Opposing roles of p190RhoGAP and Ect2 RhoGEF in regulating cytokinesis

Abstract

Evidence suggests that p190RhoGAP (p190), a GTPase activating protein (GAP) specific for Rho, plays a role in cytokinesis. First, ectopic expression of p190 induces a multinucleated cellular phenotype. Second, endogenous p190 localizes to the cleavage furrow of dividing cells. Lastly, its levels are reduced in late mitosis by ubiquitin-mediated proteasomal degradation, consistent with the idea that low levels of p190 and high levels of active Rho are required for completion of cytokinesis. As with p190, RhoA and the RhoGEF, ECT2, have been localized to the cleavage furrow. These findings raise the question of whether p190 and ECT2 cooperate antagonistically to regulate the activity of Rho and contraction of the actomyosin ring during cytokinesis. Here we demonstrate ECT2 can, in a dose-dependent manner, reduce multinucleation induced by p190. Furthermore, endogenous p190 and ECT2 colocalize at the cleavage furrow of dividing cells and stably associate with one another in co-immunoprecipitation assays. Functional and physical interactions between p190 and ECT2 are reflected in the levels of Rho activity, as assessed by Rho pull-down assays. Together, these results suggest that co-regulation of Rho activity by p190RhoGAP and ECT2 in the cleavage furrow determines whether cells properly complete cytokinesis.

Fig. 1. Coexpression of ECT2 RhoGEF reduces p190-induced multinucleation

(A) Coexpression of ECT2 reduces p190-induced multinucleation in a dose-dependent manner. Varied amounts of wild type, FLAG-tagged ECT2 plasmid (as indicated) were co-transfected with a constant amount of HA-tagged p190 plasmid into Hela cells as described in Methods. Forty-eight hrs later, cells were analyzed by immunofluorescence for the presence of HA-p190- and Flag-ECT2- expressing cells that displayed a multinucleated phenotype. Data are presented as the Mean ± SEM for n≥3. * represents p<0.05. (B) Full length and constitutively active FLAG-ECT2 reduce multinucleation in HA-p190-overexpressing cells. Equal moles of plasmids encoding wild type or mutant FLAG-ECT2 were co-expressed with HA-p190 in HeLa cells, and transfected cells were analyzed as in Panel A. Data are presented as Mean ± SEM for n≥3. * represents p<0.05. (C) Dominant negative ECT2 induces multinucleation. Equal moles of plasmids encoding wild type or mutant Flag ECT2 were transfected into Hela cells, and transfected cells were analyzed as in Panel A. Data are presented as the Mean ± SEM for n≥3. * represents p<0.05. (D) Western blotting of whole cell lysates from HA-p190 and FLAG-ECT2 co-expressing cells analyzed in Panel B. Thirty µg whole cell lysates were immunoblotted with anti-FLAG antibody to detect levels of E (ECT2), EN (ECT2N), and EC (ECT2C), and with anti-HA antibody to detect p190.

Fig. 2. p190 and ECT2 form stable complexes

(A) Western blotting of endogenous or ectopically expressed p190 and ECT2 in whole cell lysates. HeLa cells were left untransfected or transfected with 0.5 nM HA-p190 or transfected with FLAG-ECT2 plasmids alone, and synchronized in mitosis (M) as described in Methods or left as cycling (C) populations. Thirty µg whole cell lysates were immunoblotted with anti-FLAG or anti-ECT2 antibody to detect levels of ECT2 or with anti-HA or anti-p190 antibody to detect p190. (B) Reciprocal co-immunoprecipitation of p190 and ECT2 in singly transfected cells. HA-p190 or Flag-ECT2 plasmids were individually transfected into Hela cells, as indicated. C (cycling ~95% in interphase) and M (mitotic after 2 hr release from prometaphase) cells were collected, and 1mg whole cell lysate was immunoprecipitated with mouse anti-p190 (UBI) or rabbit anti-ECT2 (Santa Cruz), and immunoblotted with anti-ECT2 (Santa Cruz) or anti-p190 (Becton Dickinson) antibodies, as described in Methods. ECT2 and p190 preferentially and reciprocally co-immunoprecipitated from mitotic cells. (C) Co-immunoprecipitation of endogenous p190 with endogenous ECT2. Untransfected HeLa cells were synchronized in mitosis (M) or left as a cycling population and immunoprecipitated as in Panel B. P190 specifically co-immunoprecipitated with ECT2 in both cycling and mitotic cells. However, reciprocal co-precipitation was not observed in this experiment (see text).

Fig. 3. The C-terminal half of ECT2 preferentially interacts with p190

(A) Co-immunoprecipitation of endogenous p190 with ECT2 variants. HeLa cells were transfected with wild type FLAG-ECT2, FLAG-ECT2N, or FLAG-ECT2C and synchronized at mitosis (M) or left as a cycling (C) population. One mg protein lysate was immunoprecipitated with mouse anti-p190 antibody (UBI). Immunoprecipitates were immunoblotted with mouse anti-p190 antibody (Becton-Dickinson) or anti-FLAG antibody. FLAG-ECT2, FLAG-ECT2N, and FLAG-ECT2C co-immunoprecipitated with p190 from both cycling and mitotic cell extracts, with preferential association observed between p190 and ECT2C. (B) Western blotting of endogenous p190 or FLAG-tagged, wild type ECT2, ECT2N, and ECT2C overexpressing cells. Thirty µg whole cell lysates from cells in Panel A were immunoblotted with anti-FLAG antibody to detect levels of ECT2 or with anti-p190 (Becton Dickinson) to detect p190.

Fig. 4. Colocalization of endogenous p190 and ECT2 during mitosis

Hela cells were synchronized in mitosis, released for various lengths of time, and collected at each mitotic phase: inter- (interphase), pro- (prophase), meta- (metaphase), ana-(anaphase), telo- (telophase), and cytokinesis. Endogenous p190 and ECT2 were immunostained with mouse anti-p190 and rabbit-ECT2 antibody, respectively, and analyzed by immunofluorescence microscopy as described in Methods. Co-localization of p190 and ECT2 was observed in the cleavage furrow.

Fig. 5. Rho activity inversely correlates with incidence of multinucleation

HeLa cells were nucleofected with vector or designated plasmids, synchronized, and released for 1.5 hrs. At this time, cell lysates were prepared and used for Rho-pull-down assays, as described in Methods. (A) Representative Western immunoblot of GTP-bound RhoA pulled down with GST-RBD agarose beads (upper panel) and total Rho in 30 µg whole cell lysate (lower panel). (B) Western immunoblots in Panel A and two additional experiments were quantified by the Alpha EC program. Data are presented as the Mean ratio of RhoGTP/Total RhoA ± SEM, n=3 in each cell population. *represents a statistically significant difference from vector control (p-value<0.05).