Regulation of the interaction between glycogen synthase kinase 3 and the Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen.
The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein stabilizes beta-catenin by the novel mechanism of binding to the negative regulator, glycogen synthase kinase 3 (GSK-3), and depleting cytoplasmic GSK-3 levels. The two domains of LANA required for interaction with GSK-3 were further characterized. Evidence for similarity between the C-terminal LANA interaction domain and the axin GSK-3 interaction domain was obtained using GSK-3 and LANA mutants. GSK-3(F291L), which does not interact with axin, also failed to bind to LANA, and a mutation in the axin homology domain of LANA, L1132P, destroyed binding to GSK-3. The N-terminal LANA interaction domain was found to mediate interaction by acting as a substrate for GSK-3. GSK-3(R96A), a priming pocket mutant, did not bind to LANA, suggesting that LANA was a primed GSK-3 substrate. Phosphorylation of endogenous LANA precipitated from primary effusion lymphoma cells was inhibited by the GSK-3 inhibitor LiCl. GST-LANA(1-340) was phosphorylated by GSK-3, and mitogen-activated protein kinase (MAPK) and casein kinase I functioned as priming kinases in vitro. Mutation of consensus GSK-3 sites revealed that sites between LANA amino acids 219 and 268 were important for GSK-3 phosphorylation. Immunoprecipitation assays revealed that loss of GSK-3 phosphorylation of this N-terminal domain correlated with loss of GSK-3 interaction. Although LANA-associated GSK-3 actively phosphorylated LANA, GSK-3 coprecipitated with LANA was unable to phosphorylate an exogenous peptide substrate. LANA sequestration of GSK-3 may explain the ability of KSHV-infected cells to tolerate increased levels of nuclear GSK-3.
Fujimuro
Masahiro
M
Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan.
Liu
Jianyong
J
Zhu
Jian
J
Yokosawa
Hideyoshi
H
Hayward
S Diane
SD
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D006367:HeLa Cells; D019288:Herpesvirus 8, Human / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
D000956:Antigens, Viral; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen; D038362:Glycogen Synthase Kinase 3