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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 (A) and autoradiogram (B) of immunoprecipitated Cx43m after PP2A digestion. (A) Cx43 was immunoprecipitated from human arterial endothelial cells using polyclonal  rabbit anti-Cx43 antiserum (CT-360). The immunoprecipitates  from mitotic cells were treated for 1 h at 30°C with PP2A (0.25  U/μl), in the absence (lane 1) or presence (lane 2) of phosphatase  inhibitors, and analyzed by Western blotting. A nonmitotic sample was also immunoprecipitated for comparison (lane 3). Also  shown on the Western blot are whole cell lysates from nonmitotic  (lane 4) and mitotic (lane 5) cells. The broad band above the  Cx43 protein is the immunoprecipitating rabbit antibody that is  recognized by the HRP-conjugated goat anti–rabbit IgG antiserum. (B) Mitotic HUVEC cells were labeled with [32P]orthophosphate for 2 h before immunoprecipitation. The immunoprecipitates were treated either with PP2A or with dilution buffer only,  under conditions similar to those described above. Samples were  analyzed by SDS-PAGE and autoradiography. Lane 1, sample  treated with dilution buffer only; lane 2, sample treated with  PP2A, which removes virtually all of the 32P label from the immunoprecipitated Cx43m.
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Figure 3: Western blot (A) and autoradiogram (B) of immunoprecipitated Cx43m after PP2A digestion. (A) Cx43 was immunoprecipitated from human arterial endothelial cells using polyclonal rabbit anti-Cx43 antiserum (CT-360). The immunoprecipitates from mitotic cells were treated for 1 h at 30°C with PP2A (0.25 U/μl), in the absence (lane 1) or presence (lane 2) of phosphatase inhibitors, and analyzed by Western blotting. A nonmitotic sample was also immunoprecipitated for comparison (lane 3). Also shown on the Western blot are whole cell lysates from nonmitotic (lane 4) and mitotic (lane 5) cells. The broad band above the Cx43 protein is the immunoprecipitating rabbit antibody that is recognized by the HRP-conjugated goat anti–rabbit IgG antiserum. (B) Mitotic HUVEC cells were labeled with [32P]orthophosphate for 2 h before immunoprecipitation. The immunoprecipitates were treated either with PP2A or with dilution buffer only, under conditions similar to those described above. Samples were analyzed by SDS-PAGE and autoradiography. Lane 1, sample treated with dilution buffer only; lane 2, sample treated with PP2A, which removes virtually all of the 32P label from the immunoprecipitated Cx43m.

Mentions: Serine phosphorylation has been shown to correlate with gap junction assembly (Musil et al., 1990; Musil and Goodenough, 1991) as well as with 12-O-tetradecanoylphorbol13-acetate and EGF stimulation (Brissette et al., 1991; Lau et al., 1992; Warn-Cramer et al., 1996). In addition to serine phosphorylation, Cx43 can be modified on tyrosine residue(s) by src, which has been shown to inhibit GJIC (Crow et al., 1990; Filson et al., 1990; Swenson et al., 1990). Because src can be activated by a key initiator of mitosis, M-phase–promoting factor (Morgan et al., 1989; Shenoy et al., 1989), the possibility of Cx43 being phosphorylated by src on tyrosine residue(s) during mitosis was investigated by Western analyses of immunoprecipitated, unlabeled Cx43 protein with an anti–tyrosine phosphate antiserum. Although both mitotic and nonmitotic Cx43 were precipitated by the polyclonal anti-Cx43 antiserum CT360, neither form of Cx43 reacted with the polyclonal anti– tyrosine phosphate antiserum on Western blots (data not shown). Absence of phosphotyrosine was further corroborated by the observation that the Ser/Thr-specific protein phosphatase PP2A, like alkaline phosphatase, was able to shift the Cx43m species (Fig. 3 A, lane 2) to the species of lowest relative molecular mass (highest mobility) (Fig. 3 A, lane 1) as well as remove virtually all 32P-labeled phosphate from immunoprecipitated Cx43m (Fig. 3 B, lane 2). In comparison to Cx43 from nonmitotic cells, both immunoprecipitated and in whole cell lysate (Fig. 3 A, lanes 3 and 4, respectively), the Cx43m species (both immunoprecipitated and in whole cell lysate) appears to have a slightly higher relative molecular mass (Fig. 3 A, lanes 2 and 5, respectively), consistent with Cx43 banding patterns seen in Fig. 1. Furthermore, phosphoamino acid analysis by thin layer electrophoresis of 32P-labeled mitotic Cx43 hydrolyzed by treatment with HCl revealed the presence of only phosphoserine (data not shown). Taken together, these data suggest that the phosphorylated form of Cx43 unique to mitotic cells arises most likely by phosphorylation of a serine residue(s), although limits in the sensitivity of the techniques used might not have detected minor phosphorylation of tyrosine or threonine. However, additional serine phosphorylation has also been observed in mitotic Rat-1 fibroblasts, either transformed with v-src or overexpressing c-src, (Lau, A., personal communication).


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 (A) and autoradiogram (B) of immunoprecipitated Cx43m after PP2A digestion. (A) Cx43 was immunoprecipitated from human arterial endothelial cells using polyclonal  rabbit anti-Cx43 antiserum (CT-360). The immunoprecipitates  from mitotic cells were treated for 1 h at 30°C with PP2A (0.25  U/μl), in the absence (lane 1) or presence (lane 2) of phosphatase  inhibitors, and analyzed by Western blotting. A nonmitotic sample was also immunoprecipitated for comparison (lane 3). Also  shown on the Western blot are whole cell lysates from nonmitotic  (lane 4) and mitotic (lane 5) cells. The broad band above the  Cx43 protein is the immunoprecipitating rabbit antibody that is  recognized by the HRP-conjugated goat anti–rabbit IgG antiserum. (B) Mitotic HUVEC cells were labeled with [32P]orthophosphate for 2 h before immunoprecipitation. The immunoprecipitates were treated either with PP2A or with dilution buffer only,  under conditions similar to those described above. Samples were  analyzed by SDS-PAGE and autoradiography. Lane 1, sample  treated with dilution buffer only; lane 2, sample treated with  PP2A, which removes virtually all of the 32P label from the immunoprecipitated Cx43m.
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Figure 3: Western blot (A) and autoradiogram (B) of immunoprecipitated Cx43m after PP2A digestion. (A) Cx43 was immunoprecipitated from human arterial endothelial cells using polyclonal rabbit anti-Cx43 antiserum (CT-360). The immunoprecipitates from mitotic cells were treated for 1 h at 30°C with PP2A (0.25 U/μl), in the absence (lane 1) or presence (lane 2) of phosphatase inhibitors, and analyzed by Western blotting. A nonmitotic sample was also immunoprecipitated for comparison (lane 3). Also shown on the Western blot are whole cell lysates from nonmitotic (lane 4) and mitotic (lane 5) cells. The broad band above the Cx43 protein is the immunoprecipitating rabbit antibody that is recognized by the HRP-conjugated goat anti–rabbit IgG antiserum. (B) Mitotic HUVEC cells were labeled with [32P]orthophosphate for 2 h before immunoprecipitation. The immunoprecipitates were treated either with PP2A or with dilution buffer only, under conditions similar to those described above. Samples were analyzed by SDS-PAGE and autoradiography. Lane 1, sample treated with dilution buffer only; lane 2, sample treated with PP2A, which removes virtually all of the 32P label from the immunoprecipitated Cx43m.
Mentions: Serine phosphorylation has been shown to correlate with gap junction assembly (Musil et al., 1990; Musil and Goodenough, 1991) as well as with 12-O-tetradecanoylphorbol13-acetate and EGF stimulation (Brissette et al., 1991; Lau et al., 1992; Warn-Cramer et al., 1996). In addition to serine phosphorylation, Cx43 can be modified on tyrosine residue(s) by src, which has been shown to inhibit GJIC (Crow et al., 1990; Filson et al., 1990; Swenson et al., 1990). Because src can be activated by a key initiator of mitosis, M-phase–promoting factor (Morgan et al., 1989; Shenoy et al., 1989), the possibility of Cx43 being phosphorylated by src on tyrosine residue(s) during mitosis was investigated by Western analyses of immunoprecipitated, unlabeled Cx43 protein with an anti–tyrosine phosphate antiserum. Although both mitotic and nonmitotic Cx43 were precipitated by the polyclonal anti-Cx43 antiserum CT360, neither form of Cx43 reacted with the polyclonal anti– tyrosine phosphate antiserum on Western blots (data not shown). Absence of phosphotyrosine was further corroborated by the observation that the Ser/Thr-specific protein phosphatase PP2A, like alkaline phosphatase, was able to shift the Cx43m species (Fig. 3 A, lane 2) to the species of lowest relative molecular mass (highest mobility) (Fig. 3 A, lane 1) as well as remove virtually all 32P-labeled phosphate from immunoprecipitated Cx43m (Fig. 3 B, lane 2). In comparison to Cx43 from nonmitotic cells, both immunoprecipitated and in whole cell lysate (Fig. 3 A, lanes 3 and 4, respectively), the Cx43m species (both immunoprecipitated and in whole cell lysate) appears to have a slightly higher relative molecular mass (Fig. 3 A, lanes 2 and 5, respectively), consistent with Cx43 banding patterns seen in Fig. 1. Furthermore, phosphoamino acid analysis by thin layer electrophoresis of 32P-labeled mitotic Cx43 hydrolyzed by treatment with HCl revealed the presence of only phosphoserine (data not shown). Taken together, these data suggest that the phosphorylated form of Cx43 unique to mitotic cells arises most likely by phosphorylation of a serine residue(s), although limits in the sensitivity of the techniques used might not have detected minor phosphorylation of tyrosine or threonine. However, additional serine phosphorylation has also been observed in mitotic Rat-1 fibroblasts, either transformed with v-src or overexpressing c-src, (Lau, A., personal communication).

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