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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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S364 of Cx43 is phosphorylated in unstimulated WTCx43/KO fibroblasts. (A) HPLC elution profile for 32P-labeled Cx43 peptides isolated from cells expressing WTCx43 (•) or S364PCx43 (□). The elution position of certain peptides is indicated. Amino acids 346–382 of Cx43 and tryptic cleavage sites are depicted above the profile (arrows). Asterisks denote sites that may only be partially cleaved by trypsin. (B) Autoradiogram depicting in vitro phosphorylation of Cx GST fusion proteins. (Lane 1) Purified GST CTCx56 phosphorylated by PKA. (Lane 2) Purified GST CTCx43 phosphorylated by of PKA. (Lane 3) Purified GST CTCx43 phosphorylated by PKC. (C) Phosphorylation of Cx43 after treatment with 8Br-cAMP. Autoradiogram showing results of two separate experiments in which confluent monolayers of WT fibroblasts (10-3) were loaded with radiolabeled [32P]i in the presence or absence of 8Br-cAMP (Materials and methods). (1 and 3) Untreated. (2 and 4) Treated with 8Br-cAMP.
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fig4: S364 of Cx43 is phosphorylated in unstimulated WTCx43/KO fibroblasts. (A) HPLC elution profile for 32P-labeled Cx43 peptides isolated from cells expressing WTCx43 (•) or S364PCx43 (□). The elution position of certain peptides is indicated. Amino acids 346–382 of Cx43 and tryptic cleavage sites are depicted above the profile (arrows). Asterisks denote sites that may only be partially cleaved by trypsin. (B) Autoradiogram depicting in vitro phosphorylation of Cx GST fusion proteins. (Lane 1) Purified GST CTCx56 phosphorylated by PKA. (Lane 2) Purified GST CTCx43 phosphorylated by of PKA. (Lane 3) Purified GST CTCx43 phosphorylated by PKC. (C) Phosphorylation of Cx43 after treatment with 8Br-cAMP. Autoradiogram showing results of two separate experiments in which confluent monolayers of WT fibroblasts (10-3) were loaded with radiolabeled [32P]i in the presence or absence of 8Br-cAMP (Materials and methods). (1 and 3) Untreated. (2 and 4) Treated with 8Br-cAMP.

Mentions: In an attempt to ascertain which residue(s) of the S364–S365-containing peptide was phosphorylated, WTCx43/ or S364PCx43/KO fibroblasts were metabolically labeled with [32P]i, Cx43 was immunoprecipitated, and tryptic peptides were analyzed by HPLC. The WTCx43-expressing cells incorporated 3,400 cpm, and the S364PCx43 cells incorporated 1,200 cpm that could be recovered in tryptic fragments. HPLC separation of labeled Cx43 from WTCx43 cells revealed peaks of radioactivity eluting at 8, 21–22, and 24 min, whereas separation of labeled S364PCx43 revealed peaks at 8 and 20–21 min, consistent with WTCx43 cells, and several other smaller peaks (Fig. 4 A). Comparison of common peaks showed a 29-fold greater incorporation of 32P in the 8-min fraction from WTCx43 as related to the same fraction from S364PCx43, whereas the 20–21-min fractions contained much closer to equal levels of radioactivity. We have previously shown that when the sequence A367-R370 was phosphorylated at S368, it eluted at 8 min using this HPLC system (Lampe et al., 2000). We expect P363S(32P)SR to also elute in the 8-min fraction, as its charge is nearly identical and its mass only differs from phosphorylated A367-R370 by 26 Da.


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)

S364 of Cx43 is phosphorylated in unstimulated WTCx43/KO fibroblasts. (A) HPLC elution profile for 32P-labeled Cx43 peptides isolated from cells expressing WTCx43 (•) or S364PCx43 (□). The elution position of certain peptides is indicated. Amino acids 346–382 of Cx43 and tryptic cleavage sites are depicted above the profile (arrows). Asterisks denote sites that may only be partially cleaved by trypsin. (B) Autoradiogram depicting in vitro phosphorylation of Cx GST fusion proteins. (Lane 1) Purified GST CTCx56 phosphorylated by PKA. (Lane 2) Purified GST CTCx43 phosphorylated by of PKA. (Lane 3) Purified GST CTCx43 phosphorylated by PKC. (C) Phosphorylation of Cx43 after treatment with 8Br-cAMP. Autoradiogram showing results of two separate experiments in which confluent monolayers of WT fibroblasts (10-3) were loaded with radiolabeled [32P]i in the presence or absence of 8Br-cAMP (Materials and methods). (1 and 3) Untreated. (2 and 4) Treated with 8Br-cAMP.
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fig4: S364 of Cx43 is phosphorylated in unstimulated WTCx43/KO fibroblasts. (A) HPLC elution profile for 32P-labeled Cx43 peptides isolated from cells expressing WTCx43 (•) or S364PCx43 (□). The elution position of certain peptides is indicated. Amino acids 346–382 of Cx43 and tryptic cleavage sites are depicted above the profile (arrows). Asterisks denote sites that may only be partially cleaved by trypsin. (B) Autoradiogram depicting in vitro phosphorylation of Cx GST fusion proteins. (Lane 1) Purified GST CTCx56 phosphorylated by PKA. (Lane 2) Purified GST CTCx43 phosphorylated by of PKA. (Lane 3) Purified GST CTCx43 phosphorylated by PKC. (C) Phosphorylation of Cx43 after treatment with 8Br-cAMP. Autoradiogram showing results of two separate experiments in which confluent monolayers of WT fibroblasts (10-3) were loaded with radiolabeled [32P]i in the presence or absence of 8Br-cAMP (Materials and methods). (1 and 3) Untreated. (2 and 4) Treated with 8Br-cAMP.
Mentions: In an attempt to ascertain which residue(s) of the S364–S365-containing peptide was phosphorylated, WTCx43/ or S364PCx43/KO fibroblasts were metabolically labeled with [32P]i, Cx43 was immunoprecipitated, and tryptic peptides were analyzed by HPLC. The WTCx43-expressing cells incorporated 3,400 cpm, and the S364PCx43 cells incorporated 1,200 cpm that could be recovered in tryptic fragments. HPLC separation of labeled Cx43 from WTCx43 cells revealed peaks of radioactivity eluting at 8, 21–22, and 24 min, whereas separation of labeled S364PCx43 revealed peaks at 8 and 20–21 min, consistent with WTCx43 cells, and several other smaller peaks (Fig. 4 A). Comparison of common peaks showed a 29-fold greater incorporation of 32P in the 8-min fraction from WTCx43 as related to the same fraction from S364PCx43, whereas the 20–21-min fractions contained much closer to equal levels of radioactivity. We have previously shown that when the sequence A367-R370 was phosphorylated at S368, it eluted at 8 min using this HPLC system (Lampe et al., 2000). We expect P363S(32P)SR to also elute in the 8-min fraction, as its charge is nearly identical and its mass only differs from phosphorylated A367-R370 by 26 Da.

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