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.
Highlights
The gap junctional protein connexin 43 (Cx43) has a 1–3 h half-life, and both the proteasome and lysosome have been implicated in Cx43 turnover
When expressed in cells that did not express wild-type Cx43, this mutant version trafficked to the plasma membrane, formed gap junctions, and responded to proteasomal inhibitors in a manner similar to wild-type Cx43, i.e. junctions became larger in immunofluorescence studies, and slower migrating Cx43 was observed in immunoblots, essentially demonstrating that direct Cx43 ubiquitination was not necessary to observe the effects of proteasomal inhibition on gap junction size
After trying and failing to discover either sites of Cx43 ubiquitination or a functional need for ubiquitination in the cell types we analyzed, we hypothesized that another ubiquitinated protein that was degraded by the proteasome might regulate gap junctional Cx43 retention and/or removal
Summary
The gap junctional protein connexin 43 (Cx43) has a 1–3 h half-life, and both the proteasome and lysosome have been implicated in Cx43 turnover. When transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved 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. 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. A clear polyubiquitin ladder co-labeled with Cx43 and ubiquitin antibodies has not been shown nor has a specific lysine acceptor for ubiquitin been identified in Cx43 that when
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