Identification of Rab11 as a small GTPase binding protein for the Evi5 oncogene

Abstract

The Evi5 oncogene has recently been shown to regulate the stability and accumulation of critical G(1) cell cycle factors including Emi1, an inhibitor of the anaphase-promoting complex/cyclosome, and cyclin A. Sequence analysis of the amino terminus of Evi5 reveals a Tre-2, Bub2, Cdc16 domain, which has been shown to be a binding partner and GTPase-activating protein domain for the Rab family of small Ras-like GTPases. Here we describe the identification of Evi5 as a candidate binding protein for Rab11, a GTPase that regulates intracellular transport and has specific roles in endosome recycling and cytokinesis. By yeast two-hybrid analysis, immunoprecipitation, and Biacore analysis, we demonstrate that Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. However, Evi5 displays no activation of Rab11 GTPase activity in vitro. Evi5 colocalizes with Rab11 in vivo, and overexpression of Rab11 perturbs the localization of Evi5, redistributing it into Rab11-positive recycling endosomes. Interestingly, in vitro binding studies show that Rab11 effector proteins including FIP3 compete with Evi5 for binding to Rab11, suggesting a partitioning between Rab11-Evi5 and Rab11 effector complexes. Indeed, ablation of Evi5 by RNA interference causes a mislocalization of FIP3 at the abscission site during cytokinesis. These data demonstrate that Evi5 is a Rab11 binding protein and that Evi5 may cooperate with Rab11 to coordinate vesicular trafficking, cytokinesis, and cell cycle control independent of GTPase-activating protein function.

Fig. 1.

Evi5 binds Rab11a and Rab11b in a GTP-dependent manner. (A) Recombinant GST-tagged Rab11 and MBP-tagged Evi5N proteins bind in vitro. GST or GST–Rab11a was immobilized on glutathione-Sepharose and then tested for its ability to bind MBP–Evi5N in the presence of either GDP or GMP-PNP (nonhydrolyzable GTP analog). Captured proteins were detected by immunoblotting with antibodies against Evi5 and GST. (B) Evi5 specifically binds to the active forms of Rab11a and Rab11b by surface plasmon resonance. MBP–Evi5N was immobilized on a Biacore chip and tested for binding to various Rab and ARF GTPases. GTPases were preloaded with either GDP or GMP-PNP to assay the nucleotide dependency of the interaction. (C) Recombinant Evi5 binds activated Rab11 expressed in mammalian cells. GST–Rab fusion proteins were transiently expressed in 293T cells and captured on glutathione-Sepharose beads before addition of MBP–Evi5N. Bead-bound proteins were detected by immunoblotting with antibodies against Evi5 and GST as in A. (D) Evi5 does not display significant GAP activity toward Rab11 in vitro. Bacterially expressed GST–Rab proteins were preloaded with [α-³²P]GTP and incubated with MBP or MBP–Evi5N protein at 30°C. Rab GTPase activity is expressed as the percentage of total nucleotide converted to GDP after incubation with Evi5 or control protein.

Fig. 2.

Evi5 and Rab11 bind in vivo. (A) Endogenous Evi5 and Rab11a proteins colocalize in the pericentriolar region of the cell. U2OS cells were processed for immunofluorescence analysis by using affinity-purified anti-Evi5 and anti-Rab11 antibodies, as well as Hoechst to mark DNA. (B) Endogenous Evi5 and Rab11a proteins form a complex in vivo. Equal amounts of 293T cell lysate were subjected to immunoprecipitation by using either rabbit IgG or antibody against Rab11a. Captured immune complexes were eluted with sample buffer and subjected to SDS/PAGE and immunoblotting with anti-Evi5 antibody.

Fig. 3.

Expressed wild type and constitutively active Rab11 recruits endogenous Evi5 to Rab11-positive endosomes. U2OS cells were transfected with GFP–Rab11 wild type or inactive S25N mutant, fixed with 4% PFA, and stained with anti-Evi5 antibody as well as Hoechst to mark the DNA. White arrows highlight sample regions of colocalization, which appear yellow in the merged panel.

Fig. 4.

Evi5 regulates Rab11 effector function in vitro and in vivo. (A) FIP3 competes with Evi5 for binding to Rab11a. Glutathione-Sepharose beads were incubated with GST–Rab11a GMP-PNP and then washed. The Rab11a beads were then incubated with a fixed amount of MBP–Evi5N protein and an increasing molar ratio of FIP3 protein. After washing the beads, bound proteins were eluted with SDS sample buffer and analyzed by SDS/PAGE followed by silver staining. (B) Evi5 depletion causes mislocalization of FIP3 during late cytokinesis. HeLa cells stably expressing GFP–FIP3 were transfected with control or Evi5 siRNAs for 48 h and then processed for immunofluorescence analysis by using antibodies against MKLP1 to mark the midbody and Hoechst to mark the DNA. In each panel, the midbody of the dividing cell (outlined in white) is magnified and shown in Right for additional clarity.