TY - JOUR
T1 - ATG5 cancer mutations and alternative mRNA splicing reveal a conjugation switch that regulates ATG12–ATG5-ATG16L1 complex assembly and autophagy
AU - Wible, Daric J.
AU - Chao, Hsueh Ping
AU - Tang, Dean G.
AU - Bratton, Shawn B.
N1 - Funding Information:
The authors wish to thank Drs. Noburu Mizushima (University of Tokyo, JP), Masaaki Komatsu (Tokyo Metropolitan Institute of Medical Science, JP), Jayanta Debnath (University of California, San Francisco, USA) and Shizou Akira (Osaka University, JP) for providing Atg5−/−, Atg7−/−, Atg12−/−, and Atg16l1Δ/Δ MEFs, respectively52–55. This work was supported by an NIEHS Toxicology Training Grant fellowship (T32ESO7274) to D.J.W.; a DOD grant (W81XWH-16-1-0575) to D.G.T.; and NCI/NIGMS grants (CA129521 and GM096101) and an Institutional Research Grant (IRG) from The University of Texas MD Anderson Cancer Center (UTMDACC) to S.B.B. This study utilized the UTMDACC Protein Array and Analysis Core (PAAC) Facility (CPRIT PR130432), as well as the UTMDACC Flow Cytometry and Cellular Imaging Core Facility (FCCICF)-Smithville and the UTMDACC Functional Genomics Core (FGC) Facility, which are supported by an NIH/NCI Cancer Center Support Grant (P30 CA016672).
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85071155028&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85071155028&partnerID=8YFLogxK
U2 - 10.1038/s41421-019-0110-1
DO - 10.1038/s41421-019-0110-1
M3 - Article
C2 - 31636955
AN - SCOPUS:85071155028
SN - 2056-5968
VL - 5
JO - Cell Discovery
JF - Cell Discovery
IS - 1
M1 - 42
ER -