During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.

The ubiquitin ligase Parkin and autophagic adapter protein p62 are known to function in a common pathway controlling mitochondrial autophagy (mitophagy). However, the evidence supporting that p62 is directly recruited by ubiquitinated proteins remains undetermined. Here, we demonstrate that mitochondrial fission factor (Mff ) associates with Parkin and carbonyl cyanide m-chlorophenyl hydrazone treatment significantly increases the affinity of Parkin with Mff. After recruitment to depolarized mitochondria, Parkin mediates poly-ubiquitination of Mff at lysine 251. Replacement of lysine 251 by arginine (K251R) totally abrogates Parkin-stimulated ubiquitination of Mff. Subsequently, the ubiquitinated Mff promotes its association with p62. Mff knockout interferes with p62 translocation to damaged mitochondria. Only re-transfection of Mff WT, but not K251R mutant, rescues this phenotype. Furthermore, loss of Mff results in failure of Parkin translocation and final clearance of damaged mitochondria. Thus, our data reveal functional links among Mff, p62, and the selective autophagy of mitochondria, which are implicated in the pathogenesis of neurodegeneration diseases.

Aggrephagy, the aggresome-related protein degradation system, represents a protective cellular response to shuttle misfolded proteins into the microtubule-organizing center for degradation through the autophagic pathway during stress conditions, including heat shock, oxidative stress and proteasome inhibition. In response to proteasome failure, many genes are transcriptionally activated to facilitate ubiquitinated proteins to be cleared via the aggrephagy pathway. Although many regulators involved in aggresome formation have been identified, the mechanism how transcriptional activation promotes aggresome formation remains unknown. Here, we have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) accumulated in the nucleus and activated the transcription of sequestosome-1 (p62) during proteasome inhibition in 293 cells. Loss of Nrf2 resulted in failure of aggresome formation and cell death; whereas overexpression of p62 alleviated Nrf2 knockdown-induced aggresome formation defects and promoted cell survival. Notably, blocking Nrf2 activation using a p38/MAPK inhibitor prevented proteasome inhibitor-induced aggresome formation. These findings suggested that Nrf2 may be a critical regulator of aggresome formation, which protects cells from proteasome dysfunction-induced stress.

Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. The expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.

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