps9, a Guanine Nucleotide Exchange Factor for FolVps21, Is Essential for Fungal Development and Pathogenicity in Fusarium oxysporum f. sp. lycopersici.

The soil-borne, asexual fungus Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of tomato wilt disease. Autophagy plays a crucial role in the development and virulence of Fol. The Fol endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity. Rab GTPases and the regulators are highly conserved in regulating autophagy and endocytosis in most eukaryotes. Identification and characterization of additional Rab regulators in fungal pathogens should facilitate the understanding of the autophagy and endocytosis in different filamentous fungi. Here, we have identified and characterized the yeast VPS9 homolog FolVPS9 in Fol. Targeted gene deletion showed that FolVPS9 is important for growth, conidiation and virulence in Fol. Cytological examination revealed that FolVps9 co-localized with FolVps21 (a marker of early endosome) and played a critical role in endocytosis and autophagosome degradation. Pull-down assays showed that FolVps9 interacted with FolVps21, which was also important for development and plant infection in Fol. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays revealed that FolVps9 specifically interacts with the GDP-bound form of FolVps21. Furthermore, a constitutively active form of FolVps21 (Q72L) was able to rescue defects of ΔFolvps9 and ΔFolvps21 mutants. In summary, our study provides solid evidence that FolVps9 acts as a FolVps21 guanine nucleotide exchange factor (GEFs) to modulate endocytosis and autophagy, thereby controlling vegetative growth, asexual development and pathogenicity in Fol.

Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations

Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal “stalk” protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

The RBG-1-RBG-2 complex modulates autophagy activity by regulating lysosomal biogenesis and function in C. elegans.

Vici syndrome is a severe and progressive multisystem disease caused by mutations in the EPG5 gene. In patient tissues and animal models, loss of EPG5 function is associated with defective autophagy caused by accumulation of non-degradative autolysosomes, but very little is known about the mechanism underlying this cellular phenotype. Here, we demonstrate that loss of function of the RBG-1-RBG-2 complex ameliorates the autophagy defect in C. elegansepg-5 mutants. The suppression effect is independent of the complex's activity as a RAB-3 GAP and a RAB-18 GEF. Loss of rbg-1 activity promotes lysosomal biogenesis and function, and also suppresses the accumulation of non-functional autolysosomes in epg-5 mutants. The mobility of late endosome- and lysosome-associated RAB-7 is reduced in epg-5 mutants, and this defect is rescued by simultaneous loss of function of rbg-1 Expression of the GDP-bound form of RAB-7 also promotes lysosomal biogenesis and suppresses the autophagy defect in epg-5 mutants. Our study reveals that the RBG-1-RBG-2 complex acts by modulating the dynamics of membrane-associated RAB-7 to regulate lysosomal biogenesis, and provides insights into the pathogenesis of Vici syndrome.

Oncogenic G12D mutation alters local conformations and dynamics of K-Ras

K-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and α3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-RasG12D-driven cancers.

PIP2 Influences the Conformational Dynamics of Membrane-Bound KRAS4b.

KRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. The activation of KRAS4b occurs only after recruitment of the regulatory proteins to the plasma membrane, thus making the role of the phospholipid bilayer an integral part of the signaling mechanism. Phospholipids, primarily with anionic headgroups, interact with both the membrane-anchoring hypervariable (HVR) region and the G-domain (catalytic domain) and influence the orientation of KRAS4b on the membrane surface, potentially playing a key role in the regulation of activation. Although there has been significant research focused on the role of the anionic phosphatidylserine, less effort has been spent on the role of the important signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Using instrumentation to measure the fluorescence anisotropy decay of site specifically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Nanodiscs over a wide frequency range, we quantitate the binding of KRAS4b to Nanodiscs containing either 30% phosphatidylserine (PS) or 10% l-α-phosphatidylinositol 4,5-bisphosphate by measuring the rotational correlation time of the Nanodisc-KRAS4b complex. We find that KRAS4b binds significantly tighter to Nanodiscs containing PIP2 but that at any level of binding saturation of KRAS4b, both 30% PS and 10% PIP2 containing Nanodiscs display similar rotational correlation times. This shows that the overall hydrodynamic radii of the KRAS4b-Nanodisc complexes are similar regardless of the incorporated anionic lipid. Atomic force microscopy is used to visualize KRAS4b when bound to individual Nanodiscs. Clean images are observed with the PIP2-doped Nanodiscs, but significantly blurred images are obtained when the anionic lipid is PS. This suggests that KRAS4b is not only more tightly bound overall with PIP2 as the anionic lipid but also less mobile on the bilayer surface. Microsecond molecular dynamics simulations of KRAS4b on PS- and PIP2-containing membranes show that the dynamics of the G-domain at the bilayer surface are significantly altered in the presence of PIP2, due to the formation of long-lived salt bridges with basic residues on the G-domain. The orientation and dynamics of KRAS4b on the membrane are critical to understanding the mechanisms of oncoprotein signaling, and our results with the GDP-bound form show subtle differences from that published for GTP-KRAS4b.

In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation.

The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5, Rab9 and Rab11. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

Structural and functional investigation of the Small Ribosomal Subunit Biogenesis GTPase A (RsgA) from Pseudomonasaeruginosa.

The Small Ribosomal Subunit Biogenesis GTPase A (RsgA) is a bacterial assembly factor involved in the late stages of the 30S subunit maturation. It is a multidomain GTPase in which the central circularly permutated GTPase domain is flanked by an OB domain and a Zn-binding domain. All three domains participate in the interaction with the 30S particle thus ensuring an efficient coupling between catalytic activity and biological function. Invivo studies suggested the relevance of rsgA in bacterial growth and cellular viability, but other pleiotropic roles of RsgA are also emerging. Here, we report the 3D structure of RsgA from Pseudomonasaeruginosa (PaRsgA) in the GDP-bound form. We also report a biophysical and biochemical characterization of the protein in both the GDP-bound and its nucleotide-free form. In particular, we report a kinetic analysis of the RsgA binding to GTP and GDP. We found that PaRsgA is able to bind both nucleotides with submicromolar affinity. The higher affinity towards GDP (KD =0.011μm) with respect to GTP (KD =0.16μm) is mainly ascribed to a smaller GDP dissociation rate. Our results confirm that PaRsgA, like most other GTPases, has a weak intrinsic enzymatic activity (kCAT =0.058min-1 ). Finally, the biological role of RsgA in P.aeruginosa was investigated, allowing us to conclude that rsgA is dispensable for P.aeruginosa growth but important for drug resistance and virulence in an animal infection model. DATABASES: Coordinates and structure factors for the protein structure described in this manuscript have been deposited in the Protein Data Bank (https://www.rcsb.org) with the accession code 6H4D.

Phosphorylated Rho-GDP directly activates mTORC2 kinase towards AKT through dimerization with Ras-GTP to regulate cell migration.

mTORC2 plays critical roles in metabolism, cell survival, and actin cytoskeletal dynamics via phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae Dictyostelium cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling the supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by chemoattractant-induced phosphorylation of Rho-GDP at serine 192 by GSK-3. Furthermore, Rho-GDP rescued defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of serine 192. Thus, in contrast to the prevailing view that GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signaling is activated by Rho-GDP.

IRR is involved in glucose-induced endocytosis after insulin secretion

Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic β-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3 generation and controls endocytosis after insulin secretion.

SMURF1-mediated ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration

Rho GTPase-activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small GTP-binding protein RhoA (GTP-RhoA) to its inactive GDP-bound form and is a putative tumor suppressor gene associated with cell growth and migration. Here, the involvement of ARHGAP26 in ovarian cancer cell proliferation and migration was investigated. In this study, low ARHGAP26 expression was observed in ovarian cancer tissues and was associated with a poor overall survival and higher β-catenin expression in patients with ovarian cancer. A2780 and HEY cells with ARHGAP26 upregulation showed decreased cell proliferation, migration, and invasion, along with decreased GTP-RhoA, β-catenin, VEGF, MMP2, and MMP7 expression. ARHGAP26 upregulation in A2780 cells also inhibited lung metastasis in vivo. SKOV3 cells with ARHGAP26 downregulation demonstrated an inverse effect, which was inhibited by ARHGAP26 overexpression or DKK1, an antagonist of the β-catenin pathway. SMURF1, an E3 ubiquitin ligase, interacted with and induced ubiquitination of ARHGAP26. ARHGAP26 upregulation in SKOV3 cells significantly inhibited SMURF1 upregulation-induced cell migration and invasion. Overall, SMURF1-mediated ubiquitination of ARHGAP26 may promote invasion and migration of ovarian cancer cells via the β-catenin pathway.

Proportional subcellular localization of Arabidopsis thaliana RabA1a

ABSTRACTSubcellular localization of trafficking proteins in a single cell affects the assembly of trafficking machinery between organelles and vesicles throughout the targeting pathway. RabGTPase is one of the regulators to direct specific targeting of cargo molecules depending on GDP/GTP bound status. We have recently determined the crystal structures of GDP-bound inactive and both GTP- and GppNHp-bound active forms of Arabidopsis RabA1a. It is notable that the switch regions of RabA1a exhibit conformational changes derived by GDP or GTP binding. However, it was not clear that where the GDP- or GTP-bound RabA1a is localized at the subcellular level in a cell. Here we demonstrate that the distinct proportion of subcellular localization of RabA1a depends on its site-specific mutation as the GDP- or GTP-bound form. RabA1a proteins located at the plasma membrane, endosomes, and cytosol. While the GDP-bound form of RabA1aS27N located more at endosomes than the plasma membrane compared to the proportions of RabA1a wild-type, and the GTP-bound RabA1aQ72L located mainly at the plasma membrane in comparison to RabA1a wild-type and RabA1aS27N. These distinct proportional localizations of RabA1a enable a cognate interaction between inactive/active RabA1 and effector molecules to direct specific targeting of its cargo molecules.

Structural Factors Controlling Orientation of KRAS G-Domain Membrane Binding

Ras proteins are small GTPases involved in key cell signaling pathways regulating cell growth, proliferation and division. Overactivity of Ras proteins has been a hallmark of diverse forms of cancer in humans. Interconversion between the active (GTP-bound) and inactive (GDP-bound) forms is mediated by two key protein partners that bind and interact with Ras on the surface of the membrane: hydrolysis of GTP, and inactivation of Ras, is mediated by GAP, while exchange of GDP by GTP is facilitated by GEF, reactivating Ras. While the G-domain, the globular domain which hydrolyzes GTP, of Ras isoforms is highly conserved sequentially and structurally, the difference lies in the hypervariable region (HVR). The HVR plays a crucial role in anchoring the G-domain into the cytosolic membrane. We used K-Ras as it is the most common isoform whose mutations lead to a number of cancer type. The orientation of the globular domain on the membrane is essential to its ability to interact with downstream effectors. We simulated the GTP- and GDP-bound domains on HMMM membrane composed of 70:30 PS/PC lipids. The G-domain initial orientation in solution was varied by 25-30 degrees with respect to the membrane, to avoid convergence of results due to initial placement bias. While the differences in structure are small in the GTP- and GDP- bound domains, they bind different effectors, therefore their individual orientation on the membrane is important. Our simulations of over 3.5 microseconds show the G-domain, in the absence of the linker samples many conformations on the surface of the membrane, contrasting results from simulations where the protein was bound to the membrane by the HVR, converging to two distinct binding modes. Our findings suggest the linker highly constrains the orientations available for the G-domain to sample.