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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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GJ assembly following the expression of vector, S364PCx43, S364ECx43, S364ACx43, or WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either S364PCx43 or S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with M257Cx43 or S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (M257Cx43) or 7 (S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous WTCx43 and transfected M257Cx43 (arrowhead, first panel) membranes were labeled with NH2-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.
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fig6: GJ assembly following the expression of vector, S364PCx43, S364ECx43, S364ACx43, or WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either S364PCx43 or S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with M257Cx43 or S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (M257Cx43) or 7 (S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous WTCx43 and transfected M257Cx43 (arrowhead, first panel) membranes were labeled with NH2-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.

Mentions: Because homomeric S364PCx43 and S364ACx43 GJs in KO fibroblasts were not positively regulated by 8Br-cAMP, we questioned whether these mutants, coexpressed with WTCx43, would affect the regulation of GJ assembly. Thus, WT fibroblasts were transiently transfected with S364PCx43, S364ECx43, S364ACx43, or pIRES vector alone, and assembly was studied. Transfection with each of these mutants increased both GJ communication after assembly (Fig. 6 A) and Cx43 levels on westerns (Fig. 6 B) by at least twofold, indicating that the mutants were strongly expressed and were forming functional GJs. In keeping with the experiments utilizing stably transfected KO fibroblasts, the S364PCx43-transfected WT fibroblasts did not display enhanced assembly, whereas control pIRES-transfected and S364ECx43-transfected WT fibroblasts did (Fig. 6, A and C). In contrast to the results with S364ACx43/KO fibroblasts, enhanced assembly was observed between the S364ACx43/WT fibroblasts (Fig. 6 C). However, levels of assembly due to endogenous Cx43 in S364ACx43/WT fibroblasts were not as significantly augmented by S364ACx43 as they were by the other two mutants (Fig. 6 C), in spite of similar levels of expression by Western immunoblotting (unpublished data). Note that transfection of WTCx43 into the WT fibroblasts doubled both basal and enhanced GJ assembly (Fig 6 C).


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)

GJ assembly following the expression of vector, S364PCx43, S364ECx43, S364ACx43, or WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either S364PCx43 or S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with M257Cx43 or S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (M257Cx43) or 7 (S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous WTCx43 and transfected M257Cx43 (arrowhead, first panel) membranes were labeled with NH2-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.
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fig6: GJ assembly following the expression of vector, S364PCx43, S364ECx43, S364ACx43, or WTCx43 in WT fibroblasts. (A) Comparison of GJ assembly between WT fibroblasts (10-3) transfected with either S364PCx43 or S364ECx43 and treated with or without 8Br-cAMP for the entire experiment (*, injected cell). (B) Example of immunoblots of cell homogenates following two different assembly experiments using either WT fibroblasts transiently transfected with M257Cx43 or S364PCx43. Cells were transfected 48 h prior to experimentation with either vector alone (pIRES; 1, 2, 5, and 6) or mutant construct (3, 4, 7, and 8). Cells were dissociated and recovered for 4 h 15 or 23 min, reaggregated for 15 (M257Cx43) or 7 (S364PCx43) min, and treated either without (1 and 3) or with (2 and 4) 8Br-cAMP for the entire GJ assembly experiment. To detect endogenous WTCx43 and transfected M257Cx43 (arrowhead, first panel) membranes were labeled with NH2-terminal Cx43 antibody (1:8,000) detected with goat anti–rabbit peroxidase antibody (1:10,000). To detect mutant and WTCx43 expression (second panel), membranes were labeled with Cx43 CT antibody (1:8,000; Sigma-Aldrich) detected with goat anti–rabbit peroxidase antibody (1:10,000). (C) Compilation of trials in which WT fibroblasts were transfected with either vector alone, mutant, or WT Cx43 and GJ assembly monitored without (A) or with (B) 8Br-cAMP.
Mentions: Because homomeric S364PCx43 and S364ACx43 GJs in KO fibroblasts were not positively regulated by 8Br-cAMP, we questioned whether these mutants, coexpressed with WTCx43, would affect the regulation of GJ assembly. Thus, WT fibroblasts were transiently transfected with S364PCx43, S364ECx43, S364ACx43, or pIRES vector alone, and assembly was studied. Transfection with each of these mutants increased both GJ communication after assembly (Fig. 6 A) and Cx43 levels on westerns (Fig. 6 B) by at least twofold, indicating that the mutants were strongly expressed and were forming functional GJs. In keeping with the experiments utilizing stably transfected KO fibroblasts, the S364PCx43-transfected WT fibroblasts did not display enhanced assembly, whereas control pIRES-transfected and S364ECx43-transfected WT fibroblasts did (Fig. 6, A and C). In contrast to the results with S364ACx43/KO fibroblasts, enhanced assembly was observed between the S364ACx43/WT fibroblasts (Fig. 6 C). However, levels of assembly due to endogenous Cx43 in S364ACx43/WT fibroblasts were not as significantly augmented by S364ACx43 as they were by the other two mutants (Fig. 6 C), in spite of similar levels of expression by Western immunoblotting (unpublished data). Note that transfection of WTCx43 into the WT fibroblasts doubled both basal and enhanced GJ assembly (Fig 6 C).

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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Related in: MedlinePlus