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PMID: 10871288 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

The Journal of cell biology ·Vol. 149 ·No. 7 ·2000-06-26 ·Pages 1503-12

Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG, Lau AF

Abstract

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

MeSH Terms
Animals Cell Communication/physiology Cells, Cultured Connexin 43/metabolism Gap Junctions/metabolism,physiology Mice Mice, Knockout Mutagenesis, Site-Directed/physiology Neoplasms/metabolism,physiopathology Phosphorylation Protein Kinase C/metabolism Sequence Analysis, Protein Serine/metabolism
Chemicals
Connexin 43 Serine Protein Kinase C
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Lampe P D
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. plampe@fhcrc.org
TenBroek E M
Burt J M
Kurata W E
Johnson R G
Lau A F
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.149.7.1503
Email
plampe@fhcrc.org
Published
2000-06-26
Pages
1503-12
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2175134
Subset
IM
Grants
NIGMS NIH HHS · R01 GM055632 · United States
NIGMS NIH HHS · GM55632 · United States
NIGMS NIH HHS · GM46277 · United States
NHLBI NIH HHS · HL58732 · United States
External Links
PubMed source DOI: 10.1083/jcb.149.7.1503 PMC Full Text View journal details
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