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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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Spatial distribution of Cx43 in mitotic and nonmitotic HX1 cells. Transmitted light images (A and C) of mitotic cells immunolabeled for Cx43 (B and D) show the spatial distribution of Cx43. Note the punctate labeling of Cx43 in intracellular compartments (arrows). (E) Reconstructed image of Cx43 in mitotic HX1 cells. Mitotic cells immunolabeled for Cx43 were optically sectioned and three  images collected at 1-μm intervals were superimposed to illustrate a succession of Cx43-positive cellular locations (arrows). The mitotic  cell in the insert is a reconstruction of five images collected at 1-μm intervals. (F) A confocal microscopic image for Cx43 in nonmitotic  HX1 cells revealed punctate labeling at sites of cell–cell apposition with little intracellular labeling. Bars, 10 μm.
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Figure 5: Spatial distribution of Cx43 in mitotic and nonmitotic HX1 cells. Transmitted light images (A and C) of mitotic cells immunolabeled for Cx43 (B and D) show the spatial distribution of Cx43. Note the punctate labeling of Cx43 in intracellular compartments (arrows). (E) Reconstructed image of Cx43 in mitotic HX1 cells. Mitotic cells immunolabeled for Cx43 were optically sectioned and three images collected at 1-μm intervals were superimposed to illustrate a succession of Cx43-positive cellular locations (arrows). The mitotic cell in the insert is a reconstruction of five images collected at 1-μm intervals. (F) A confocal microscopic image for Cx43 in nonmitotic HX1 cells revealed punctate labeling at sites of cell–cell apposition with little intracellular labeling. Bars, 10 μm.

Mentions: To determine the distribution pattern of Cx43 during mitosis, mitotic cells were immunolabeled for Cx43 and analyzed on a confocal microscope. The transmitted light images (Fig. 5, A and C) of mitotic cells in comparison with the corresponding confocal immunofluorescent images (Fig. 5, B and D) show that Cx43 was often localized to intracellular compartments when optical sections were taken through the center of the cell. Moreover, optical slice reconstructions revealed a substantial amount of redistributed Cx43 in mitotic cells (Fig. 5 E, arrows) as well as an overall increase in intracellular cytoplasmic staining. Conversely, nonmitotic cells that remained on the substrate after mitotic shake-off showed the typical distribution of Cx43, principally at locations of cell–cell contact (Fig. 5 F).


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)

Spatial distribution of Cx43 in mitotic and nonmitotic HX1 cells. Transmitted light images (A and C) of mitotic cells immunolabeled for Cx43 (B and D) show the spatial distribution of Cx43. Note the punctate labeling of Cx43 in intracellular compartments (arrows). (E) Reconstructed image of Cx43 in mitotic HX1 cells. Mitotic cells immunolabeled for Cx43 were optically sectioned and three  images collected at 1-μm intervals were superimposed to illustrate a succession of Cx43-positive cellular locations (arrows). The mitotic  cell in the insert is a reconstruction of five images collected at 1-μm intervals. (F) A confocal microscopic image for Cx43 in nonmitotic  HX1 cells revealed punctate labeling at sites of cell–cell apposition with little intracellular labeling. Bars, 10 μm.
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getmorefigures.php?uid=PMC2139857&req=5

Figure 5: Spatial distribution of Cx43 in mitotic and nonmitotic HX1 cells. Transmitted light images (A and C) of mitotic cells immunolabeled for Cx43 (B and D) show the spatial distribution of Cx43. Note the punctate labeling of Cx43 in intracellular compartments (arrows). (E) Reconstructed image of Cx43 in mitotic HX1 cells. Mitotic cells immunolabeled for Cx43 were optically sectioned and three images collected at 1-μm intervals were superimposed to illustrate a succession of Cx43-positive cellular locations (arrows). The mitotic cell in the insert is a reconstruction of five images collected at 1-μm intervals. (F) A confocal microscopic image for Cx43 in nonmitotic HX1 cells revealed punctate labeling at sites of cell–cell apposition with little intracellular labeling. Bars, 10 μm.
Mentions: To determine the distribution pattern of Cx43 during mitosis, mitotic cells were immunolabeled for Cx43 and analyzed on a confocal microscope. The transmitted light images (Fig. 5, A and C) of mitotic cells in comparison with the corresponding confocal immunofluorescent images (Fig. 5, B and D) show that Cx43 was often localized to intracellular compartments when optical sections were taken through the center of the cell. Moreover, optical slice reconstructions revealed a substantial amount of redistributed Cx43 in mitotic cells (Fig. 5 E, arrows) as well as an overall increase in intracellular cytoplasmic staining. Conversely, nonmitotic cells that remained on the substrate after mitotic shake-off showed the typical distribution of Cx43, principally at locations of cell–cell contact (Fig. 5 F).

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.

Show MeSH
Related in: MedlinePlus