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

• Raf/MEK/ERK activation affects cellular levels of BiP, LC3B, and SQSTM1 at mRNA and protein levels.
• Raf/MEK/ERK regulates BiP and SQSTM1 at the transcription level.
• BiP is necessary for Raf/MEK/ERK to regulate LC3B at the protein, but not mRNA, level.
• Raf/MEK/ERK can thus regulate certain autophagy machinery via non-canonical mechanisms
• This signaling may be important for determining cellular capacity for autophagy.

While cellular LC3B 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 LC3B and SQSTM1 levels. In different cell types, ΔRaf-1:ER- or B-RafV600E-mediated MEK/ERK activation increased LC3B-I, LC3B-II, and SQSTM1/p62 levels, which was accompanied by increased BiP/GRP78 expression. Use of the autophagy inhibitors chloroquine and bafilomycin A1, or RNA interference of ATG7, suggested that these increases in LC3B 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 LC3B 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/MEK/ERK to regulate LC3B 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.