Active JNK2-mediated β-catenin degradation occurred through the proteasome system and GSK3β.

Abstract

<p>(A) HEK293T cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Forty-four hours after transfection, 25 µM MG132 was added to the indicated samples (lane 2 and 4). Four hours later cells were harvested for immunoblotting analysis to detect the expression of HA-β-catenin and p-JNK. (B) Blocking GSK3β activity by LiCl reduced β-catenin expression inhibition by activated JNK2. pcDNA3-HA-β-catenin was transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Thirty-six hours after transfection, half of the cultures were treated overnight with 30 mM LiCl (lane 2 and 4) and then harvested for immunoblotting analysis to detect the expression of HA-β-catenin, phospho-Ser-9 GSK3β, and p-JNK. (C) Mutant β-catenin was resistant to activated JNK2 induced degradation. Wild-type β-catenin (HA- β-catenin) (lanes 1 and 2) or various β-catenin mutants (HA-S33F β-catenin, lanes 3 and 4; HA-S33Y β-catenin, lanes 5 and 6; HA-S37A β-catenin, lanes 7 and 8) were transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 2,4,6,8) or empty vector (lanes 1,3,5,7). 48 hours after transfection, cells were harvested for immunoblotting analysis to determine the protein levels of HA-β-catenin. β-actin served as loading control.</p