Analysis of Connexin Phosphorylation Sites

Cooper, Cynthia D.; Solan, Joell L.; Dolejsi, Mary Kay; Lampe, Paul D.

doi:10.1006/meth.1999.0937

Cited by 54 publications

(37 citation statements)

References 36 publications

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“…HisCx43CT cDNA was prepared by PCR by adding a hexahistidine epitope tag to amino acids 236 -382 of Cx43 as previously described (17). The insert was cloned in pGEX-2T (which added a GST epitope tag), transformed into DH5␣ E. coli, expressed after isopropyl-1-thio-␤-D-galactopyranoside induction (1 mM isopropyl-1-thio-␤-D-galactopyranoside), and purified on glutathione-agarose (Sigma) as previously described (17,30).…”

Section: Methodsmentioning

confidence: 99%

“…The insert was cloned in pGEX-2T (which added a GST epitope tag), transformed into DH5␣ E. coli, expressed after isopropyl-1-thio-␤-D-galactopyranoside induction (1 mM isopropyl-1-thio-␤-D-galactopyranoside), and purified on glutathione-agarose (Sigma) as previously described (17,30). Purified GST-HisCx43CT was digested with 2 units of thrombin for 4 h at 25-30°C to remove the GST epitope tag followed by dialysis against 30 mM ammonium bicarbonate, pH 7.7.…”

Section: Methodsmentioning

confidence: 99%

“…These approaches have implicated roles for protein kinase C and mitogen-activated protein kinase in Cx43 phosphorylation and acute gating of gap junction channels (15,16). Based on several studies, Cx43 undergoes multiple phosphorylation events because at least five phosphorylated serines have been detected on Cx43 isolated from unstimulated cells (17). These uncharacterized phosphorylation events have been correlated with changes in assembly, acquisition of Triton X-100 insolubility, and degradation of Cx43 gap junction channels and, hence, could play critical roles in regulating gap junctional communication.…”

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confidence: 99%

“…P i Metabolic Labeling of NRK Cultures-Cell cultures were metabolically labeled as previously described (17,31). Briefly, cells were labeled for 4 h at 37°C in the presence or absence of CK1 inhibitors CKI-7 (50 or 100 M) or IC261 (2.5 M).…”

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confidence: 99%

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Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly

Cooper

Lampe

2002

Journal of Biological Chemistry

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183

167

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Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330.32 P i -Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for ␦ or ⑀ isoforms. These data suggest CK1␦ could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly

Cooper

Lampe

2002

Journal of Biological Chemistry

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183

167

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“…Moreover, the altered amino acid transforms the phosphorylation locus and further affects protein synthesis, transport, fabrication and signal transduction. Therefore, the function of vascular endothelial cells is affected to different extents, thus contributing to individual differences in CAD susceptibility (6).…”

Section: Introductionmentioning

confidence: 99%

Lack of association between the connexin 37 C1019T gene polymorphism and coronary artery disease in a Chinese population: Meta-analysis of 2,206 subjects

Qian

Zhou

2013

Biomedical Reports

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Abstract. The connexin 37 (Cx37) C1019T gene polymorphism has been suggested to be correlated with increased coronary artery disease (CAD) risk, but research results remain inconsistent. To explore the relationship between the Cx37 C1019T gene polymorphism and CAD in a Chinese population, the current meta-analysis of 6 individual studies involving 1,244 CAD patients and 962 controls was conducted. The pooled odds ratios (ORs) as well as the corresponding 95% confidence intervals (CIs) were estimated using a random-or fixed-effect model. No significant association was found between Cx37 C1019T gene polymorphism and CAD in the Chinese population under the allelic (OR=0.96; 95% CI=0.59-1.56, P=0.87), recessive (OR=0.77, 95% CI=0.28-2.08, P=0.60), dominant (OR=0.990, 95% CI=0.773-1.266, P=0.934), additive (OR=1.000, 95% CI=0.736-1.359, P=1.000), homozygous (OR=1.062, 95% CI=0.598-1.887, P=0.836) or heterozygous (OR=1.017, 95% CI=0.802-1.291, P=0.888) genetic models. Cx37 C1019T gene polymorphism was not suggested to be associated with CAD susceptibility in the Chinese population. In conclusion, no association was found between Cx37 C1019T gene polymorphism and CAD in the Chinese population.

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Establishing the Tumor Microenvironment

Betof

Dewhirst

2010

Tumor Microenvironment

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Analysis of Connexin Phosphorylation Sites

Cited by 54 publications

References 36 publications

Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly

Casein Kinase 1 Regulates Connexin-43 Gap Junction Assembly

Lack of association between the connexin 37 C1019T gene polymorphism and coronary artery disease in a Chinese population: Meta-analysis of 2,206 subjects

Establishing the Tumor Microenvironment

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