Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels

While cellular LOB and SQSTM1 levels serve as key autophagy markers, their regulation by different signaling pathways requires better understanding. Here, we report the mechanisms by which the Raf/MEK/ERK pathway regulates cellular LOB and SQSTM1 levels. In different cell types, Delta Raf-1 :ER- or B-Raf(V600E)-mediated MEK/ERK activation increased LOB-I, LOB-II, and SQSTM1 /p62 levels, which was accompanied by increased BiP/GRP78 expression. Use of the autophagy inhibitors chloroquine and bafilomycin Al, or RNA interference of ATG7, suggested that these increases in LOB and SQSTM1 levels were in part attributed to altered autophagic flux. However, intriguingly, these increases were also attributed to their increased expression. Upon Raf/MEK/ERK activation, mRNA levels of LOB and SQSTM1 were also increased, and subsequent luciferase reporter analyses suggested that SQSTM1 upregulation was mediated at transcription level. Under this condition, transcription of BiP/GRP78 was also increased, which was necessary for Raf/MEKIERK to regulate LOB at the protein, but not mRNA, level. This suggests that BiP has a role in regulating autophagy machinery when Raf/MEK/ERK is activated. In conclusion, these results suggest that, under a Raf/MEK/ERK-activated condition, the steady-state cellular levels of LC3B and SQSTM1 can also be determined by their altered expression wherein BiP is utilized as an effector of the signaling. (C) 2014 Elsevier Inc. All rights reserved.