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

Tatsumoto T, Xie X, Blumenthal R, Okamoto I, Miki T - J. Cell Biol. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.

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.

Show MeSH
(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.
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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.

Mentions: To examine the function of ECT2 as a Rho GEF, we expressed ECT2 as a FLAG epitope-tagged protein in COS cells immunoprecipitated with anti-FLAG mAb, and used immunoprecipitated protein for exchange assays. Full-length ECT2 efficiently stimulated nucleotide exchange on three representative members of the Rho GTPases, RhoA, Rac1, and Cdc42 in vitro, whereas similar immunoprecipitates from vector transfectants showed no significant activity (Fig. 1 b, left panel). In contrast to the activity on Rho GTPases, ECT2 did not exhibit significant exchange activity on two Ras family GTPases, H-Ras and Rap1A (data not shown).


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

Tatsumoto T, Xie X, Blumenthal R, Okamoto I, Miki T - J. Cell Biol. (1999)

(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.
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Related In: Results  -  Collection

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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.
Mentions: To examine the function of ECT2 as a Rho GEF, we expressed ECT2 as a FLAG epitope-tagged protein in COS cells immunoprecipitated with anti-FLAG mAb, and used immunoprecipitated protein for exchange assays. Full-length ECT2 efficiently stimulated nucleotide exchange on three representative members of the Rho GTPases, RhoA, Rac1, and Cdc42 in vitro, whereas similar immunoprecipitates from vector transfectants showed no significant activity (Fig. 1 b, left panel). In contrast to the activity on Rho GTPases, ECT2 did not exhibit significant exchange activity on two Ras family GTPases, H-Ras and Rap1A (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Tumor Biology Section, Basic Research Laboratory, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.

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.

Show MeSH