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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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The mitotic form of  Cx43 is alkaline phosphatase  sensitive. Control human arterial endothelial cell lysates (A)  or mitotic cell lysates (M) were  treated with alkaline phosphatase (AP) in the presence  or absence of inhibitors (I).  Samples were resolved by  SDS-PAGE and immunoblotted for Cx43. Note that the mitotic form of Cx43 (Cx43m) was  specifically sensitive to alkaline  phosphatase.
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Figure 2: The mitotic form of Cx43 is alkaline phosphatase sensitive. Control human arterial endothelial cell lysates (A) or mitotic cell lysates (M) were treated with alkaline phosphatase (AP) in the presence or absence of inhibitors (I). Samples were resolved by SDS-PAGE and immunoblotted for Cx43. Note that the mitotic form of Cx43 (Cx43m) was specifically sensitive to alkaline phosphatase.

Mentions: As shown in Fig. 2 (lane M + AP), alkaline phosphatase treatment eliminated the gel shift of Cx43 seen in mitotic cells (lane M), suggesting that the mitosis-specific modification of Cx43 is a phosphorylation phenomenon. Furthermore, the phosphatase-mediated conversion of Cx43m to a Cx43 species of lower relative molecular mass was inhibited by phosphatase inhibitors (lane M + AP + I). Nonmitotic cells (lane A) had the well-characterized pattern of Cx43 species at relative molecular masses lower than Cx43m.


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)

The mitotic form of  Cx43 is alkaline phosphatase  sensitive. Control human arterial endothelial cell lysates (A)  or mitotic cell lysates (M) were  treated with alkaline phosphatase (AP) in the presence  or absence of inhibitors (I).  Samples were resolved by  SDS-PAGE and immunoblotted for Cx43. Note that the mitotic form of Cx43 (Cx43m) was  specifically sensitive to alkaline  phosphatase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139857&req=5

Figure 2: The mitotic form of Cx43 is alkaline phosphatase sensitive. Control human arterial endothelial cell lysates (A) or mitotic cell lysates (M) were treated with alkaline phosphatase (AP) in the presence or absence of inhibitors (I). Samples were resolved by SDS-PAGE and immunoblotted for Cx43. Note that the mitotic form of Cx43 (Cx43m) was specifically sensitive to alkaline phosphatase.
Mentions: As shown in Fig. 2 (lane M + AP), alkaline phosphatase treatment eliminated the gel shift of Cx43 seen in mitotic cells (lane M), suggesting that the mitosis-specific modification of Cx43 is a phosphorylation phenomenon. Furthermore, the phosphatase-mediated conversion of Cx43m to a Cx43 species of lower relative molecular mass was inhibited by phosphatase inhibitors (lane M + AP + I). Nonmitotic cells (lane A) had the well-characterized pattern of Cx43 species at relative molecular masses lower than Cx43m.

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