title: Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels.
abstract: 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-Raf(V600E)-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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Jin-Hwan
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Seung-Keun
authors.initials: SK
authors.lastname: Hong
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Pui-Kei
authors.initials: PK
authors.lastname: Wu
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Alexsia L
authors.initials: AL
authors.lastname: Richards
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: William T
authors.initials: WT
authors.lastname: Jackson
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Electronic address: jipark@mcw.edu.
authors.forename: Jong-In
authors.initials: JI
authors.lastname: Park
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D017209:Apoptosis; D001343:Autophagy; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D000091342:Endoplasmic Reticulum Chaperone BiP; D005347:Fibroblasts / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071456:Sequestosome-1 Protein; D048490:raf Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D048490:raf Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human
keywords: Autophagy; BiP/GRP78; ERK1/2; LC3; Raf; SQSTM1