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A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Xie H, Laird DW, Chang TH, Hu VW - J. Cell Biol. (1997)

Bottom Line: This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m).Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A.The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The George Washington University, DC 20037, USA.

ABSTRACT
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

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Western blot analyses revealed that Cx43 in mitotic cells  from HUVEC strains HX1 and H101 and rat vascular smooth  muscle cells had slower mobility, with a relative molecular mass  of 47–48. Lanes 1 and 2, control nonmitotic and colcemid-arrested  mitotic HX1 cells, respectively; lanes 3 and 4, control nonmitotic  and nocodazole-arrested mitotic HX1 cells, respectively; lanes 5  and 6, mitotic HX1 and H101 HUVEC cells, respectively; lanes 7  and 8, nonmitotic and mitotic rat vascular smooth muscle cells,  respectively.
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Figure 1: Western blot analyses revealed that Cx43 in mitotic cells from HUVEC strains HX1 and H101 and rat vascular smooth muscle cells had slower mobility, with a relative molecular mass of 47–48. Lanes 1 and 2, control nonmitotic and colcemid-arrested mitotic HX1 cells, respectively; lanes 3 and 4, control nonmitotic and nocodazole-arrested mitotic HX1 cells, respectively; lanes 5 and 6, mitotic HX1 and H101 HUVEC cells, respectively; lanes 7 and 8, nonmitotic and mitotic rat vascular smooth muscle cells, respectively.

Mentions: Western blot analyses with a monoclonal antibody against Cx43 revealed a higher relative molecular mass isoform of Cx43 in mitotic cells (Fig. 1, lanes 2, 4, 5, 6, and 8). This unique species, designated Cx43m, was estimated at 47–48 Mr and was not restricted to a single cell type inasmuch as two strains of HUVEC (strains HX1 and H101; Fig. 1, lanes 5 and 6, respectively) as well as rat vascular smooth muscle cells (Fig. 1, lanes 7 and 8) exhibited this mitosisspecific modification. As shown in Fig. 1, mitotic cells contained predominantly one Cx43 species of higher relative molecular mass, while control cells contained up to three species of lower relative molecular mass. This mobility shift is unlikely to be the direct result of treatment with a specific mitotic inhibitor in that both colcemid (lane 2) and nocodazole (lane 4) arrest gave rise to Cx43 bands of similar mobility, and unsynchronized control cells that were treated for a few hours with nocodazole contained only the faster mobility forms of Cx43 (data not shown). Moreover, the mobility shift of Cx43 observed in mitotic cells is unrelated to a general rounding-up effect per se since the control cells were detached from the substratum with trypsin (causing the cells to round-up) before being lysed with the SDS gel-loading buffer.


A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Xie H, Laird DW, Chang TH, Hu VW - J. Cell Biol. (1997)

Western blot analyses revealed that Cx43 in mitotic cells  from HUVEC strains HX1 and H101 and rat vascular smooth  muscle cells had slower mobility, with a relative molecular mass  of 47–48. Lanes 1 and 2, control nonmitotic and colcemid-arrested  mitotic HX1 cells, respectively; lanes 3 and 4, control nonmitotic  and nocodazole-arrested mitotic HX1 cells, respectively; lanes 5  and 6, mitotic HX1 and H101 HUVEC cells, respectively; lanes 7  and 8, nonmitotic and mitotic rat vascular smooth muscle cells,  respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139857&req=5

Figure 1: Western blot analyses revealed that Cx43 in mitotic cells from HUVEC strains HX1 and H101 and rat vascular smooth muscle cells had slower mobility, with a relative molecular mass of 47–48. Lanes 1 and 2, control nonmitotic and colcemid-arrested mitotic HX1 cells, respectively; lanes 3 and 4, control nonmitotic and nocodazole-arrested mitotic HX1 cells, respectively; lanes 5 and 6, mitotic HX1 and H101 HUVEC cells, respectively; lanes 7 and 8, nonmitotic and mitotic rat vascular smooth muscle cells, respectively.
Mentions: Western blot analyses with a monoclonal antibody against Cx43 revealed a higher relative molecular mass isoform of Cx43 in mitotic cells (Fig. 1, lanes 2, 4, 5, 6, and 8). This unique species, designated Cx43m, was estimated at 47–48 Mr and was not restricted to a single cell type inasmuch as two strains of HUVEC (strains HX1 and H101; Fig. 1, lanes 5 and 6, respectively) as well as rat vascular smooth muscle cells (Fig. 1, lanes 7 and 8) exhibited this mitosisspecific modification. As shown in Fig. 1, mitotic cells contained predominantly one Cx43 species of higher relative molecular mass, while control cells contained up to three species of lower relative molecular mass. This mobility shift is unlikely to be the direct result of treatment with a specific mitotic inhibitor in that both colcemid (lane 2) and nocodazole (lane 4) arrest gave rise to Cx43 bands of similar mobility, and unsynchronized control cells that were treated for a few hours with nocodazole contained only the faster mobility forms of Cx43 (data not shown). Moreover, the mobility shift of Cx43 observed in mitotic cells is unrelated to a general rounding-up effect per se since the control cells were detached from the substratum with trypsin (causing the cells to round-up) before being lysed with the SDS gel-loading buffer.

Bottom Line: This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m).Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A.The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The George Washington University, DC 20037, USA.

ABSTRACT
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

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