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Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

TenBroek EM, Lampe PD, Solan JL, Reynhout JK, Johnson RG - J. Cell Biol. (2001)

Bottom Line: Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP.Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts.Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, MN 55108, USA. tenbr001@tc.umn.edu

ABSTRACT
The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

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Comparison of cells expressing WTCx43 or M257Cx43. (A) A representative experiment in which established monolayers of WTCx43/N2A and M257Cx43/N2A cells were treated with or without 8Br-cAMP for 4.5 h and coupling measured by microinjection of Lucifer yellow dye. (B) A representative experiment comparing de novo GJ assembly between WTCx43/N2A and M257Cx43/N2A cells treated for the entire experiment with or without 8Br-cAMP. *0.001 < P < 0.01
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fig3: Comparison of cells expressing WTCx43 or M257Cx43. (A) A representative experiment in which established monolayers of WTCx43/N2A and M257Cx43/N2A cells were treated with or without 8Br-cAMP for 4.5 h and coupling measured by microinjection of Lucifer yellow dye. (B) A representative experiment comparing de novo GJ assembly between WTCx43/N2A and M257Cx43/N2A cells treated for the entire experiment with or without 8Br-cAMP. *0.001 < P < 0.01

Mentions: Because M257Cx43 did not form significant GJs in the fibroblast, N2A cells that had been previously transfected with WTCx43 or M257Cx43 were also examined. Prior to transfection, N2A cells have a very low level of electrical coupling measured by double whole-cell patch clamp that is reportedly the result of Cx45 expression (Veenstra et al., 1992). We detected no Lucifer yellow dye coupling between parental N2A cells (0.18 ± 0.4; n = 10), and 8Br-cAMP treatment (4.5 h) failed to stimulate coupling (0 ± 0; n = 10). Confluent monolayers of WTCx43/N2A and M257Cx43/N2A, cells were found to be equally well dye coupled, and treatment of established WTCx43/N2A monolayers with 8Br-cAMP for 4.5 h increased coupling three- to fourfold, whereas coupling between the M257Cx43/N2As remained at control levels (Fig. 3 A). When the same cells were dissociated, recovered, and reaggregated in the presence or absence of 8Br-cAMP, GJ assembly between the WTCx43/N2As was significantly increased, but that between the M257Cx43/N2As remained unchanged (Fig. 3 B). The M257Cx43/N2A cells also assembled GJs more slowly than the WTCx43/N2A cells did, in spite of communicating equally well in established monolayers (Fig. 3, compare A and B). Thus, as was found using the less well-coupled M257Cx43/KO fibroblasts, these data show that the Cx43 CT affects the kinetics of basal GJ assembly and that the CT is required for enhanced GJ assembly.


Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

TenBroek EM, Lampe PD, Solan JL, Reynhout JK, Johnson RG - J. Cell Biol. (2001)

Comparison of cells expressing WTCx43 or M257Cx43. (A) A representative experiment in which established monolayers of WTCx43/N2A and M257Cx43/N2A cells were treated with or without 8Br-cAMP for 4.5 h and coupling measured by microinjection of Lucifer yellow dye. (B) A representative experiment comparing de novo GJ assembly between WTCx43/N2A and M257Cx43/N2A cells treated for the entire experiment with or without 8Br-cAMP. *0.001 < P < 0.01
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199346&req=5

fig3: Comparison of cells expressing WTCx43 or M257Cx43. (A) A representative experiment in which established monolayers of WTCx43/N2A and M257Cx43/N2A cells were treated with or without 8Br-cAMP for 4.5 h and coupling measured by microinjection of Lucifer yellow dye. (B) A representative experiment comparing de novo GJ assembly between WTCx43/N2A and M257Cx43/N2A cells treated for the entire experiment with or without 8Br-cAMP. *0.001 < P < 0.01
Mentions: Because M257Cx43 did not form significant GJs in the fibroblast, N2A cells that had been previously transfected with WTCx43 or M257Cx43 were also examined. Prior to transfection, N2A cells have a very low level of electrical coupling measured by double whole-cell patch clamp that is reportedly the result of Cx45 expression (Veenstra et al., 1992). We detected no Lucifer yellow dye coupling between parental N2A cells (0.18 ± 0.4; n = 10), and 8Br-cAMP treatment (4.5 h) failed to stimulate coupling (0 ± 0; n = 10). Confluent monolayers of WTCx43/N2A and M257Cx43/N2A, cells were found to be equally well dye coupled, and treatment of established WTCx43/N2A monolayers with 8Br-cAMP for 4.5 h increased coupling three- to fourfold, whereas coupling between the M257Cx43/N2As remained at control levels (Fig. 3 A). When the same cells were dissociated, recovered, and reaggregated in the presence or absence of 8Br-cAMP, GJ assembly between the WTCx43/N2As was significantly increased, but that between the M257Cx43/N2As remained unchanged (Fig. 3 B). The M257Cx43/N2A cells also assembled GJs more slowly than the WTCx43/N2A cells did, in spite of communicating equally well in established monolayers (Fig. 3, compare A and B). Thus, as was found using the less well-coupled M257Cx43/KO fibroblasts, these data show that the Cx43 CT affects the kinetics of basal GJ assembly and that the CT is required for enhanced GJ assembly.

Bottom Line: Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP.Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts.Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, St. Paul, MN 55108, USA. tenbr001@tc.umn.edu

ABSTRACT
The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Show MeSH
Related in: MedlinePlus