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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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Stimulation of GJ assembly after the elevation of cAMP. Two separate, representative experiments (dark vs. light bars) comparing GJ assembly between untreated WTCx43/KO fibroblasts (clone 22C-3) and cells treated with 8Br-cAMP, with both 8Br-cAMP and H89, or with H89 alone. (Dark bars) When present, H89 was added at recovery. (Light bars) When present, H89 was added 15 min before reaggregation.
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fig1: Stimulation of GJ assembly after the elevation of cAMP. Two separate, representative experiments (dark vs. light bars) comparing GJ assembly between untreated WTCx43/KO fibroblasts (clone 22C-3) and cells treated with 8Br-cAMP, with both 8Br-cAMP and H89, or with H89 alone. (Dark bars) When present, H89 was added at recovery. (Light bars) When present, H89 was added 15 min before reaggregation.

Mentions: Established WT fibroblasts expressing endogenous WTCx43 typically display a two- to fourfold enhancement of GJ communication after exposure to cell permeant 8Br-cAMP for 3–4 h (e.g., clone 10-8 average recipient cells for control: 18.5 ± 0.31, n = 10; 8Br-cAMP: 37.0 ± 2.69, n = 10). A complement to the WT fibroblast, the KO fibroblast, was used for the majority of these studies, as it likely provides a similar physiological milieu suitable for the reintroduction and study of Cx43 (Fig. 1). Lucifer yellow dye transfer is absent between untransfected KO fibroblasts, and whole-cell conductance values are as low, if not lower, than those of other cells (Kwak et al., 1995; Martyn et al., 1997).


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)

Stimulation of GJ assembly after the elevation of cAMP. Two separate, representative experiments (dark vs. light bars) comparing GJ assembly between untreated WTCx43/KO fibroblasts (clone 22C-3) and cells treated with 8Br-cAMP, with both 8Br-cAMP and H89, or with H89 alone. (Dark bars) When present, H89 was added at recovery. (Light bars) When present, H89 was added 15 min before reaggregation.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Stimulation of GJ assembly after the elevation of cAMP. Two separate, representative experiments (dark vs. light bars) comparing GJ assembly between untreated WTCx43/KO fibroblasts (clone 22C-3) and cells treated with 8Br-cAMP, with both 8Br-cAMP and H89, or with H89 alone. (Dark bars) When present, H89 was added at recovery. (Light bars) When present, H89 was added 15 min before reaggregation.
Mentions: Established WT fibroblasts expressing endogenous WTCx43 typically display a two- to fourfold enhancement of GJ communication after exposure to cell permeant 8Br-cAMP for 3–4 h (e.g., clone 10-8 average recipient cells for control: 18.5 ± 0.31, n = 10; 8Br-cAMP: 37.0 ± 2.69, n = 10). A complement to the WT fibroblast, the KO fibroblast, was used for the majority of these studies, as it likely provides a similar physiological milieu suitable for the reintroduction and study of Cx43 (Fig. 1). Lucifer yellow dye transfer is absent between untransfected KO fibroblasts, and whole-cell conductance values are as low, if not lower, than those of other cells (Kwak et al., 1995; Martyn et al., 1997).

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