(A–B) Knockdown of MEKK3 or p38δ impaired the interaction of TRAF6 with p62 in response to amino acids. shNT, shMEKK3, or shp38δ HEK293T cells were deprived of amino acids for 50 min and then restimulated with amino acids for 30 min. Cell lysates and p62 immunoprecipitates were analyzed by western blot for the indicated proteins.
(C–E) MEKK3, p38δ, and p62 phosphorylation were required for polyubiquitination of mTOR in response to amino acids. shNT, shMEKK3, and shp38δ HEK293T cells or cells stably expressing FLAG-p62WT or FLAG-p62T269/S272AA were treated as in (A). Cell lysates and mTOR immunoprecipitates were immunoblotted for the indicated proteins.
Results are representative of three experiments.