Abstract
The TRE2 oncoprotein is structurally related to the RabGAP (GTPase-activating protein) family. However, TRE2 seems enzymatically inactive. Two regions are important for its lack of GAP activity. First, the TBC domain, forming the catalytically active domain of RabGAPs, is non-functional in the oncoprotein. Also involved in TRE2 inactivity is the 93-aa region flanking the TBC domain on the C-terminal side. In order to identify the residues responsible for non-functionality, we performed hydrophobic cluster analysis of the oncoprotein sequence, combined with secondary structure prediction, receptor-binding domain analysis, and a tilted peptide calculation. These analyses were complemented with site-directed and random mutagenesis experiments. On the basis of our data, we hypothesize that the lack of secondary structure of the region flanking the TBC domain in TRE2 may explain why this region plays a role in the lack of GAP activity, even when a potentially functional TBC domain is present.
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References
Adams A, Gottschling DE, Kaiser CA et al (1997) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York
Albert S, Gallwitz D (2000) Msb4p, a protein involved in Cdc42p-dependent organization of the actin cytoskeleton, is a Ypt/Rab-specific GAP. Biol Chem 381:453–456
Albert S, Will E, Gallwitz D (1999) Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases. EMBO J 18:5216–5225
Ausubel FM, Brent R, Kingston RE et al (1987) Current protocols in molecular biology. Wiley, New York
Bach S, Bouchat O, Portetelle D et al (2000) Co-deletion of the MSB3 and MSB4 coding regions affects bipolar budding and perturbs the organization of the actin cytoskeleton. Yeast 16:1015–1023
Beukers MW, Ijzerman AP (2005) Techniques: how to boost GPCR mutagenesis studies using yeast. Trends Pharmacol Sci 26:533–539
Bizimungu C, Vandenbol M (2005) At least two regions of the oncoprotein Tre2 are involved in its lack of GAP activity. Biochem Biophys Res Commun 335:883–890
Bizimungu C, De Neve N, Burny A et al (2003) Expression in a RabGAP yeast mutant of two human homologues, one of which is an oncogene. Biochem Biophys Res Commun 310:498–504
Brasseur R (2000) Tilted peptides: a motif for membrane destabilization (hypothesis). Mol Membr Biol 17:31–40
Brasseur R, Vandenbranden M, Cornet B et al (1990) Orientation into the lipid bilayer of an asymmetric amphipathic helical peptide located at the N-terminus of viral fusion proteins. Biochim Biophys Acta 1029:267–273
Dechamps C, Bach S, Portetelle D et al (2006) The Tre2 oncoprotein, implicated in Ewing’s sarcoma, interacts with two components of the cytoskeleton. Biotechnol Lett 28:223–231
Gaboriaud C, Bissery V, Benchetrit T et al (1987) Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences. FEBS Lett 224:149–155
Gallet X, Charloteaux B, Thomas A et al (2000) A fast method to predict protein interaction sites from sequences. J Mol Biol 302:917–926
Gao XD, Albert S, Tcheperegine SE et al (2003) The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization. J Cell Biol 162:635–646
Haas AK, Fuchs E, Kopajtich R et al (2005) A GTPase-activating protein controls Rab5 function in endocytic trafficking. Nat Cell Biol 7:887–893
Inga A, Resnick MA (2001) Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactivate tumor p53 mutants. Oncogene 20:3409–3419
Lanzetti L, Rybin V, Malabarba MG et al (2000) The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408:374–377
Lanzetti L, Palamidessi A, Areces L et al (2004) Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature 429:309–314
Masuda-Robens JM, Kutney SN, Qi H et al (2003) The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling. Mol Cell Biol 23:2151–2161
Nakamura T, Hillova J, Mariage-Samson R et al (1992) A novel transcriptional unit of the tre oncogene widely expressed in human cancer cells. Oncogene 7:733–741
Pandey N, Ganapathi M, Kumar K et al (2004) Comparative analysis of protein unfoldedness in human housekeeping and non-housekeeping proteins. Bioinformatics 20:2904–2910
Paulding CA, Ruvolo M, Haber DA (2003) The Tre2 (USP6) oncogene is a hominoid-specific gene. Proc Natl Acad Sci U S A 100:2507–2511
Rak A, Fedorov R, Alexandrov K et al (2000) Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins. EMBO J 19:5105–5113
Seabra MC, Wasmeier C (2004) Controlling the location and activation of Rab GTPases. Curr Opin Cell Biol 16:451–457
Segev N (2001) Ypt/rab gtpases: regulators of protein trafficking. Sci STKE 2001:RE11
Tcheperegine SE, Gao XD, Bi E (2005) Regulation of cell polarity by interactions of Msb3 and Msb4 with Cdc42 and polarisome components. Mol Cell Biol 25:8567–8580
Thomas A, Allouche M, Basyn F et al (2005) Role of the lid hydrophobicity pattern in pancreatic lipase activity. J Biol Chem 280:40074–40083
Will E, Gallwitz D (2001) Biochemical characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating protein from yeast. J Biol Chem 276:12135–12139
Acknowledgements
We wish to thank M. Breban for her excellent help in random mutagenesis and M. Prévot for his technical assistance in yeast genetics (Unité de Biologie Animale et Microbienne, Gembloux, Belgium). This work was supported by the Belgian “Fonds National de la Recherche Scientifique” (FNRS). C. Bizimungu is a recipient of an “FNRS-Télévie” grant.
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Bizimungu, C., Thomas, A., Brasseur, R. et al. Mutational analysis of the TRE2 oncogene encoding an inactive RabGAP. Biotechnol Lett 29, 1927–1937 (2007). https://doi.org/10.1007/s10529-007-9475-6
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DOI: https://doi.org/10.1007/s10529-007-9475-6