title: Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.
abstract: The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.
authors.affiliation: Molecular Diagnostics Program, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
authors.forename: Clarence A
authors.initials: CA
authors.lastname: Dunn
authors.forename: Vivian
authors.initials: V
authors.lastname: Su
authors.forename: Alan F
authors.initials: AF
authors.lastname: Lau
authors.forename: Paul D
authors.initials: PD
authors.lastname: Lampe
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation / Q000502:physiology; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D018031:Connexin 43; D051057:Proto-Oncogene Proteins c-akt