Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein.
The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.
Park
Darren J
DJ
Natural Products and Cancer Biology Program, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.
Freitas
Tracey A
TA
Wallick
Christopher J
CJ
Guyette
Carrie V
CV
Warn-Cramer
Bonnie J
BJ
D048948:14-3-3 Proteins / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002450:Cell Communication; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D046912:Multiprotein Complexes; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D051381:Rats; D012694:Serine / Q000378:metabolism; D013045:Species Specificity
D048948:14-3-3 Proteins; D018031:Connexin 43; D046912:Multiprotein Complexes; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt