Accumulation of GTP-bound RhoA during cytokinesis and a critical role of ECT2 in this accumulation

We developed a new pull-down assay for GTP-Rho and examined its level during cell cycle. HeLa cells were arrested in the S phase by thymidine and were enriched in the prometaphase, metaphase, telophase, and G(1) phase by collecting at 0, 45, 90, and 180 min after the release from the nocodazole arrest, respectively. The level of GTP-Rho did not change significantly from the S phase to the prometaphase, but increased thereafter, peaking in the telophase, and returned to the original level in the G(1) phase. The GDP-GTP exchange activity for Rho measured in cell lysates in parallel increased also during the mitosis with a peak in the metaphase. Using this system, we examined a role of ECT2, an exchanger for Rho GTPases, suggested to be involved in cytokinesis (Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki., T. (1999) J. Cell. Biol, 147, 921-928). Expression of the dominant negative form of ECT2 completely suppressed both the rise of GTP-Rho in the telophase and the increased GDP-GTP exchange activity in the mitotic cell extracts. These results suggest a critical role of ECT2 in Rho activation during cytokinesis.