Tre-2/Bub2/Cdc16 Research Articles

Identification of TBC7 having TBC domain as a novel binding protein to TSC1–TSC2 complex

TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1–TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1–TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.

Identification of genes with correlated patterns of variations in DNA copy number and gene expression level in gastric cancer

To identify DNA copy number changes that had a direct influence on mRNA expression in gastric cancer, cDNA microarray-based comparative genomic hybridization (aCGH) and gene expression profiling were performed using 17 K cDNA microarrays. A set of 158 genes showing Pearson correlation coefficients over 0.6 between DNA copy number changes and mRNA expression level variations was selected. In an independent gene expression profiling of 60 tissue samples, the 158 genes were able to distinguish most of the normal and tumor tissues in an unsupervised hierarchical clustering, suggesting that the differential expression patterns displayed by this specific group of genes are most likely based on the gene copy number changes. Furthermore, 43 statistically significant ( P < 0.01) genes were selected that correctly distinguished all of the tissue samples. The copy number changes detected by aCGH can be verified by fluorescence in situ hybridization and real-time polymerase chain reaction. The selected genes include those that were previously identified as being tumor suppressors or deleted in various tumors, including GATA binding protein 4 (GATA4), monoamine oxidase A (MAOA), cyclin C (CCNC), and oncogenes including malignant fibrous histiocytoma amplified sequence 1 (MFHAS1/MASL1), high mobility group AT-hook 2 (HMGA2), PPAR binding protein (PPARBP), growth factor receptor-bound protein 7 (GRB7), and TBC1 (tre-2, BUB2, cdc16) domain family, member 1 (TBC1D1).

Identification of EPI64 as a GTPase-activating Protein Specific for Rab27A

Small GTPase Rab27A plays a pivotal role in melanosome transport in melanocytes and in secretion by various secreting cells. Because the GTP- or GDP-locked mutant of Rab27A causes perinuclear aggregation of melanosomes, appropriate GTP-GDP cycling of Rab27A is essential for melanosome transport, and certain guanine nucleotide exchange factors and GTPase-activating proteins (GAPs) of Rab27A must be present in melanocytes. However, no such regulators of Rab27A have ever been identified. In this study we developed novel methods of rapidly screening 40 different TBC (Tre2/Bub2/Cdc16) proteins, putative Rab-GAPs, for Rab27A-GAP by: (i) searching for TBC proteins that induce melanosome aggregation in melanocytes; (ii) trapping GTP-Rab27A with a Rab27A effector domain (i.e. the SHD of Slac2-a) in cultured cells that express both Rab27A and TBC proteins; and (iii) measuring in vitro Rab27A-GAP activity. These methods allowed us to identify EPI64, previously characterized as an EBP50-binding protein that contains an orphan TBC domain, as a specific Rab27A-GAP. We further showed that mutations in the catalytic domain of EPI64 caused complete loss of its ability to induce melanosome aggregation. This is the first report of screening for Rab27A-GAP based on functional interactions, and our screening methods can be applied for other uncharacterized TBC proteins.

Screening for target Rabs of TBC (Tre‐2/Bub2/Cdc16) domain‐containing proteins based on their Rab‐binding activity

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A- and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.

P.427 The hepatitis E virus ORF3 protein modulates endocytic trafficking and the acute phase protein response

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.

Calcium/Calmodulin Regulates Ubiquitination of the Ubiquitin-specific Protease TRE17/USP6

The TRE17 (USP6/TRE-2) oncogene induces tumorigenesis in both humans and mice. However, little is known regarding its regulation or mechanism of transformation. TRE17 encodes a TBC (Tre-2/Bub2/Cdc16)/Rab GTPase-activating protein homology domain at its N terminus and a ubiquitin-specific protease at its C terminus. In the current study, we identified the ubiquitous calcium (Ca2+)-binding protein calmodulin (CaM) as a novel binding partner for TRE17. CaM bound directly to TRE17 in a Ca2+-dependent manner both in vitro and in vivo. The CaM-binding site was mapped to two hydrophobic motifs near the C terminus of the TBC domain. Point mutations within these motifs significantly reduced the interaction of TRE17 with CaM. We further found that TRE17 is monoubiquitinated and promotes its own deubiquitination in vivo. CaM binding-deficient mutants of TRE17 exhibited significantly reduced monoubiquitination, suggesting that binding of Ca2+/CaM to TRE17 promotes this modification. Consistent with this notion, treatment of cells with the CaM inhibitor W7 reduced levels of TRE17 monoubiquitination. Interestingly, the calcium ionophore A23187 induced accumulation of a polyubiquitinated TRE17 species. The effect of A23187 was attenuated in CaM binding-deficient mutants of TRE17. Taken together, these studies indicate a role for Ca2+/CaM in regulating ubiquitination through direct interaction with TRE17.

TBC domain family, member 15 is a novel mammalian Rab GTPase-activating protein with substrate preference for Rab7

Ypt/Rabs are Ras-related GTPases that function as key regulators of intracellular vesicular trafficking. Their slow intrinsic rates of GTP hydrolysis are catalyzed by GTPase-activating proteins (GAPs). Ypt/Rab-GAPs constitute a family of proteins that contain a TBC (Tre-2/Bub2/Cdc16) domain. Only three of the 51 family members predicted in the human genome are confirmed Ypt/Rab-GAPs. Here, we report the identification and characterization of a novel mammalian Ypt/Rab-GAP, TBC domain family, member 15 (TBC1D15). TBC1D15 is ubiquitously expressed and localized predominantly to the cytosol. The TBC domain of TBC1D15 exhibits relatively high homology with that of Gyp7p, a yeast Ypt/Rab-GAP. Furthermore, TBC1D15 stimulates the intrinsic GTPase activity of Rab7, and to a lesser extent Rab11, but is essentially inactive towards Rab4 or Rab6. These data increase the number of mammalian TBC domain family members with demonstrated Rab-GAP activity to four, and suggest that TBC1D15 may be involved in Rab7-mediated late endosomal trafficking.

The TBC (Tre-2/Bub2/Cdc16) Domain Protein TRE17 Regulates Plasma Membrane-Endosomal Trafficking through Activation of Arf6

TBC (Tre-2/Bub2/Cdc16) domains are predicted to encode GTPase-activating proteins (GAPs) for Rab family G proteins. While approximately 50 TBC proteins are predicted to exist in humans, little is known about their substrate specificity. Here we show that TRE17 (also called Tre-2 and USP6), a founding member of the TBC family, targets the Arf family GTPase Arf6, which regulates plasma membrane-endosome trafficking. Surprisingly, TRE17 does not function as a GAP for Arf6 but rather promotes its activation in vivo. TRE17 associates directly with Arf6 in its GDP- but not GTP-bound state. Mapping experiments pinpoint the site of interaction to the TBC domain of TRE17. Forced expression of TRE17 promotes the localization of Arf6 to the plasma membrane, leading to Arf6 activation, presumably due to facilitated access to membrane-associated guanine nucleotide exchange factors (GEFs). Furthermore, TRE17 cooperates with Arf6 GEFs to induce GTP loading of Arf6 in vivo. Finally, short interfering RNA-mediated loss of TRE17 leads to attenuated Arf6 activation. These studies identify TRE17 as a novel regulator of the Arf6-regulated plasma membrane recycling system and reveal an unexpected function for TBC domains.

Candidate Genes Required for Embryonic Development: A Comparative Analysis of Distal Mouse Chromosome 14 and Human Chromosome 13q22

Mice homozygous for the Ednrbs-1Acrg deletion arrest at embryonic day 8.5 from defects associated with mesoderm development. To determine the molecular basis of this phenotype, we initiated a positional cloning of the Acrg minimal region. This region was predicted to be gene-poor by several criteria. From comparative analysis with the syntenic human locus at 13q22 and gene prediction program analysis, we found a single cluster of four genes within the 1.4-to 2-Mb contig over the Acrg minimal region that is flanked by a gene desert. We also found 130 highly conserved nonexonic sequences that were distributed over the gene cluster and desert. The four genes encode the TBC (Tre-2, BUB2, CDC16) domain-containing protein KIAA0603, the ubiquitin carboxy-terminal hydrolase L3 (UCHL3), the F-box/PDZ/LIM domain protein LMO7,and a novel gene. On the basis of their expression profile during development, all four genes are candidates for the Ednrbs-1Acrg embryonic lethality. Because we determined that a mutant of Uchl3 was viable, three candidate genes remain within the region.