Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

Abstract

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity

“…21,22,60,61 K63-ubiquitination is particularly associated with the autophagic clearance of protein aggregates. 27 These findings provide converging lines of evidence that are consistent with a striking up-regulation of protein sequestration and autophagy as an in vivo response of the respiratory epithelium to inhaled diacetyl vapors.…”

“…27 These findings provide converging lines of evidence that are consistent with a striking up-regulation of protein sequestration and autophagy as an in vivo response of the respiratory epithelium to inhaled diacetyl vapors. 21,30,62 Diacetyl is highly reactive and has a well-established ability to damage proteins. 18 Diacetyl is particularly reactive with the amino acid arginine.…”

“…Indeed, autophagy is more frequently a protective biological response than a major contributor to a pathological response. 21,33,79 Because the reactive a-dicarbonyl group is implicated in diacetyl-induced protein damage, we originally hypothesized that DCXR would provide protection from protein damage through metabolism of diacetyl to acetoin, which does not have the reactive a-dicarbonyl group. 36 Consistent with this hypothesis, we did observe a significantly increased number of cells with ubiquitin puncta and K63-ubiquitin puncta in airway epithelium of DCXR knockout compared with wild-type mice in the 200 ppm exposure group in the dose-response experiment.…”

“…Unfolded and misfolded proteins are often cytotoxic. 20,21 Misfolded proteins tend to form aggregates. Aggregated proteins are extraordinarily toxic to cells, generally inaccessible to the proteasome, and sometimes form recognizable ubiquitin-containing inclusions or puncta within cells.…”

“…21,30 However, excessive autophagy of proteins can lead to cell death and potentially causes ubiquitin and amino acid depletion. This is important because ubiquitin plays an essential role in both protein quality control and regulating proteins important in signaling cascades.…”