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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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The effects of cycloheximide on GJ assembly. Representative experiments comparing GJ assembly between WT fibroblasts (A) (clone 10-3) untreated (1) or treated with 8Br-cAMP (2), cycloheximide (3), or both cycloheximide and 8Br-cAMP (4). Cells were recovered for 4 h 23 min in suspension and reaggregated for 7 min (light bars) or recovered for 3.5 h in suspension and reaggregated for 1 h (dark bars). (Inset) Western immunoblot of WT fibroblasts after 1-h assembly (dark bars). (B) Immunolocalization of Cx43 using Cx43IF1 mouse monoclonal antibody (1:10), detected by goat anti–mouse Alexafluor 568 (1:500). WT fibroblasts were recovered for 3.5 h in suspension and reaggregated for 1 h before fixation. When present, 8Br-cAMP and/or cycloheximide were added at the beginning of recovery. (A) control; (B) 8Br-cAMP; (C) cycloheximide; (D) cycloheximide + 8Br-cAMP.
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fig2: The effects of cycloheximide on GJ assembly. Representative experiments comparing GJ assembly between WT fibroblasts (A) (clone 10-3) untreated (1) or treated with 8Br-cAMP (2), cycloheximide (3), or both cycloheximide and 8Br-cAMP (4). Cells were recovered for 4 h 23 min in suspension and reaggregated for 7 min (light bars) or recovered for 3.5 h in suspension and reaggregated for 1 h (dark bars). (Inset) Western immunoblot of WT fibroblasts after 1-h assembly (dark bars). (B) Immunolocalization of Cx43 using Cx43IF1 mouse monoclonal antibody (1:10), detected by goat anti–mouse Alexafluor 568 (1:500). WT fibroblasts were recovered for 3.5 h in suspension and reaggregated for 1 h before fixation. When present, 8Br-cAMP and/or cycloheximide were added at the beginning of recovery. (A) control; (B) 8Br-cAMP; (C) cycloheximide; (D) cycloheximide + 8Br-cAMP.

Mentions: To determine whether elevated cAMP stimulated the synthesis of Cx43, Western immunoblotting and densitometry were used to monitor Cx43 levels in cell homogenates from assembly experiments. 8Br-cAMP treatment during the assembly assay elevated total Cx43 in WT fibroblasts by 26% on average (n = 6; 4.5-h treatment time) (Fig. 2 A, inset). In contrast, expression levels were not significantly elevated by 8Br-cAMP in WTCx43- or mutant Cx43-transfected KO fibroblasts after correcting for loading differences (unpublished data; see Fig. 5 B).


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)

The effects of cycloheximide on GJ assembly. Representative experiments comparing GJ assembly between WT fibroblasts (A) (clone 10-3) untreated (1) or treated with 8Br-cAMP (2), cycloheximide (3), or both cycloheximide and 8Br-cAMP (4). Cells were recovered for 4 h 23 min in suspension and reaggregated for 7 min (light bars) or recovered for 3.5 h in suspension and reaggregated for 1 h (dark bars). (Inset) Western immunoblot of WT fibroblasts after 1-h assembly (dark bars). (B) Immunolocalization of Cx43 using Cx43IF1 mouse monoclonal antibody (1:10), detected by goat anti–mouse Alexafluor 568 (1:500). WT fibroblasts were recovered for 3.5 h in suspension and reaggregated for 1 h before fixation. When present, 8Br-cAMP and/or cycloheximide were added at the beginning of recovery. (A) control; (B) 8Br-cAMP; (C) cycloheximide; (D) cycloheximide + 8Br-cAMP.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: The effects of cycloheximide on GJ assembly. Representative experiments comparing GJ assembly between WT fibroblasts (A) (clone 10-3) untreated (1) or treated with 8Br-cAMP (2), cycloheximide (3), or both cycloheximide and 8Br-cAMP (4). Cells were recovered for 4 h 23 min in suspension and reaggregated for 7 min (light bars) or recovered for 3.5 h in suspension and reaggregated for 1 h (dark bars). (Inset) Western immunoblot of WT fibroblasts after 1-h assembly (dark bars). (B) Immunolocalization of Cx43 using Cx43IF1 mouse monoclonal antibody (1:10), detected by goat anti–mouse Alexafluor 568 (1:500). WT fibroblasts were recovered for 3.5 h in suspension and reaggregated for 1 h before fixation. When present, 8Br-cAMP and/or cycloheximide were added at the beginning of recovery. (A) control; (B) 8Br-cAMP; (C) cycloheximide; (D) cycloheximide + 8Br-cAMP.
Mentions: To determine whether elevated cAMP stimulated the synthesis of Cx43, Western immunoblotting and densitometry were used to monitor Cx43 levels in cell homogenates from assembly experiments. 8Br-cAMP treatment during the assembly assay elevated total Cx43 in WT fibroblasts by 26% on average (n = 6; 4.5-h treatment time) (Fig. 2 A, inset). In contrast, expression levels were not significantly elevated by 8Br-cAMP in WTCx43- or mutant Cx43-transfected KO fibroblasts after correcting for loading differences (unpublished data; see Fig. 5 B).

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