Fig. 2

Aida Inhibits Axin-Meditated JNK Activation by Disrupting Homodimerization of Axin (A) Aida inhibits Axin-mediated JNK activation. HEK293T cells were transfected with FLAG-tagged JNK together with other plasmids as indicated. Following immunoprecipitation of FLAG-JNK, JNK kinase activities were assayed using GST-c-Jun as substrate. The amount of the kinase and the kinase activity in each immunoprecipitate were quantified by immunoblotting using anti-JNK antibody and anti-phospho-c-Jun antibody, respectively. (B) AidaΔAxin that cannot bind to Axin fails to attenuate Axin-mediated JNK activation. (C) AxinΔAida-mediated JNK activation could not be attenuated by Aida. (D) Zebrafish Aida inhibits Axin-induced JNK activation. An experiment similar to (A) was performed using zebrafish axin and aida instead of their mouse counterparts. The immunokinase assay was performed as described above. (E) The siRNA against Aida enhances AP-1 activity. HEK293T cells were transfected with AP-1 luciferase reporter together with empty pSUPER vector, pSUPER-Aida, or control RNAi. At 32 hr posttransfection, relative luciferase activity was measured. (F) Aida has no effect on Wnt signaling as assessed with LEF-1 luciferase reporter. HEK293T cells were transfected with 0.5 μg of LEF-1 luciferase reporter plasmid together with 1.5 μg of Axin, Aida alone, or both. Aida did not affect basal, wnt-1-induced, or Axin-downregulated reporter expression. (G) Aida disrupts Axin homodimerization as determined by using two differently tagged Axins, HA-Axin and FLAG-Axin. HEK293T cells were transfected with HA-Axin and FLAG-Axin together with increasing amounts of Aida (0 μg, 1 μg, and 3 μg). When HA-tagged Axin was immunoprecipitated by anti-HA, the amount of coprecipitated FLAG-tagged Axin was negatively correlated with the increasing amounts of cotransfected Aida (left panel); similarly, decreasing amounts of HA-tagged Axin were coprecipitated with FLAG-Axin by the anti-FLAG antibody in the presence of increasing amounts of Aida (middle panel). The right panel shows similar expression levels of HA- and FLAG-tagged Axin among the three samples, and it shows that Aida levels were proportionally increased when increasing amounts of DNA were transfected. (H) AidaΔAxin has no effect on Axin homodimerization. Coimmunoprecipitated HA-Axin levels in anti-FLAG immunoprecipitates were not changed in the presence of increasing amounts of AidaΔAxin.