Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control.

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38ß dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control.

p38 mitogen-activated protein kinases (MAPKs) regulate early endocytic trafficking, but their effects on late endocytic trafficking remain unclear. Herein, we report that the pyridinyl imidazole p38 MAPK inhibitors, SB203580 and SB202190, induce a rapid but reversible Rab7-dependent accumulation of large cytoplasmic vacuoles. While SB203580 did not induce canonical autophagy, phosphatidylinositol 3-phosphate [PI(3)P] accumulated on vacuole membranes, and inhibition of the class III PI3-kinase (PIK3C3/VPS34) suppressed vacuolation. Ultimately, vacuolation resulted from the fusion of ER/Golgi-derived membrane vesicles with late endosomes and lysosomes (LELs), combined with an osmotic imbalance in LELs that led to severe swelling and a decrease in LEL fission. Since PIKfyve inhibitors induce a similar phenotype by preventing the conversion of PI(3)P to PI(3,5)P2, we performed in vitro kinase assays and found that PIKfyve activity was unexpectedly inhibited by SB203580 and SB202190, corresponding to losses in endogenous PI(3,5)P2 levels in treated cells. However, vacuolation was not entirely due to 'off-target' inhibition of PIKfyve by SB203580, as a drug-resistant p38α mutant suppressed vacuolation. Moreover, genetic deletion of both p38α and p38ß rendered cells dramatically more sensitive to PIKfyve inhibitors, including YM201636 and apilimod. In subsequent 'washout' experiments, the rate of vacuole dissolution upon the removal of apilimod was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, p38 MAPKs act epistatically to PIKfyve to promote LEL fission; and pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs.

ATG5 cancer mutations and alternative mRNA splicing reveal a conjugation switch that regulates ATG12-ATG5-ATG16L1 complex assembly and autophagy.

Autophagy is critical for maintaining cellular homeostasis during times of stress, and is thought to play important roles in both tumorigenesis and tumor cell survival. Formation of autophagosomes, which mediate delivery of cytoplasmic cargo to lysosomes, requires multiple autophagy-related (ATG) protein complexes, including the ATG12-ATG5-ATG16L1 complex. Herein, we report that a molecular ATG5 "conjugation switch", comprised of competing ATG12 and ubiquitin conjugation reactions, integrates ATG12-ATG5-ATG16L1 complex assembly with protein quality control of its otherwise highly unstable subunits. This conjugation switch is tightly regulated by ATG16L1, which binds to free ATG5 and mutually protects both proteins from ubiquitin conjugation and proteasomal degradation, thereby instead promoting the irreversible conjugation of ATG12 to ATG5. The resulting ATG12-ATG5 conjugate, in turn, displays enhanced affinity for ATG16L1 and thus fully stabilizes the ATG12-ATG5-ATG16L1 complex. Most importantly, we find in multiple tumor types that ATG5 somatic mutations and alternative mRNA splicing specifically disrupt the ATG16L1-binding pocket in ATG5 and impair the essential ATG5-ATG16L1 interactions that are initially required for ATG12-ATG5 conjugation. Finally, we provide evidence that ATG16L2, which is overexpressed in several cancers relative to ATG16L1, hijacks the conjugation switch by competing with ATG16L1 for binding to ATG5. While ATG16L2 stabilizes ATG5 and enables ATG12-ATG5 conjugation, this endogenous dominant-negative inhibitor simultaneously displaces ATG16L1, resulting in its proteasomal degradation and a block in autophagy. Thus, collectively, our findings provide novel insights into ATG12-ATG5-ATG16L1 complex assembly and reveal multiple mechanisms wherein dysregulation of the ATG5 conjugation switch inhibits autophagy.

Reciprocity in ROS and autophagic signaling.

Reactive oxygen species (ROS) are important signaling molecules that mediate oxidative stress and cellular damage when improperly regulated. ROS and oxidative stress can activate autophagy, which generally serves as a cytoprotective negative feedback mechanism to selectively eliminate sources of ROS, including mitochondria and peroxisomes. In this review we describe the mechanisms by which ROS directly and indirectly activate autophagy, and conversely, how selective autophagy suppresses the formation of ROS. Furthermore, we highlight what appear to be contradictory examples in which ROS suppress, rather than activate, autophagy; and where selective autophagy promotes, rather than inhibits ROS production, thereby contributing to cell death. Given that ROS are implicated in cancer, diabetes, atherosclerosis, neurodegenerative diseases and ischemia/reperfusion injury, a deeper understanding of the connections linking ROS and autophagy is greatly needed.