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

Jin-Hwan Kim1, Seung-Keun Hong1, Pui-Kei Wu1

  • 1Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Experimental Cell Research
|August 17, 2014
PubMed

Insights

The Raf/MEK/ERK pathway influences autophagy markers microtubule-associated protein 1A/1B-light chain 3 (LC3B) and sequestosome 1 (SQSTM1) levels. This pathway alters autophagic flux and increases LC3B and SQSTM1 expression, with BiP acting as a key effector.

Area of Science:

  • Cell Biology
  • Molecular Signaling
  • Autophagy Research

Background:

  • Cellular levels of autophagy markers LC3B and SQSTM1 are crucial for monitoring autophagic activity.
  • Understanding the regulatory mechanisms of these markers by signaling pathways is essential.

Purpose of the Study:

  • To elucidate how the Raf/MEK/ERK signaling pathway regulates the cellular levels of LC3B and SQSTM1.
  • To investigate the role of BiP/GRP78 in this regulatory process.

Main Methods:

  • Utilized cell-based assays with engineered Raf/MEK/ERK pathway activators (ΔRaf-1:ER, B-Raf(V600E)).
  • Employed autophagy inhibitors (chloroquine, bafilomycin A1) and ATG7 RNA interference.
  • Assessed mRNA and protein levels of LC3B, SQSTM1, and BiP/GRP78.
  • Conducted luciferase reporter assays to analyze transcriptional regulation.

Main Results:

  • Raf/MEK/ERK activation increased LC3B-I, LC3B-II, and SQSTM1/p62 protein levels.
  • Increased BiP/GRP78 expression was observed concurrently with LC3B and SQSTM1 changes.
  • Autophagic flux was altered, but increased gene expression of LC3B and SQSTM1 also contributed to higher protein levels.
  • SQSTM1 upregulation occurred at the transcriptional level.
  • BiP/GRP78 upregulation was necessary for the Raf/MEK/ERK pathway to regulate LC3B protein levels.

Conclusions:

  • The Raf/MEK/ERK pathway modulates autophagy markers LC3B and SQSTM1 not only by affecting autophagic flux but also by increasing their expression.
  • BiP/GRP78 plays a critical role as an effector in the Raf/MEK/ERK-mediated regulation of LC3B protein levels.
  • Altered expression contributes to steady-state levels of LC3B and SQSTM1 under activated Raf/MEK/ERK signaling.
Keywords:
AutophagyBiP/GRP78ERK1/2LC3RafSQSTM1

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