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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 is a Cdk1 target.(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.
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pone-0057532-g003: Gas2l3 is a Cdk1 target.(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.

Mentions: Geminin, Tome-1, and Gas2l3 degradation was also examined in somatic cell extracts with high APC/CCdc20 activity made from 293 cells arrested in late mitosis by the overexpression of D-box mutant full-length Cyclin B1 [25]. This extract system, described here for the first time, can be referred to as the human equivalent of the X. laevis mitotic extracts, in which APC/CCdc20 is highly active (for further details, see the Materials and Methods section). Geminin is quickly degraded in this system but is stabilized in the presence of Ubch10DN – the dominant negative derivate of the E2 enzyme UbcH10– thus excluding the presence of any non-specific proteolytic activity in these extracts (Figure 2B). In contrast, Tome-1 remains stable irrespective of the presence of UbcH10DN (Figure 2B), and shifts to the high-mobility form, as in X. laevis mitotic extracts (Figure 2A). Like Tome-1, Gas2l3 is stable in late mitosis extracts (Figure 2B) but is degraded in G1-phase cell extracts containing the active APC/CCdh1 complex (Figure 1E). Additionally, the high-mobility forms of Gas2l3, as well as those of the two known Cdk1 targets Tome-1 and Securin [24], [28], are undetectable in the presence of the Cdk1 inhibitors RO-3306 and roscovitine (Figure 3A), suggesting that Gas2l3 is a target of Cdk1. Mass-spec analysis of gel-extracted proteins from 293 cells overexpressing Myc-tagged Gas2l3 and either non-degradable Cyclin B1 (late mitosis [M]) or empty vector (unsynchronized [US]), revealed two Cdk1 sites in Gas2l3. Moreover, these two peptides were found to be phosphorylated only in the late mitotic extracts (Figure 3B). Taken together, these results show that Gas2l3 is targeted for degradation by the APC/CCdh1 complex, but not by the APC/CCdc20 complex, and is a Cdk1 substrate.


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 is a Cdk1 target.(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057532-g003: Gas2l3 is a Cdk1 target.(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.
Mentions: Geminin, Tome-1, and Gas2l3 degradation was also examined in somatic cell extracts with high APC/CCdc20 activity made from 293 cells arrested in late mitosis by the overexpression of D-box mutant full-length Cyclin B1 [25]. This extract system, described here for the first time, can be referred to as the human equivalent of the X. laevis mitotic extracts, in which APC/CCdc20 is highly active (for further details, see the Materials and Methods section). Geminin is quickly degraded in this system but is stabilized in the presence of Ubch10DN – the dominant negative derivate of the E2 enzyme UbcH10– thus excluding the presence of any non-specific proteolytic activity in these extracts (Figure 2B). In contrast, Tome-1 remains stable irrespective of the presence of UbcH10DN (Figure 2B), and shifts to the high-mobility form, as in X. laevis mitotic extracts (Figure 2A). Like Tome-1, Gas2l3 is stable in late mitosis extracts (Figure 2B) but is degraded in G1-phase cell extracts containing the active APC/CCdh1 complex (Figure 1E). Additionally, the high-mobility forms of Gas2l3, as well as those of the two known Cdk1 targets Tome-1 and Securin [24], [28], are undetectable in the presence of the Cdk1 inhibitors RO-3306 and roscovitine (Figure 3A), suggesting that Gas2l3 is a target of Cdk1. Mass-spec analysis of gel-extracted proteins from 293 cells overexpressing Myc-tagged Gas2l3 and either non-degradable Cyclin B1 (late mitosis [M]) or empty vector (unsynchronized [US]), revealed two Cdk1 sites in Gas2l3. Moreover, these two peptides were found to be phosphorylated only in the late mitotic extracts (Figure 3B). Taken together, these results show that Gas2l3 is targeted for degradation by the APC/CCdh1 complex, but not by the APC/CCdc20 complex, and is a Cdk1 substrate.

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