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

Abstract

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.

Keywords: BHD; FLCN; GABARAP; MAP1LC3B; SQSTM1; ULK1; autophagy.

Figure 1. SQSTM1 levels are elevated in FLCN-deficient cells and BHD-tumor derived tissue. (A) Control HK2 cells (non-target (NT) shRNA) and those with stable knockdown of FLCN were starved for 4 h in KRB or grown in normal media and SQSTM1 levels were analyzed. Data are mean ± SEM of 3 independent experiments. (B) HA-FLCN was re-expressed (with coexpressed HA-SQSTM1) in FLCN knockdown HK2 cells and HA-SQSTM1 levels were analyzed by western blot. Data are mean ± SEM of 3 independent experiments. ***P < 0.001. (C) A tumor sample (T) showing mixed histology of clear and chromophobe cells, and surrounding normal tissue (N) from a BHD patient was stained for SQSTM1. Scale bar: 100 μm.

Figure 2. FLCN is a positive driver of autophagy. (A) HK2 control and FLCN knockdown cells were transfected with the ptfMAP1LC3B vector, fixed, and examined by confocal microscopy. Representative maximal Z-projection images of cells showing the RFP-GFP-MAP1LC3B puncta are shown in the upper panel. Scale bar: 10 μm. Red and yellow puncta were scored across 3 independent experiments (at least 30 cells per cell line in total) and are plotted below the images, mean ± SEM (B) Control HK2 cells and those with stable knockdown of FLCN were treated with 100 μM chloroquine (CQ) for the indicated times. Samples were probed for conversion of MAP1LC3B (graphed in panel below, mean ± S.E.M.) and GABARAP expression. (C) As for (B) but in HEK293 cells with transient knockdown of FLCN expression. For all graphs *P < 0.05, **P < 0.01.

Figure 3. FLCN interacts with GABARAP, which is enhanced in the presence of FNIP1/2. (A) A network of GABARAP interactors, as determined by mass spectrometry. (B) GST alone or GST-GABARAP was used as bait, and bound endogenous FLCN was detected by western blot. (C) Bacterially expressed GST, GST-MAP1LC3B, and GST-GABARAP was used as bait for lysates with or without overexpression of HA-FLCN. Following GST purification, bound HA-FLCN was detected by western blot. (D) V5-FLCN, HA-MAP1LC3B or HA-GABARAP were expressed in HEK293 cells with FNIP proteins where indicated. Following an HA immunoprecipitation, V5-FLCN was detected by western blot. Total blots represent 30% of IP input. (E) MDCK cells were transfected with EGFP-FLCN-WT, myc-FNIP2 and HA-GABARAP. Cells were stained with mouse-anti-HA and polyclonal FNIP2 antibody. Scale bar: 20 μm. (F) Myc-FNIP2 was expressed and immunoprecipitated from HEK293 cells. Endogenous FLCN and GABARAP bound to FNIP2 were detected by western blotting. Total blots represent 1% of IP input.

Figure 4. Expression of wild-type ULK1 induces phosphorylation of FLCN. (A) Untagged FLCN was coexpressed with V5-tagged wild-type (WT) or kinase-dead (KD) ULK1 as indicated in HEK293 cells, and subjected to V5 immunoprecipitation. FLCN bound to ULK1 was detected by western blotting. Total blots represent 40% of IP input. (B) Incorporation of [³²P] into HA-FLCN in vivo was determined in the presence and absence of ULK1. (C) An in vitro kinase assay was performed using wild-type (WT) or kinase-dead (KD) ULK1 against purified ATG13 and FLCN. Phosphorylation was determined by [³²P] incorporation.

Figure 5. Mass spectrometry reveals 3 ULK1-mediated phosphorylation sites on FLCN. (A–C) Mass spectrometry (LC-MS/MS) was used to determine the phosphorylated residues of FLCN coexpressed with ULK1. (D) A multispecies alignment of FLCN proteins using Clustal Omega shows that the Ser406, Ser537 and Ser542 phosphorylation sites are well conserved between species. (E) Cartoon representation of the mapped phosphorylation sites on the crystal structure of the FLCN C-terminal domain (PDB Id: 3V42). The insets show a closer view of the serine residues, which are represented as sticks.

Figure 6. ULK1 modulates the FLCN-GABARAP interaction. (A) V5-FLCN and myc-FNIP2 bound to HA-GABARAP in the presence or absence of wild-type (WT) or kinase-dead (KD) V5-ULK1 were determined by immunoprecipitating HA-GABARAP and detecting bound proteins by western blot. Total blots represent 20% of IP input. (B) Cells were transfected with control or ULK1 shRNA, along with V5-FLCN, myc-FNIP2, and HA-GABARAP and grown in complete DMEM. Following an HA immunoprecipitation, V5-FLCN and myc-FNIP2 were detected by western blot. Total blots represent 20% of IP input. The graphs show relative binding of V5-FLCN to HA-GABARAP and myc-FNIP2 to HA-GABARAP as determined by densitometry across 4 independent experiments, mean ± SEM *P < 0.05. (C) Bacterially expressed GST, GST-MAP1LC3B or GST-GABARAP was used as bait for lysates containing HA-FLCN (WT or 3A mutant). Following GST purification, bound HA-FLCN was detected by western blot. GST loading controls are shown for each pulldown. (D) Wild-type (WT) FLCN, or the serine-to-alanine (3A) FLCN mutant, were re-expressed in FLCN-deficient HK2 cells, along with HA-SQSTM1. HA-SQSTM1 levels were determined by western blot and densitometry of SQSTM1 levels from 3 independent experiments are plotted in the graph, mean ± SEM *P < 0.05, ***P < 0.001.

Figure 7. Patient tumors and patient-derived FLCN mutations show autophagy defects. (A) Kidney tumor tissues from a BHD patient showing mixed histology of clear cell and chromophobe cells were stained for SQSTM1, GABARAP and MAP1LC3B and compared with normal kidney. Scale bar: 50 μm. (B) HA-FLCN (wild-type or patient-derived mutants) was coexpressed with V5-tagged wild-type (WT) ULK1 in HEK293 cells, and subjected to V5 immunoprecipitation. FLCN bound to ULK1 was detected by western blotting. Total blots represent 40% of IP input. (C) HA-GABARAP was coexpressed with untagged-FLCN (wild-type or patient-derived mutants) and myc-FNIP2 in HEK293 cells, and subjected to HA immunoprecipitation. FLCN bound to GABARAP was detected by western blotting. Total blots represent 5% of IP input. (D) Untagged FLCN (wild type or mutants) was re-expressed (with coexpressed HA-SQSTM1) in FLCN knockdown HK2 cells and HA-SQSTM1 levels were analyzed by western blot. Data are mean ± SEM of 5 independent experiments. *P < 0.05, **P < 0.01.