GSK3 beta-dependent phosphorylation of the alpha NAC coactivator regulates its nuclear translocation and proteasome-mediated degradation.
c-Jun is an immediate-early gene whose degradation by the proteasome pathway is required for an efficient transactivation. In this report, we demonstrated that the c-Jun coactivator, nascent polypeptide associated complex and coactivator alpha (alphaNAC) was also a target for degradation by the 26S proteasome. The proteasome inhibitor lactacystin increased the metabolic stability of alphaNAC in vivo, and lactacystin, MG-132, or epoxomicin treatment of cells induced nuclear translocation of alphaNAC. We have shown that the ubiquitous kinase glycogen synthase kinase 3beta (GSK3beta) directly phosphorylated alphaNAC in vitro and in vivo. Inhibition of the endogenous GSKappa3beta activity resulted in the stabilization of this coactivator in vivo. We identified the phosphoacceptor site in the C-terminal end of the coactivator, on position threonine 159. We demonstrated that the inhibition of GSK3beta activity by treatment of cells with the inhibitor 5-iodo-indirubin-3'-monoxime, as well as with a dominant-negative GSK3beta mutant, induced the accumulation of alphaNAC in the nuclei of cells. Mutation of the GSK3beta phosphoacceptor site on alphaNAC induced a significant increase of its coactivation potency. We conclude that GSK3beta-dependent phosphorylation of alphaNAC was the signal that directed the protein to the proteasome. The accumulation of alphaNAC caused by the inhibition of the proteasome pathway or the activity of GSK3beta contributes to its nuclear translocation and impacts on its coactivating function.
Genetics Unit, Shriners Hospital for Children, Montréal, Québec, Canada H3G 1A6.
Isabelle
I
Quélo
Omar
O
Akhouayri
Josée
J
Prud'homme
René
R
St-Arnaud
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004357:Drug Synergism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000378:metabolism / Q000502:physiology*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity / Q000235:genetics; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D018832:Molecular Chaperones; D010761:Phosphorus Radioisotopes; D015534:Trans-Activators; C096099:nascent-polypeptide-associated complex; C067713:lactacystin; D000255:Adenosine Triphosphate; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000111:Acetylcysteine