Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

Abstract

In selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a time-dependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01383-13.

ACKNOWLEDGMENTS

Wild-type and p62 gene knockout mouse embryonic fibroblasts as well as the pEGFP-p62 plasmid were gifts from Jorge Moscat (Sanford-Burnham Medical Research Institute). We thank Karla Kirkegaard (Stanford University) for the pEGFP-LC3 plasmid and Eric J. Brown (Genentech) for the pBABE-puro-mCherry-EGFP-LC3B plasmid.

This work was supported by awards P50 CA142509 to Y.D., S.C., R.Z.O., and S.G. and awards CA100866, CA93738, and CA167708 to S.G. and Y.D. from the National Institutes of Health; an award to S.G. from the Multiple Myeloma Research Foundation; and award R6238 to S.G. and Y.D. from the Leukemia and Lymphoma Society of America. Plasmid preparation was performed at the VCU Macromolecule Core Facility, supported in part by funding from NIH-NCI Cancer Center Core grant 5P30CA016059-29. Confocal microscopy and EM were performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported in part by funding from NIH-NINDS Center Core grant 5P30NS047463.