Key connexin 43 phosphorylation events regulate the gap junction life cycle.
Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in "phosphorylation" based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.
Solan
Joell L
JL
Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Lampe
Paul D
PD
D000818:Animals; D016466:CHO Cells; D002450:Cell Communication; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism
D018031:Connexin 43; D020033:Protein Isoforms; D011493:Protein Kinase C