Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis

Abstract

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

Figure 1

(a) Schematic representation of the human ECT2 protein. The human ECT2 cDNA clone was isolated from a B5/589 human epithelial cell cDNA library. The detailed structure will be published elsewhere. CLB6, a domain homologous to a yeast S phase cyclin CLB6. BRCT1 and BRCT2, BRCA1 COOH-terminal repeats. DH, Dbl-homology domain. PH, pleckstrin-homology domain. NLS, nuclear localization signals. The regions carried by ECT2-F, ECT2-N, and ECT2-C are shown. (b) Guanine nucleotide exchange activity of ECT2 on Rho GTPases. (Left panel) Exchange activity of immunoprecipitates from FLAG-ECT2–expressing cells (open symbols) or FLAG-expressing cells (filled symbols) on RhoA (circles), Rac1 (squares), or Cdc42 (triangles). Shown are representative results of at least three independent experiments. (Right panel) FLAG-ECT2 immunoprecipitates were treated with the indicated concentrations (μg/ml) of VHR at 30°C for 1 h and then subjected to exchange assays on Rac1. V, immunoprecipitates from vector alone-transfectants. VHR-treated ECT2 immunoprecipitates did not show apparent degradation. (c) G2/M-specific phosphorylation of ECT2. HeLa cells were synchronized at G1/S boundary by thymidine/aphidicolin double block. ECT2 protein was detected with anti-ECT2 antibody in the cells as they progress through the cell cycle upon release from the drug arrest (upper left panel). Lysates of cells arrested at G1 phase by aphidicolin or at M phase by nocodazole were incubated with a protein phosphatase VHR, separated by SDS-PAGE, and analyzed for ECT2 (lower left panel). DNA contents of the cells in the above samples were analyzed by flow cytometry following the release from the G1 arrest (right panel). Positions of cells in G1 phase (2N) and G2/M phase (4N) are shown by arrowheads.

Figure 2

Subcellular localization of ECT2 in interphase and mitotic cells. Endogenous ECT2 in HeLa cells was detected by affinity-purified anti-ECT2 antibodies (green). Tubulin (red) and DNA (blue) were also stained with anti–β-tubulin antibody and 4′,6-diamidino-2′-phenylindole (DAPI), respectively. Merged images are also shown at the bottom, where colocalization of ECT2 and tubulin results in yellow color. a, interphase; b, prometaphase; c, metaphase; d, anaphase; e, telophase; f, cytokinesis.

Figure 3

Effects of a dominant negative mutant of ECT2 on cytokinesis. (A) Subcellular localization of exogenously expressed ECT2 protein. The full-length (ECT2-F; panels a and b), NH₂-terminal half (ECT2-N; panels c–f), or COOH-terminal half (ECT2-C; panels g and h) of ECT2 was transiently expressed in U2OS cells as a GFP-fused protein. GFP fusion proteins were detected by green fluorescence (panels a, c, e, and g). DNA was stained with DAPI (panels b, d, f, and h). (B) The NH₂-terminal domain of ECT2 acts as a dominant negative mutant for cytokinesis. Cells were transfected with GFP-fused ECT2-F, ECT2-N, or ECT2-C, or GFP vector alone. GFP-expressed cells are visualized by green fluorescence (panels a and c). DNA was stained with DAPI (panels b and d). Arrowheads indicate multinucleated cells. Bars, 20 μm. (Right panel) GFP-expressing multinucleated cells were scored under immunofluorescent microscopy 72 h after transfection. Data are average of three independent experiments.

Figure 4

Inhibition of cytokinesis by microinjection of anti-ECT2 antibodies. (A) Affinity-purified anti-ECT2 antibodies specific to the NH₂-terminal domain (αECT2-N), or the DH domain (αECT2-DH) were microinjected into unsynchronized cultures of HeLa cells. Injected cells were identified by immunostaining with anti–rabbit IgG antibody (panels a, d, and g). Cells were also stained for actin with phalloidine (panels b, e, and h) and for DNA with DAPI (panels c, f, and i) to determine the periphery of the cells and morphology of nuclei, respectively. Panels a–c show the morphology of cells injected with control antibody, where two daughter cells were observed. Microinjection of cells with αECT2-DH (panels d–f) or αΕCT2-N (panels g–i) resulted in single cells with two nuclei 24 h after injection. Longer incubation (48 h) also resulted in cells with more than three nuclei (data not shown). Bars, 10 μm. (B) Number of single interphase cells, normally divided cells, binucleated cells, and multinucleated cells are shown 24 or 48 h after microinjection of the designated antibodies. The last lane includes tri- and tetranucleated cells.