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Gas2l3, a novel constriction site-associated protein whose regulation is mediated by the APC/C Cdh1 complex.

Pe'er T, Lahmi R, Sharaby Y, Chorni E, Noach M, Vecsler M, Zlotorynski E, Steen H, Steen JA, Tzur A - PLoS ONE (2013)

Bottom Line: Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis.Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C(Cdh1) complex, but not by the APC/C(Cdc20) complex, and is phosphorylated by Cdk1 in mitosis.We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

View Article: PubMed Central - PubMed

Affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

ABSTRACT
Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C(Cdh1) complex, but not by the APC/C(Cdc20) complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

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Gas2l3 degradation is mediated by APC/CCdh1, but not by APC/CCdc20.(A) Incubation of IVT-generated Tome-1, Geminin, Gas2l3, and Gas2l3-DM4 in X. laevis interphase egg extracts (right) supplemented with recombinant Cdh1 (+) or buffer (-), or in extracts driven into mitosis (left) by preincubation with recombinant non-degradable Cyclin B1 protein [Δ90 (23)]. (B) APC/CCdc20-active extracts were made from 293 cells arrested in late mitosis by overexpression of His-tagged non-degradable hCyclin B1. Degradation of IVT-generated positive (Geminin) and negative (Tome-1) controls and of Gas2l3 was assayed in extracts supplemented with mock (-) or dominant negative UbcH10 (Ubch10DN). Time-dependent degradation was assayed by SDS-PAGE and autoradiography.
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pone-0057532-g002: Gas2l3 degradation is mediated by APC/CCdh1, but not by APC/CCdc20.(A) Incubation of IVT-generated Tome-1, Geminin, Gas2l3, and Gas2l3-DM4 in X. laevis interphase egg extracts (right) supplemented with recombinant Cdh1 (+) or buffer (-), or in extracts driven into mitosis (left) by preincubation with recombinant non-degradable Cyclin B1 protein [Δ90 (23)]. (B) APC/CCdc20-active extracts were made from 293 cells arrested in late mitosis by overexpression of His-tagged non-degradable hCyclin B1. Degradation of IVT-generated positive (Geminin) and negative (Tome-1) controls and of Gas2l3 was assayed in extracts supplemented with mock (-) or dominant negative UbcH10 (Ubch10DN). Time-dependent degradation was assayed by SDS-PAGE and autoradiography.

Mentions: The degradation of Gas2l3 was assayed in both mitotic and interphase frog egg extracts. Geminin, an APC/CCdc20 and APC/CCdh1 substrate, and Tome-1, an APC/CCdh1 substrate, were used as controls for the extracts’ activities and specificities. Geminin disappeared quickly in mitotic extracts unlike Tome-1, which remained stable and shifted to a high-mobility form, as previously reported (Figure 2A, left panels) [24]. Geminin is not degraded nor is Tome-1 modified in interphase extracts, thus excluding the possibility of contamination by mitotic APC/CCdc20-active extracts (Figure 2A, right panels). Gas2l3 was stable in mitotic extracts (Figure 2A, left panels), but degraded in a D-box–dependent manner in interphase extracts supplemented with recombinant Cdh1 (Figure 2A, right panels).


Gas2l3, a novel constriction site-associated protein whose regulation is mediated by the APC/C Cdh1 complex.

Pe'er T, Lahmi R, Sharaby Y, Chorni E, Noach M, Vecsler M, Zlotorynski E, Steen H, Steen JA, Tzur A - PLoS ONE (2013)

Gas2l3 degradation is mediated by APC/CCdh1, but not by APC/CCdc20.(A) Incubation of IVT-generated Tome-1, Geminin, Gas2l3, and Gas2l3-DM4 in X. laevis interphase egg extracts (right) supplemented with recombinant Cdh1 (+) or buffer (-), or in extracts driven into mitosis (left) by preincubation with recombinant non-degradable Cyclin B1 protein [Δ90 (23)]. (B) APC/CCdc20-active extracts were made from 293 cells arrested in late mitosis by overexpression of His-tagged non-degradable hCyclin B1. Degradation of IVT-generated positive (Geminin) and negative (Tome-1) controls and of Gas2l3 was assayed in extracts supplemented with mock (-) or dominant negative UbcH10 (Ubch10DN). Time-dependent degradation was assayed by SDS-PAGE and autoradiography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585356&req=5

pone-0057532-g002: Gas2l3 degradation is mediated by APC/CCdh1, but not by APC/CCdc20.(A) Incubation of IVT-generated Tome-1, Geminin, Gas2l3, and Gas2l3-DM4 in X. laevis interphase egg extracts (right) supplemented with recombinant Cdh1 (+) or buffer (-), or in extracts driven into mitosis (left) by preincubation with recombinant non-degradable Cyclin B1 protein [Δ90 (23)]. (B) APC/CCdc20-active extracts were made from 293 cells arrested in late mitosis by overexpression of His-tagged non-degradable hCyclin B1. Degradation of IVT-generated positive (Geminin) and negative (Tome-1) controls and of Gas2l3 was assayed in extracts supplemented with mock (-) or dominant negative UbcH10 (Ubch10DN). Time-dependent degradation was assayed by SDS-PAGE and autoradiography.
Mentions: The degradation of Gas2l3 was assayed in both mitotic and interphase frog egg extracts. Geminin, an APC/CCdc20 and APC/CCdh1 substrate, and Tome-1, an APC/CCdh1 substrate, were used as controls for the extracts’ activities and specificities. Geminin disappeared quickly in mitotic extracts unlike Tome-1, which remained stable and shifted to a high-mobility form, as previously reported (Figure 2A, left panels) [24]. Geminin is not degraded nor is Tome-1 modified in interphase extracts, thus excluding the possibility of contamination by mitotic APC/CCdc20-active extracts (Figure 2A, right panels). Gas2l3 was stable in mitotic extracts (Figure 2A, left panels), but degraded in a D-box–dependent manner in interphase extracts supplemented with recombinant Cdh1 (Figure 2A, right panels).

Bottom Line: Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis.Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C(Cdh1) complex, but not by the APC/C(Cdc20) complex, and is phosphorylated by Cdk1 in mitosis.We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

View Article: PubMed Central - PubMed

Affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

ABSTRACT
Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C(Cdh1) complex, but not by the APC/C(Cdc20) complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

Show MeSH
Related in: MedlinePlus