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Abbreviations: BBC3/PUMA, BCL2 binding component 3; BCL2L11/BIM, BCL2-like 11; CA, constitutively active; CDKN1A/ p21, cyclin-dependent kinase inhibitor 1A (p21, Cip1); CHUK/IKKa, conserved helix-loop-helix ubiquitous kinase; CMA, chaperone-mediated autophagy; CHX, cycloheximide; DOX, doxorubicin; IKBKB/IKKb, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase b; HSPA8/HSC70, heat shock 70kDa protein 8; LAMP2A, lysosomal-associated membrane protein type 2A; NFKB1/NF-kB, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; NFKBIA/IkBa, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, a; PNS, postnuclear supernatant; shRNA, short hairpin RNA; TNF, tumor necrosis factor; TP53/p53, tumor protein 53.Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that dictate the cell fate choice between autophagy and apoptosis remain largely unknown. Here we report that the proapoptotic protein BBC3/PUMA (BCL2 binding component 3) is a bona fide substrate of chaperone-mediated autophagy (CMA). BBC3 associates with HSPA8/HSC70 (heat shock 70kDa protein 8), leading to its lysosome translocation and uptake. Inhibition of CMA results in stabilization of BBC3, which in turn sensitizes tumor cells to undergo apoptosis. We further demonstrate that upon TNF (tumor necrosis factor) treatment, IKBKB/IKKb (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase b)-mediated BBC3 Ser10 phosphorylation is crucial for BBC3 stabilization via blocking its degradation by CMA. Mechanistically, Ser10 phosphorylation facilitates BBC3 translocation from the cytosol to mitochondria. BBC3 stabilization resulting from either Ser10 phosphorylation or CMA inhibition potentiates TNF-induced apoptotic cell death. Our findings thus reveal that the selective degradation of BBC3 underlies the prosurvival role of CMA and define a previously unappreciated proapoptotic role of IKBKB that acts through phosphorylation-mediated stabilization of BBC3, thereby promoting TNFtriggered apoptosis.

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32-ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis. Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics. 1 It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations. 2-5 Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues. 6-9 Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis. 1 Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation. 10,11 Notably, p32 was found to form protein complex with a variety of molecules 7,12,13 and has been suggested that it may act as a multifunctional chaperone protein. [12][13][14] ULK1 has a crucial role in mitophagy induction. 15 Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation 16,17 and phosphorylation [18][19][20] have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy. 21 Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a ...

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32-ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis. Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics.1 It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations.2-5 Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues.6-9 Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis.1 Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation.10,11 Notably, p32 was found to form protein complex with a variety of molecules 7,12,13 and has been suggested that it may act as a multifunctional chaperone protein. [12][13][14] ULK1 has a crucial role in mitophagy induction. 15 Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation 16,17 and phosphorylation [18][19][20] have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy. 21 Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a defec...

Unc-51 like kinase-1 (Ulk1) is essential for autophagy induction. We recently reported that p32/C1QBP is crucial for the induction of autophagic flux upon starvation and the clearance of damaged mitochondria through the regulation of Ulk1 stability and kinase activity. Our results underscore the importance of p32 in fine-tuning the stress response and mitochondrial homeostasis via Ulk1.

No abstract