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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 cell cycle-regulated protein whose degradation is mediated by the APC/C.(A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled (35S) methionine. In vitro transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) 35S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).
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pone-0057532-g001: Gas2l3 is a cell cycle-regulated protein whose degradation is mediated by the APC/C.(A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled (35S) methionine. In vitro transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) 35S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).

Mentions: We found the gene encoding for growth arrest-specific 2-like protein 3 (Gas2l3) to be a putative cell cycle regulator in vivo using gene expression analysis of proliferating vs. resting murine B cells [20]. Gas2l3 belongs to the Gas2 subfamily of microtubule/Actin cross-linking factor proteins [13], [14], [15]. Of the Gas2 family, the GAS2, GAS2L1, and GAS2L3 genes were analyzed in the GSE4142 dataset [20] and out of the three, only GAS2L3, much like the mitotic cyclin A2 (CCNA2), is highly expressed in the two proliferating B-cell types (germinal center [GC] and plasmablasts) compared to the two resting B-cell types (naïve and memory). In comparison, GAS2L1 is highly expressed only in GC cells, and the expression of GAS2 is roughly equal in the four B-cell types (Figure 1A). Expression analysis of GAS2L3 in synchronous HeLa S3 (S3) cells has also shown that this gene is periodically expressed in proliferating cells in a manner similar to that of the mitotic Cyclin A2 (Figure 1B).


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 cell cycle-regulated protein whose degradation is mediated by the APC/C.(A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled (35S) methionine. In vitro transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) 35S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585356&req=5

pone-0057532-g001: Gas2l3 is a cell cycle-regulated protein whose degradation is mediated by the APC/C.(A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled (35S) methionine. In vitro transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) 35S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).
Mentions: We found the gene encoding for growth arrest-specific 2-like protein 3 (Gas2l3) to be a putative cell cycle regulator in vivo using gene expression analysis of proliferating vs. resting murine B cells [20]. Gas2l3 belongs to the Gas2 subfamily of microtubule/Actin cross-linking factor proteins [13], [14], [15]. Of the Gas2 family, the GAS2, GAS2L1, and GAS2L3 genes were analyzed in the GSE4142 dataset [20] and out of the three, only GAS2L3, much like the mitotic cyclin A2 (CCNA2), is highly expressed in the two proliferating B-cell types (germinal center [GC] and plasmablasts) compared to the two resting B-cell types (naïve and memory). In comparison, GAS2L1 is highly expressed only in GC cells, and the expression of GAS2 is roughly equal in the four B-cell types (Figure 1A). Expression analysis of GAS2L3 in synchronous HeLa S3 (S3) cells has also shown that this gene is periodically expressed in proliferating cells in a manner similar to that of the mitotic Cyclin A2 (Figure 1B).

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