RabGAP Domain Research Articles

TBC1 domain-containing proteins are frequently involved in triple-negative breast cancers in connection with the induction of a glycolytic phenotype

Metabolic plasticity is a hallmark of cancer, and metabolic alterations represent a promising therapeutic target. Since cellular metabolism is controlled by membrane traffic at multiple levels, we investigated the involvement of TBC1 domain-containing proteins (TBC1Ds) in the regulation of cancer metabolism. These proteins are characterized by the presence of a RAB-GAP domain, the TBC1 domain, and typically function as attenuators of RABs, the master switches of membrane traffic. However, a number of TBC1Ds harbor mutations in their catalytic residues, predicting biological functions different from direct regulation of RAB activities. Herein, we report that several genes encoding for TBC1Ds are expressed at higher levels in triple-negative breast cancers (TNBC) vs. other subtypes of breast cancers (BC), and predict prognosis. Orthogonal transcriptomics/metabolomics analysis revealed that the expression of prognostic TBC1Ds correlates with elevated glycolytic metabolism in BC cell lines. In-depth investigations of the three top hits from the previous analyses (TBC1D31, TBC1D22B and TBC1D7) revealed that their elevated expression is causal in determining a glycolytic phenotype in TNBC cell lines. We further showed that the impact of TBC1D7 on glycolytic metabolism of BC cells is independent of its known participation in the TSC1/TSC2 complex and consequent downregulation of mTORC1 activity. Since TBC1D7 behaves as an independent prognostic biomarker in TNBC, it could be used to distinguish good prognosis patients who could be spared aggressive therapy from those with a poor prognosis who might benefit from anti-glycolytic targeted therapies. Together, our results highlight how TBC1Ds connect disease aggressiveness with metabolic alterations in TNBC. Given the high level of heterogeneity among this BC subtype, TBC1Ds could represent important tools in predicting prognosis and guiding therapy decision-making.

TBC1D24 regulates recycling of clathrin-independent cargo proteins mediated by tubular recycling endosomes

Many plasma membrane proteins enter cells by clathrin-independent endocytosis (CIE). Rab family small GTPases play pivotal roles in CIE and following intracellular trafficking of cargo proteins. Here, we provide evidence that TBC1D24, which contains an atypical Rab GAP domain, facilitates formation of tubular recycling endosomes (TREs) that are a hallmark of the CIE cargo trafficking pathway in HeLa cells. Overexpression of TBC1D24 in HeLa cells dramatically increased TREs loaded with CIE cargo proteins, while deletion of TBC1D24 impaired TRE formation and delayed the recycling of CIE cargo proteins back to the plasma membrane. We also found that TBC1D24 binds to Rab22A, through which TBC1D24 regulates TRE-mediated CIE cargo recycling. These findings provide insight into regulatory mechanisms for CIE cargo trafficking.

1325-P: Effectiveness of Comprehensive Gene Panel-Based Next-Generation Sequencing with Phenotype-Driven Bioinformatics Analysis for Diagnosis of Atypical Diabetes

Atypical diabetes is considered to be different from the common forms of diabetes (types 1 and 2); however, it may share pathogenetic and phenotypic features with the common forms of diabetes. Targeted next-generation sequencing focused on genes causing monogenic diabetes has been reported to be useful for diagnosis of a few subtypes of atypical diabetes caused by genetic defects, such as MODY. In this study, considering the clinically heterogeneous nature of atypical diabetes, we developed a comprehensive gene panel containing 383 monogenic diabetes causing genes and genes associated with common forms of diabetes, and conducted targeted next-generation sequencing in 3 patients with previously confirmed and 10 patients with suspected atypical diabetes. In addition, phenotype-driven bioinformatics analysis using genetic and phenotypic information was performed to identify causal variants from sequencing data. A total of 11 mutations including 2 gross deletions were identified, which led to diagnosis of four previously undiagnosed patients and identification of potentially pathogenic variants of interest in other three patients. Of note, a novel missense mutation of Rab GAP domain of TBC1D4 was identified in a diabetic patient with severe insulin resistance. The TBC1D4 locus has been reported to be associated with type 2 diabetes and insulin resistance. In silico structural analysis suggested that the mutation would cause pathogenesis by functional and structural modification of TBC1D4, insulin-dependent phosphorylation of which is necessary for GLUT4 translocation mediated glucose uptake in skeletal muscle and adipose tissues. The results from this study indicate that comprehensive genetic screening analyzing genes associated with common and rare subtypes of diabetes can identify causal variants efficiently, thereby aiding early diagnosis of atypical diabetes. Disclosure J. Hosoe: None. H. Kadowaki: Research Support; Spouse/Partner; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Spouse/Partner; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. F. Miya: None. M. Takakura: None. H. Waki: Research Support; Self; Astellas Pharma Inc., Kyowa Hakko Kirin Co., Ltd., MSD K.K., Nippon Boehringer Ingelheim Co Ltd, Novo Nordisk Foundation, Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, The Cell Science Research Foundation. T. Sasako: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk Inc. T. Kato: None. T. Tsunoda: None. N. Shojima: None. T. Yamauchi: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Kyowa Hakko Kirin Co., Ltd., Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Takeda Pharmaceutical Company Limited. T. Kadowaki: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical, Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Mitsubishi Tanabe Pharma Corporation, MSD, Nippon Boehringer Ingelheim, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

TBC1D23 is a bridging factor for endosomal vesicle capture by golgins at the trans-Golgi.

The specificity of membrane traffic involves tethers at destination organelles that selectively capture incoming transport vesicles to allow SNAREs on opposing membranes to then assemble and drive fusion1,2. Tethers include both protein complexes and long coiled-coil proteins, although how they contribute to specificity remains unclear3,4. The golgin coiled-coil proteins at the Golgi apparatus capture vesicles from different origins, but the vesicle-specific molecular cues that they recognise are unknown5–8. Vesicle tethering is typically a transient process and so challenging to interrogate in vivo. We have thus used a system where an ectopic golgin causes vesicles to accumulate in a tethered state. By applying proximity biotinylation to the golgin-captured vesicles we identify TBC1D23, an apparently catalytically inactive member of a family of Rab GTPase activating proteins (GAPs), as a vesicle-golgin adaptor that is required for endosome-to-Golgi traffic. The Rab-GAP domain of TBC1D23 binds to a conserved motif at the tip of golgin-245 and golgin-97 at the trans-Golgi, while the C-terminus binds to the WASH complex on endosome-derived vesicles. Thus TBC1D23 is a specificity determinant that links vesicle to target membrane during endosome-to-Golgi trafficking.

Modulation of membrane phosphoinositide dynamics by the phosphatidylinositide 4-kinase activity of the Legionella LepB effector.

Legionella pneumophila, the causative bacterium for Legionnaires' disease, hijacks host membrane trafficking for the maturation of the Legionella-containing vacuole (LCV). The LCV membrane mainly contains PtdIns4P, which is important for anchoring many secreted Legionella effectors onto the LCV. Here, we identify a cryptic functional domain (LepB_NTD) preceding the well-characterized RabGAP domain in the Legionella Dot/Icm type IV secretion system effector LepB. LepB_NTD alone is toxic to yeast and can disrupt the Golgi in mammalian cells. The crystal structure reveals an unexpected kinase fold and catalytic motif important for LepB_NTD function in eukaryotes. Cell biology-guided biochemical analyses uncovered a lipid kinase activity in LepB_NTD that specifically converts PtdIns3P into PtdIns(3,4)P2. PtdIns(3,4)P2 is efficiently hydrolysed into PtdIns4P by another Dot/Icm effector SidF that is known to possess phosphoinositide phosphatase activity. Consistently, SidF is capable of counteracting the cellular functions of LepB_NTD. Genetic analyses show a requirement for LepB kinase activity as well as lipid phosphatase activity of SidF for PtdIns4P biosynthesis on the LCV membrane. Our study identifies an unprecedented phosphatidylinositide 4-kinase activity from bacteria and highlights a sophisticated manipulation of host phosphoinositide metabolism by a bacterial pathogen.

Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases.

Rab small GTPases are highly conserved master regulators of membrane traffic in all eukaryotes. The same as the activation and inactivation of other small GTPases, the activation and inactivation of Rabs are tightly controlled by specific GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins), respectively. Although almost all Rab-GAPs reported thus far have a TBC (Tre-2/Bub2/Cdc16)/Rab-GAP domain in common, recent accumulating evidence has indicated the existence of a number of structurally unrelated types of Rab-GEFs, including DENN proteins, VPS9 proteins, Sec2 proteins, TRAPP complexes, heterodimer GEFs (Mon1-Ccz1, HPS1-HPS4 (BLOC-3 complex), Ric1-Rgp1 and Rab3GAP1/2), and other GEFs (e.g., REI-1 and RPGR). In this review article we provide an up-to-date overview of the structures and functions of all putative Rab-GEFs in mammals, with a special focus on their substrate Rabs, interacting proteins, associations with genetic diseases, and intracellular localizations.

TBC1D1 and TBC1D4 (AS160) RabGAP Domains are Characterized as Monomers in Solution by Analytical Ultracentrifugation

Upon food intake, the glucose level in circulating blood increases by trans-cellular transport of glucose from the intestinal lumen into the blood. However, the insulin secreted from the pancreatic β-cells directs the individual cells to absorb glucose for energy source to result in the maintenance of the normal blood glucose level. Elevated insulin in blood is sensed by the insulin receptor of the individual cells, and the propagation of the insulin signaling pathway drives the glucose uptake. The final cellular glucose uptake from the blood into the cells is performed by trafficking of vesicles containing the glucose transporter protein (e.g. GLUT4) from the intracellular pools to the cell surfaces. As in most of the vesicle budding, uncoating, docking and fusion processes, this translocation of GLUT4 vesicles are tightly regulated by Rab·GTPases (Rabs) and Rab·GTPase-activating proteins (RabGAPs). Rabs are GTPases with intrinsic activity, however RabGAPs enhance the low hydrolysis rate of Rab-bound GTP. The two major cell types that absorb glucose, which are the adipocytes and the skeletal myocytes, respectively encode TBC1D4 (also known as AS160) and TBC1D1 which are integral RabGAPs in the insulin-mediated GLUT4 vesicle trafficking event. TBC1D4 and TBC1D1 are multi-domain RabGAP proteins which consist of two putative N-termini phosphotyrosine binding (PTB) domains and a C-terminal catalytic RabGAP domain that are linked by regions containing several Akt(PKB)-mediating serine and threonine phosphorylation sites. In the basal state, TBC1D4 (or TBC1D1)-catalyzed hydrolysis of Rab-bound GTP (active) to GDP (inactive) leaves GLUT4 vesicles sequestered in an intracellular compartment. However, insulin signal results in the activation of Akt (PKB) and the consequent phosphorylation of TBC1D4 (or TBC1D1). The Akt phosphorylation of TBC1D4 (or TBC1D1) deactivates the Rab·GTP hydrolysis activity by an unknown mechanism, and concomitantly results in the increase of Rab·GTP concentrations. The active Rab·GTP in turn promotes GLUT4 vesicle translocation to the cell membrane and finally stimulates the glucose uptake from blood into the cells. X-ray crystal structures of the human TBC1D1 RabGAP domain and TBC1D4 RabGAP domain (PDB code: 3QYE & 3QYB, respectively) have been recently reported. Since, TBC1D1 and TBC1D4 share 76% sequence identity over their RabGAP domains, the secondary structure elements mostly overlapped in the two structures which were predominately α-helical with no β-sheet elements. The 17 αhelices in TBC1D1 RabGAP domain were sequentially named from α1 to α16 ending with α16'. However, the oligomeric association states of the two TBC1D1 and TBC1D4 RabGAP domains in the crystal asymmetric unit were different resulting from the presence (or absence) of a single helix α16'. TBC1D1 RabGAP domain containing the C-terminal α16' forms an asymmetric dimer with α16' of one molecule interacting with α16 of the other molecule (Figure 1). However in TBC1D4 RabGAP domain, α16' in the protein construct had to be deleted for crystallization and enhanced X-ray diffraction (Figure 2), and this results in TBC1D4 RabGAP domain being a monomer in the crystal (Figure 1). Certain is the fact that α16' mediates the resulting differences in the oligomeric states, the biological relevance of this asymmetric dimer cannot be concluded only from the association modes within the crystal. Since only two αhelices (α16' and α16) of TBC1D1 mediate the dimerization, it was suspected that the dimer is a result of crystal packing artifacts, and is non-relevant outside the context of the crystal. However in this study, we have compared the oligomeric association states of TBC1D1 and TBC1D4 RabGAP domains (Figure 2) using analytical ultracentrifugation (AUC) techniques to address the state of interaction in solution. Sedimentation equilibrium experiments were performed in a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge using 12 mm carbon-filled epoxy double-sector centerpieces and quartz windows at optical density of 0.25 at 280 nm (6.67 μM and 6.69 μM for TBC1D1 and TBC1D4 RabGAP domains, respectively). The measurements were performed at two different rotor speeds of 13,000 and 18,000 RPMs at 20 C. The proteins were in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1.5 mM TCEP. The sedimentation equilibrium data were analyzed using MLAB software (Civilized Software, Inc., Silver Spring, MD) using the following equation: C(r) = cb*exp (Ap* Mp* (r − rb) + e (1)

Crystal Structures of Human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating Protein (RabGAP) Domains Reveal Critical Elements for GLUT4 Translocation

We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no β-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met(930), corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met(930) and Leu(1019) predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.

Mice with AS160/TBC1D4-Thr649Ala Knockin Mutation Are Glucose Intolerant with Reduced Insulin Sensitivity and Altered GLUT4 Trafficking

SummaryAS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent to Thr642 in human AS160). To define physiological roles of AS160-Thr649 phosphorylation and 14-3-3 binding in glucose homeostasis, we substituted this residue by a nonphosphorylatable alanine by knockin mutation in mice. The mutant protein was expressed at normal levels, while insulin-stimulated AS160 binding to 14-3-3s was abolished in homozygous knockin mice. These animals displayed impaired glucose disposal and insulin sensitivity, which were associated with decreased glucose uptake in vivo. Insulin-stimulated glucose transport and cell surface GLUT4 content were reduced in isolated muscles, but not in adipocytes. These results provide genetic evidence that insulin-induced AS160-Thr649 phosphorylation and/or its binding to 14-3-3 play an important role in regulating whole-body glucose homeostasis, at least in part through regulating GLUT4 trafficking in muscle.

Comprehensive Screening for Novel Rab-Binding Proteins by GST Pull-Down Assay Using 60 Different Mammalian Rabs‡

The Rab family belongs to the Ras-like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S-transferase (GST) pull-down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab-binding proteins we identified, mKIAA1055/TBC1D2B (Rab22-binding protein), GAPCenA/TBC1D11 (Rab36-binding protein) and centaurin beta2/ACAP2 (Rab35-binding protein), are GTPase-activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab-GAP (Tre-2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin beta2 binds GTP-Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin beta2 did not exhibit any Rab35-GAP activity in vitro, the Rab35-binding ANKR domain of centaurin beta2 was found to be required for its plasma membrane localization and regulation of Rab35-dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.

Identification and characterization of a novel Tre‐2/Bub2/Cdc16 (TBC) protein that possesses Rab3A‐GAP activity

The Tre-2/Bub2/Cdc16 (TBC) domain is a conserved protein motif that consists of approximately 200 amino acids and is thought to function as a specific Rab-GAP domain. Although more than 40 distinct TBC domain-containing proteins have been identified in humans, the GAP activity and specificity of most TBC proteins have never been determined. In this study we developed a novel method of screening for Rab3A-GAP and identified two TBC proteins (FLJ13130 and RN-tre) whose expression in PC12 cells was associated with exclusion of endogenous Rab3A molecules from dense-core vesicles. As expression of RN-tre caused fragmentation of the Golgi, which presumably resulted in the loss of dense-core vesicles themselves, we further characterized FLJ13130 as a candidate Rab3A-GAP. The results showed that expression of FLJ13130, but not of its catalytically inactive R134K mutant, greatly reduced the amount of GTP-Rab3A in living cells and promoted the GTPase activity of Rab3A in vitro. Unexpectedly, however, FLJ13130 also promoted the GTPase activity of Rab22A, Rab27A, and Rab35, but not of Rab2A or Rab6A. Based on these results, we propose that FLJ13130 is a novel type of Rab-GAP that exhibits broad GAP specificity and inactivates several distinct Rab isoforms, including Rab3A, just near the plasma membrane.

Expression in a RabGAP yeast mutant of two human homologues, one of which is an oncogene

The yeast proteins Msb3p and Msb4p are two Ypt/Rab-specific GTPase-activating proteins (GAPs) involved in cell growth polarization. Both proteins share with a wide variety of other proteins the highly conserved TBC domain forming the catalytically active RabGAP domain. In particular, Msb3p and Msb4p are similar to the human proteins oncTre210p (the 786-amino-acid product of the human Tre2 oncogene, implicated in Ewing’s sarcoma) and RN-tre (a Rab5-GAP controlling endocytosis of the EGFR). To further understand the biochemical function of Tre2 oncogene, we expressed its cDNA and, as a control, the RN-tre cDNA, in an msb3 msb4 double mutant yeast strain. Complementation data show that RN-tre can, unlike Tre2, replace the function of the MSB3 and MSB4 genes. As two highly conserved amino acids, including the catalytic arginine, are mutated in the oncTre210p TBC domain, we restored these two amino acids and expressed the modified Tre2 cDNA in the yeast mutant.

A Method to Identify Serine Kinase Substrates: Akt PHOSPHORYLATES A NOVEL ADIPOCYTE PROTEIN WITH A Rab GTPASE-ACTIVATING PROTEIN (GAP) DOMAIN

This study describes a method for the identification of the substrates of specific serine kinases. An antibody specific for the phosphomotif generated by the kinase is used to isolate phosphorylated substrates by immunoprecipitation, and the isolated proteins are identified by tandem mass spectrometry of peptides. This method was applied to the identification of substrates for the protein kinase Akt, which specifically phosphorylates the RXRXXS/T motif. 3T3-L1 adipocytes were treated with insulin to activate Akt, and the putative Akt substrate proteins were isolated by immunoprecipitation with an antibody against the phospho form of this motif. This led to the identification of a novel 160-kDa substrate for Akt. The 160-kDa substrate for Akt, which was designated AS160, has a Rab GAP domain. Recombinant AS160 was shown to be a substrate for Akt, and two sites of phosphorylation, both in RXRXXS/T motifs, were identified by mass spectrometry and mutation. Insulin treatment of adipocytes caused AS160 to redistribute from the low density microsomes to the cytosol.