The human EVI5 gene was originally isolated through its involvement with a constitutional chromosome translocation in a patient with stage 4S neuroblastoma. Recently, it has been shown that EVI5 is a centrosomal protein in interphase cells, which relocalizes to the midbody during late phases of mitosis. Disruption of its function leads to incomplete cell division and the formation of multinucleate cells. The EVI5 protein contains a TBC (TRE2/BUB/ CDC16 homology) motif located in the N-terminal region. Proteins containing a TBC domain have been shown in some cases to act as GTPase-activating proteins (GAPs) and function through the interaction with Rab-like small G proteins. Despite the identification of over 50 TBCcontaining proteins, and over 70 Rab-like proteins, only three combinations have been shown to have Rab/GAP activity to date. In this study, using linear ion trap mass spectroscopy, we have demonstrated that EVI5 exists in a protein complex with Rab11. Further, using a specific Rab-binding assay, we have shown that EVI5 preferentially interacts with the guanosine triphosphate-bound form of Rab11, and in a GAP activity assay, we have confirmed that EVI5 functions as a GAP for the Rab11 GTPase.Oncogene ( Proteins with homology to the so-called TBC domain, consisting of an B200-amino-acid motif initially identified in the TRE2/BUB2/CDC16 genes (Richardson and Zon, 1995), are considered to function as GTPaseactivating proteins (GAPs), partnering with Rab-like small G proteins. Rab GTPases are members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins that play critical roles in the regulation of important membrane and protein trafficking events in the cell. Many of the Rab proteins are associated with fundamental biological processes such as vesicle fusion, receptor recycling, membrane transport and cytokinesis (Zerial and McBride, 2001). There are, however, over 50 human proteins with predicted TBC domains, and over 70 human Rab proteins, of which only two TBC domain-containing proteins have so far been shown to demonstrably have Rab/GAP activity. The human EVI5 protein, which was identified at the breakpoint in a constitutional chromosome translocation in a patient with stage 4S neuroblastoma (Roberts et al., 1998), contains a TBC domain near its N terminus. EVI5 has been identified in the centrosome in interphase cells (Faitar et al., 2005) and in the midbody during the terminal stages of cytokinesis. Small interfering RNA knockdown of EVI5 results in multinucleate cells because of an inability of daughter cell abscission (Faitar et al., 2006). Because of the essential role of EVI5 in cytokinesis, as well as its implicated involvement in cancer development, we used a proteomics approach and identified the Rab protein that is activated by the EVI5 TBC domain.Rab proteins function by cycling between the biologically active GTP-bound form and the guanosine diphosphate (GDP)-bound inactive form. In the active GTP-bound conformation, these proteins can directly interact with specific eff...

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The EVI5 gene was originally identified in a screen for integration sites of ectopic viruses that gave rise to tumors in mice. The product of the gene is an 810 amino acids long ubiquitously expressed protein. It carries two functional domains, a Tre2/Bub2/Cdc16 (TBC) consensus sequence in the N‐terminal region, which is known to have a GTPase activating function, and an extensive coiled coil in the C‐terminal region. The goal of the present study was to elucidate the cellular localization and function of the EVI5 protein. A highly specific antibody against the C‐terminal section of the protein was used in an immunocytochemical analysis, and demonstrated that EVI5 is a nuclear protein, that is also present in the pericentriolar region of interphase cells. EVI5 is localized to the mitotic spindle during chromosome migration, and is also found in the midzone and midbody in late mitosis and during cytokinesis. Knockdown of EVI5 using siRNA results in a high incidence of binucleate cells with evidence for incomplete septation. The role of EVI5 in cytokinesis is further supported through the demonstration of its interaction with members of the chromosome passenger complex (INCENP, aurora B kinase and survivin). Taken together, these results suggest that EVI5 is involved in the completion of cytokinesis.This work was supported by NINDS grant NS35791 from the National Institutes of Health.