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. 2016 Feb 16;113(9):E1226–E1235. doi: 10.1073/pnas.1600813113

Fig. 3.

Fig. 3.

MEKK2 stabilizes β-catenin via phosphorylation of S675. (A) GST or GST–β-catenin was incubated with purified HA-MEKK2 (WT or KD mutant), and MEKK2 kinase activity was analyzed by an in vitro kinase assay. (B) Purified HA-MEKK2 was incubated with GST or GST–β-catenin [WT or S675A (SA) mutant], and MEKK2 kinase activity was analyzed by in vitro kinase assay. (C) Vector or β-catenin (WT or S675A mutant) was transfected into C3H10T1/2 cells along with TOPflash-luciferase and Renilla in the absence or presence of MEKK2. Luciferase activity was measured after 48 h of transfection and normalized to Renilla. P < 0.05 by one-way ANOVA. P < 0.05; P values for Bonferroni-corrected Student’s t tests: **P < 0.01; ***P < 0.001. (D) β-Catenin–deficient COBs were reconstituted with Flag–β-catenin WT or S675A mutant via lentivirus-mediated delivery and cultured under osteoblast differentiation conditions for 7 d. Lysates were immunoblotted with the indicated antibodies. (E) HEK293 cells were transfected with Flag–β-catenin WT or S675A mutant along with HA-MEKK2, and β-catenin stability was determined by pulse-chase labeling with [35S]methionine followed by autoradiography. (F) HEK293 cells were cotransfected with Flag–β-catenin WT or S675A mutant along with different amounts of HA-ubiquitin and were treated with 10 µM MG132 for 8 h before lysis. Ubiquitinated β-catenin was immunoprecipitated by anti-Flag antibody–conjugated agarose and immunoblotted with anti-HA antibody. (G) An in vitro ubiquitination assay of β-catenin was performed using recombinant proteins. Activated His–β-catenin was incubated with the recombinant SCFSKP1 complex, ubiquitin, ATP, E1, and E2 in the presence or absence of recombinant MEKK2. Ubiquitinated β-catenin was immunoprecipitated by Ni-NTA agarose, subjected to SDS/PAGE, and immunoblotted with an anti-ubiquitin antibody. S675 phosphorylation of β-catenin by MEKK2 was confirmed by immunoblotting. (H) WT immortalized COBs were lysed, immunoprecipitated with anti–β-catenin or an IgG control and protein G-conjugated Dynabeads, and immunoblotted with the indicated antibodies.