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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 between S364PCx43, S364ECx43 and S364ACx43/KO fibroblasts. (A) Example of Lucifer yellow dye transfer between S364PCx43 and S364ECx43/KO fibroblasts assembled overnight into confluent monolayers (*, injected cell). (B) Representative immunoblot of S364PCx43, S364ECx43, or S364ACx43/KO homogenates after GJ assembly and treatment without (1, 3, 5) or with (2, 4, 6) 8Br-cAMP for the entire assay. To detect S364PCx43 and S364ECx43 (arrowhead), membranes were labeled with a Cx43 antibody (1:8,000; Sigma-Aldrich) and detected with peroxi- dase-conjugated secondary antibody (1:10,000). A cross-reactive product, which was not consistently detected, is indicated below NP S364ECx43 (small arrowhead). (C) Compilation of representative GJ assembly experiments from S364PCx43, S364ECx43, and S364ACx43/KO fibroblasts. Cells were recovered for 4 h, reaggregated for 30 min, and, where indicated, treated with 8Br-cAMP for the entire 4.5 h of the assembly experiment. N values are above the error bars. *0.05 < P < 0.1; **0.01 < P < 0.02.
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fig5: GJ assembly between S364PCx43, S364ECx43 and S364ACx43/KO fibroblasts. (A) Example of Lucifer yellow dye transfer between S364PCx43 and S364ECx43/KO fibroblasts assembled overnight into confluent monolayers (*, injected cell). (B) Representative immunoblot of S364PCx43, S364ECx43, or S364ACx43/KO homogenates after GJ assembly and treatment without (1, 3, 5) or with (2, 4, 6) 8Br-cAMP for the entire assay. To detect S364PCx43 and S364ECx43 (arrowhead), membranes were labeled with a Cx43 antibody (1:8,000; Sigma-Aldrich) and detected with peroxi- dase-conjugated secondary antibody (1:10,000). A cross-reactive product, which was not consistently detected, is indicated below NP S364ECx43 (small arrowhead). (C) Compilation of representative GJ assembly experiments from S364PCx43, S364ECx43, and S364ACx43/KO fibroblasts. Cells were recovered for 4 h, reaggregated for 30 min, and, where indicated, treated with 8Br-cAMP for the entire 4.5 h of the assembly experiment. N values are above the error bars. *0.05 < P < 0.1; **0.01 < P < 0.02.

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)

GJ assembly between S364PCx43, S364ECx43 and S364ACx43/KO fibroblasts. (A) Example of Lucifer yellow dye transfer between S364PCx43 and S364ECx43/KO fibroblasts assembled overnight into confluent monolayers (*, injected cell). (B) Representative immunoblot of S364PCx43, S364ECx43, or S364ACx43/KO homogenates after GJ assembly and treatment without (1, 3, 5) or with (2, 4, 6) 8Br-cAMP for the entire assay. To detect S364PCx43 and S364ECx43 (arrowhead), membranes were labeled with a Cx43 antibody (1:8,000; Sigma-Aldrich) and detected with peroxi- dase-conjugated secondary antibody (1:10,000). A cross-reactive product, which was not consistently detected, is indicated below NP S364ECx43 (small arrowhead). (C) Compilation of representative GJ assembly experiments from S364PCx43, S364ECx43, and S364ACx43/KO fibroblasts. Cells were recovered for 4 h, reaggregated for 30 min, and, where indicated, treated with 8Br-cAMP for the entire 4.5 h of the assembly experiment. N values are above the error bars. *0.05 < P < 0.1; **0.01 < P < 0.02.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199346&req=5

fig5: GJ assembly between S364PCx43, S364ECx43 and S364ACx43/KO fibroblasts. (A) Example of Lucifer yellow dye transfer between S364PCx43 and S364ECx43/KO fibroblasts assembled overnight into confluent monolayers (*, injected cell). (B) Representative immunoblot of S364PCx43, S364ECx43, or S364ACx43/KO homogenates after GJ assembly and treatment without (1, 3, 5) or with (2, 4, 6) 8Br-cAMP for the entire assay. To detect S364PCx43 and S364ECx43 (arrowhead), membranes were labeled with a Cx43 antibody (1:8,000; Sigma-Aldrich) and detected with peroxi- dase-conjugated secondary antibody (1:10,000). A cross-reactive product, which was not consistently detected, is indicated below NP S364ECx43 (small arrowhead). (C) Compilation of representative GJ assembly experiments from S364PCx43, S364ECx43, and S364ACx43/KO fibroblasts. Cells were recovered for 4 h, reaggregated for 30 min, and, where indicated, treated with 8Br-cAMP for the entire 4.5 h of the assembly experiment. N values are above the error bars. *0.05 < P < 0.1; **0.01 < P < 0.02.
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.

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Related in: MedlinePlus