MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.     

MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.     

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.     

Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations

ABSTRACT

Autophagy is a conserved adaptive cellular pathway essential to maintain a variety of physiological functions. Core components of this machinery are the six human Atg8 orthologs that initiate formation of appropriate protein complexes. While these proteins are routinely used as indicators of autophagic flux, it is presently not possible to discern their individual biological functions due to our inability to predict specific binding partners. In our attempts towards determining downstream effector functions, we developed a computational pipeline to define structural determinants of human Atg8 family members that dictate functional diversity. We found a clear evolutionary separation between human LC3 and GABARAP subfamilies and also defined a novel sequence motif responsible for their specificity. By analyzing known protein structures, we observed that functional modules or microclusters reveal a pattern of intramolecular network, including distinct hydrogen bonding of key residues (F52/Y49; a subset of HP2) that may directly modulate their interaction preferences. Multiple molecular dynamics simulations were performed to characterize how these proteins interact with a common protein binding partner, PLEKHM1. Our analysis showed remarkable differences in binding modes via intrinsic protein dynamics, with PLEKHM1-bound GABARAP complexes showing less fluctuations and higher number of contacts. We further mapped 373 genomic variations and demonstrated that distinct cancer-related mutations are likely to lead to significant structural changes. Our findings present a quantitative framework to establish factors underlying exquisite specificity of human Atg8 proteins, and thus facilitate the design of precise modulators.

Abbreviations: Atg: autophagy-related; ECs: evolutionary constraints; GABARAP: GABA type A receptor-associated protein; HsAtg8: human Atg8; HP: hydrophobic pocket; KBTBD6: kelch repeat and BTB domain containing 6; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MD: molecular dynamics; HIV-1 Nef: human immunodeficiency virus type 1 negative regulatory factor; PLEKHM1: pleckstrin homology and RUN domain containing M1; RMSD: root mean square deviation; SQSTM1/p62: sequestosome 1; WDFY3/ALFY: WD repeat and FYVE domain containing 3     

FKBP8 recruits LC3A to mediate Parkin‐independent mitophagy

Mitophagy, the selective removal of damaged or excess mitochondria by autophagy, is an important process in cellular homeostasis. The outer mitochondrial membrane (OMM) proteins NIX, BNIP3, FUNDC1, and Bcl2‐L13 recruit ATG8 proteins (LC3/GABARAP) to mitochondria during mitophagy. FKBP8 (also known as FKBP38), a unique member of the FK506‐binding protein (FKBP) family, is similarly anchored in the OMM and acts as a multifunctional adaptor with anti‐apoptotic activity. In a yeast two‐hybrid screen, we identified FKBP8 as an ATG8‐interacting protein. Here, we map an N‐terminal LC3‐interacting region (LIR) motif in FKBP8 that binds strongly to LC3A both in vitro and in vivo. FKBP8 efficiently recruits lipidated LC3A to damaged mitochondria in a LIR‐dependent manner. The mitophagy receptors BNIP3 and NIX in contrast are unable to mediate an efficient recruitment of LC3A even after mitochondrial damage. Co‐expression of FKBP8 with LC3A profoundly induces Parkin‐independent mitophagy. Strikingly, even when acting as a mitophagy receptor, FKBP8 avoids degradation by escaping from mitochondria. In summary, this study identifies novel roles for FKBP8 and LC3A, which act together to induce mitophagy.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis

Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE‐16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells. We further show that both subfamilies act differently at early stages of autophagosome biogenesis. Accordingly, our results indicate that LC3s are involved in elongation of the phagophore membrane whereas the GABARAP/GATE‐16 subfamily is essential for a later stage in autophagosome maturation.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

Structural basis for extremely strong binding affinity of giant ankyrins to LC3/GABARAP and its application in the inhibition of autophagy

The Atg8/LC3/GABARAP family of proteins binds its physiological binding partners, which function in macroautophagy (hereafter autophagy), via recognition of their short linear motif, also known as the LC3-interactiong region (LIR) or Atg8-interacting motif (AIM). The AIM/LIR motif, with the consensus sequence [W/F/Y]xx[L/I/V], utilizes the aromatic and hydrophobic residues that bind on the surface of Atg8/LC3/GABARAP. Despite modest binding affinity, this interaction is essential for efficient autophagy. Here we highlight the recent paper by Li and collaborators who discovered the structural basis for a much stronger interaction between the LIR motif-containing peptides and LC3/GABARAP. Moreover, they showed that these peptides are potent and selective inhibitors of autophagy in cultured cells and in C. elegans.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Analysis of the native conformation of the LIR/AIM motif in the Atg8/LC3/GABARAP-binding proteins

The Atg8/LC3/GABARAP family of proteins, a group that has structural homology with ubiquitin, connects with a large set of binding partners to function in macroautophagy (hereafter autophagy). This interaction occurs primarily via a conserved motif termed the LC3-interacting region (LIR), or the Atg8-interacting motif (AIM). The consensus sequence for this motif, [W/F/Y]xx[L/I/V], can be found in many proteins, but only some of them are physiological partners containing a functional LIR/AIM. Because the structure of many full-length partners has not been, or cannot be, solved, the structural context of the LIR/AIM within the native protein conformation is not obvious. Here we suggest that the functional LIR/AIM is a short linear motif (SLiM) protein-binding module, arising from an intrinsically disordered region. This finding enables the rapid elimination of some false Atg8/LC3/GABARAP-binding proteins, and connects the exponentially growing knowledge on disordered SLiMs with autophagy.     

BCL2L13 is a mammalian homolog of the yeast mitophagy receptor Atg32

Although Atg32 is essential for mitophagy in yeast, no mammalian homolog has been identified. Here, we demonstrate that BCL2L13 (BCL2-like 13 [apoptosis facilitator]) is a functional mammalian homolog of Atg32. First, we hypothesized that a mammalian mitophagy receptor will share certain molecular features with Atg32. Using the molecular profile of Atg32 as a search tool, we screened public databases for novel Atg32 functional homologs and identified BCL2L13. BCL2L13 induces mitochondrial fragmentation and mitophagy in HEK293 cells. In BCL2L13, the BH domains are important for fragmentation, whereas the WXXI motif, an LC3 interacting region, is needed for mitophagy. BCL2L13 induces mitochondrial fragmentation and mitophagy even in the absence of DNM1L/Drp1 and PARK2/Parkin, respectively. BCL2L13 is indispensable for mitochondrial damage-induced fragmentation and mitophagy. Furthermore, BCL2L13 induces mitophagy in Atg32-deficient yeast. Induction and/or phosphorylation of BCL2L13 may regulate its activity. Our findings thus open a new chapter in mitophagy research.     

自噬相关蛋白LC3Av1和LC3B在大鼠牙髓炎中的表达研究

目的 研究自噬相关蛋白LC3Av1和LC3B在大鼠实验性牙髓炎组织中的表达。方法 30只8周龄SD大鼠左侧下颌第一磨牙开髓,封入大肠埃希菌LPS（5 μg/μL）棉球,玻璃离子封闭髓腔。分别于开髓后0、6、12、24和48 h处死大鼠,分离下颌骨。组织学处理后,HE染色观察牙髓组织炎症状况,免疫组织化学染色SP法检测LC3Av1和LC3B的表达及分布。结果 正常牙髓组织中LC3Av1和LC3B少量表达。实验组中,LC3Av1在12、24和48 h时表达量较0、6 h时显著增高,差异具有统计学意义（P<0.01）;LC3B在6、12 h时表达量较0、24和48 h时增高,差异具有统计学意义（P<0.05）;两种蛋白表达分布均集中于成牙本质细胞及多细胞层中的成纤维细胞。结论 自噬相关蛋白LC3Av1和LC3B在牙髓组织中的表达随炎症发展增加,提示自噬作用可能与牙髓炎的发生发展相关。     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.     

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

Fluorescence‐based ATG8 sensors monitor localization and function of LC3/GABARAP proteins

Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin‐like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3‐interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C‐specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.     

Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process

The phagophore membrane is highly curved along the rim of the open cup, suggesting that the molecular mechanisms governing its formation and growth could rely on membrane curvature-dependent events. To this end, we recently reported that lipidation of the LC3 protein family is facilitated on highly curved membranes in vitro. We further showed that the conjugating enzyme ATG3 contains an amphipathic helix that is responsible for this membrane curvature dependency, and that the maintenance of this amphipathic structure is essential for ATG3 function in vivo.     

Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process

The phagophore membrane is highly curved along the rim of the open cup, suggesting that the molecular mechanisms governing its formation and growth could rely on membrane curvature-dependent events. To this end, we recently reported that lipidation of the LC3 protein family is facilitated on highly curved membranes in vitro. We further showed that the conjugating enzyme ATG3 contains an amphipathic helix that is responsible for this membrane curvature dependency, and that the maintenance of this amphipathic structure is essential for ATG3 function in vivo.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.