Casein kinase 1 regulates connexin-43 gap junction assembly.
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 delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.
Cooper
Cynthia D
CD
Fred Hutchinson Cancer Research Center and Department of Pathobiology, University of Washington, Seattle, Washington 98109-1024, USA.
Lampe
Paul D
PD
D000818:Animals; D047388:Casein Kinases; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D010696:Phloroglucinol / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018031:Connexin 43; C411652:IC 261; D007211:Indoles; D007527:Isoenzymes; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D010696:Phloroglucinol; D011494:Protein Kinases; D047388:Casein Kinases