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. Author manuscript; available in PMC: 2021 Mar 20.
Published in final edited form as: FEBS Lett. 2020 Nov 20;595(1):123–132. doi: 10.1002/1873-3468.13959

Ciliary localization of folliculin mediated via a kinesin-2-binding motif is required for its functions in mTOR regulation and tumor suppression

Yunlong Zhang 1, Ying Liu 2, Yu Dai 2, Yazhe Ren 1, Guangsen Bao 1, Ai Bo 2, Yu Jiang 2,*
PMCID: PMC7980781  NIHMSID: NIHMS1669813  PMID: 33064845

Abstract

Folliculin (FLCN) is a tumor suppressor protein involved in many cellular processes, including cell signaling, apoptosis and autophagy. In ciliated cells FLCN localizes to primary cilia and controls mTORC1 signaling in response to flow stress. Here, we show that the ciliary localization of FLCN requires its interaction with kinesin-2, the motor protein for anterograde intraflagellar transport. FLCN binds to kinesin-2 through a loop region in the middle of the protein. Single point mutations within the region disrupt its kinesin-2 binding and ciliary entry. The mutants lose the ability to suppress the abnormal mTORC1/2 signaling activities and anchorage-independent growth of FLCN deficient tumor cells. Those observations suggest that ciliary localization of FLCN is essential for its function as a tumor suppressor.

Keywords: FLCN, mTORC1, mTORC2, cilium, kinesin-2

Introduction

The Birt–Hogg–Dubé (BHD) syndrome is a rare autosomal dominant genetic disorder manifested by high incidences of hamartomas and cysts in multiple organs, including the kidney, lung, colon and skin [1]. The syndrome is caused by germ-line mutations in the BHD gene, which encodes the folliculin protein (FLCN), a 64 kD polypeptide that shares little sequence similarity with any other known proteins [2]. In animal models inactivation of FLCN leads to development of polycystic kidneys and renal cell carcinoma that are characteristically similar to those caused by defects in several other tumor suppressors, including tuberous sclerosis complex (TSC1/2), Von Hippel-Lindau (VHL), LKB1 and polycystic proteins (PC1/2) [3,4]. Analysis of the FLCN-deficient cells from BHD patients and animal models reveals hyperactivities of the mechanistic target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2) [5–7], which is believed to contribute to pathophysiological conditions of the BHD syndrome [4]. FLCN binds directly to two homologous proteins termed as folliculin interacting protein 1 (FNIP1) and 2 (FNIP2) [8,9]. The binding presents FLCN to AMPK as well as to the Rag GTPases [8,10,11]. The latter are responsible for tethering mTORC1 to lysosomes that is required for its activation [12]. FLCN has been shown to function as a GTPase activating protein (GAP) that negatively regulates the RagC/D GTPases, and hence mTORC1 activity [11]. However, how FLCN-deficiency leads to aberrant mTORC2 signaling activity remains unclear.

FLCN has recently been found to localize to the primary cilium [13], a unique membranous protrusion on the apical surface of a cell that is formed when the cell exits cell cycle and enters quiescent phase. The primary cilium plays a critical role in cell differentiation and tissue homeostasis by functioning as a signaling antenna for quiescent cells to sense extracellular conditions and communicate with surrounding cells [14,15]. We have previously shown that FLCN interacts directly with KIF3A/KIF3B, the motor subunits of kinesin-2 that mediates the anterograde intraflagellar transport (IFT). FLCN binds to the cargo binding domain of KIF3A/KIF3B, indicating that FLCN may be a cargo of the anterograde IFT [16]. FLCN is required for ciliary accumulation of LKB1 in response to fluidic flow that deflects the cilium. The FLCN-dependent ciliary accumulation of LKB1 correlates with flow stress-induced mTORC1 downregulation [16]. In the absence of FLCN, LKB1 is unable to concentrate in the cilium and mTORC1 activity becomes unresponsive to flow stress. These observations establish a critical role for FLCN in cilium-dependent regulation of mTORC1. However, as function of FLCN has been implicated in other cellular processes independent of the primary cilium [17], it remains unclear whether the role of FLCN in mTORC1 regulation and tumor suppression depends on its ciliary localization.

FLCN contains two putative functional domains located at its N- and C-terminal halves [18]. The N-terminal region bears a structural similarity to the Longin domain that is commonly found in proteins involved in membrane trafficking [19]. The C-terminal region contains a DENN-like sequence shared by many nucleotide exchange factors (GEFs) of small GTPases [20]. The two domains are linked by a flexible loop region in the middle of the protein [21]. Previous studies have shown that the C-terminal DENN domain is required for FLCN to interact with FNIP1, whereas both the Longin and DENN domains are involved for its interaction with FNIP2 [8,21]. To understand how FLCN interacts with kinesin-2 and whether the interaction is required for its role in mTOR regulation, we mapped the region in FLCN mediating its binding with kinesin-2. Our findings show that FLCN interacts with kinesin-2 through its loop region and that the interaction is essential for its ciliary localization and regulation of mTOR signaling.

Materials and Methods

Cell lines, antibodies and plasmids—

HEK293 cells were cultured in DMEM medium supplemented with 10% FBS and 100U ml−1 penicillin/streptomycin. FLCN null line UOK257 and its corresponding FLCN restored line UOK257-2 were kind gifts from Laura Schmidt and Marston Linehan at NCI and have been described before [22]. The cells were normally cultured in DMEM with 5% heat inactivated FBS and 100U ml−1 penicillin/streptomycin. To induce ciliation for the UOK257 cells, cells were cultured in serum starvation condition (0.5% FBS) for 3 days after cells reaching confluence. Antibodies for FLCN (Cat# 3697), FNIP2 (Cat# 57612), AKT (Cat# 9272), phospho-AKT at S473 (cat# 4051), S6 (#2317) and phospho-S6 at S235/236 (Cat# 2211), 4EBP1 (#9452) and phospho-4EBP1at S65 (#9451), RagC (#9480) antibodies were purchased from Cell Signaling Technology. Anti-KIF3A (Cat#376680) and KIF3B (Cat# sc-50456) antibodies were from Santa Cruz Biotechnology. Antibodies for HA (Cat#11666606001), Flag (Cat#F3165) and GFP (Cat#11814460001) epitopes and FNIP1 (Cat# MABS1717) were from Sigma-Aldrich. HA and Flag tagged FLCN expression vectors were created by cloning PCR amplified FLCN gene into pcDNA3.1-3xHA or 3xFlag vector. Point mutations in FLCN were generated by site-directed mutagenesis using the QuikChange Mutagenesis kit from Agilent (Cat#200523). For expression of the kinesin-2 binding domain of FLCN, a double strand oligonucleotide corresponding to the sequence encoding the 19 amino acids of the binding domain was synthesized and inserted at 3’ end of the GFP or GST gene, respectively, in the pEGFP-C1 or pGEX-6P-1 vector. All cloning and mutagenesis constructs were verified by DNA sequencing. All experiments were repeated for at least three times and representative data were shown in the figures.

Co-immunoprecipitation and immunoblotting—

Cells were lysed with lysis buffer containing 50 mM HEPES, pH7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF and 1 x protease inhibitor cocktail (Roche). Cell debris and unbroken cells were removed by centrifugation at 10, 000 g for 10 min. Clarified lysates were incubated with antibodies overnight followed by addition of Protein A conjugated agarose beads (Life Technologies). After incubation for additional 1.5 hr with agitation, beads were washed four times with lysis buffer, once with 20 mM Tris-HCl (pH7.4) and boiled for 5 min in 60 μl of 2x SDS sample buffer. Samples were subjected to SDS-PAGE. Western blotting was performed by standard protocols and developed using ECL 2 reagents (Pierce). Clean-blot IP detection kit from Thermo-Fisher Scientific (#21232) was used to detect immunoprecipitated RagC (Fig. 5C). Antibody concentrations were optimized with various dilutions to ensure that the blotting signals are linear to the levels of loaded antigens. Quantitative analysis of the blots was performed with densitometry scanning. Data from at least three independent experiments were analyzed.

Figure 5. FLCN mutants defective for ciliary localization are unable to suppress mTOR signaling.

Figure 5.

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A. UOK257 cells stably expressing control vector (Ctrl), wild type and mutants of FLCN were assayed for mTORC1-dependent phosphorylation of S6 and mTORC2-dependent phosphorylation of AKT(S473) by western blotting. B. Extracts from UOK257 cells stably expressing control vector (Ctrl), wild type and mutants of FLCN were immunoprecipitated with anti-FLCN antibody. The levels of FLCN, FNIP1 and FNIP2 in the precipitates (α-FLCN IP) and extracts (Extract) were determined by western blotting. C. Extracts from UOK257 cells stably expressing control vector (Ctrl), wild type and mutants of FLCN were immunoprecipitated with anti-RagC antibody. The levels of FLCN, FNIP2, RagC in the precipitates (α-RagC IP) and extracts (Extract) were assayed by western blotting.

Immunofluorescence microscopy—

UOK257 cells ectopically expressing HA or GFP tagged wild type or mutant FLCN proteins were grown to confluence on collagen coated glass cover slips and ciliation was induced by serum starvation for 48 hr. Under this condition, a ciliation rate of ~70% was consistently observed (data not shown) for cells expressing wild type or mutants of FLCN. The ciliated cells were fixed with 4% paraformaldehyde at room temperature and permeabilized with 0.02% saponin in PBS buffer containing 2% BSA and 1% fish skin gelatin. Cells were incubated overnight at 4°C with antibody against HA epitope or GFP followed by 1 hr incubation at room temperature with anti-tubulin antibody and 1 hr incubation with Alexa 488 and Cy3 conjugated secondary antibodies (Life Technologies). DNA was stained with DAPI (Fisher Scientific). Fluorescent samples were visualized using Olympus BX61WI Fluoview™ FV1000 confocal microscope with an Olympus PlanAbo 60x/1.45 oil objective.

Colony formation assay—

Colony formation assay was performed as described previously [23]. Briefly, UOK257 cells (5 × 103) were suspended in 1.0 ml of DMEM containing 10% FBS and 0.3% agar and overlaid on 1.0 ml pre-solidified 0.5% agar in the same medium on a six-well plate. Cells were cultured for 3-4 weeks. Colonies were stained for with crystal violet solution and counted under a dissection microscope.

Data analyses—

All experiments were repeated at least three times and representative data were shown. Quantitative comparison analyses were done with One-way ANOVA and a P value <0.05 was considered statistically significant.

Results

FLCN forms a dimer

FLCN has been shown to bind directly with the cargo binding domains of kinesin-2, indicating that FLCN may be a cargo protein for kinesin-2 [16]. The kinesin-2 in primary cilia comprises of two motor subunits, KIF3A and KIF3B, and an associated protein KAP3. Each of the motor subunits contains a cargo binding domain at its C-terminal [24]. The dimerization of the two subunits hence raises a question about how FLCN interacts with the two binding domains in kinesin-2. We tested the possibility that FLCN also forms a dimer for an effective interaction with kinesin-2. Accordingly, we co-expressed Flag and HA-tagged FLCN in HEK293 cells and assessed the dimerization of FLCN by co-immunoprecipitation. As shown in Fig. 1A, we found that Flag-FLCN co-precipitated with HA-FLCN and vice versa. This finding suggests that the tagged FLCN proteins are able to form a dimer in the cells. To confirm the result, we assessed the ability of purified recombinant FLCN for dimerization in vitro. We found that the recombinant FLCN appeared on native gel electrophoresis with a molecular weight of approximate 140 kD, which is a doubled size of FLCN (70kD), indicating that FLCN was in a dimer form (Fig. 1B).

Figure 1. FLCN forms a dimer.

Figure 1.

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A. HA- and Flag-tagged FLCN proteins were co-expressed in HEK293 cells. Cell extracts (Ext) were immunoprecipitated (IP) with control IgG, HA (α-HA) or Flag (α-Flag) antibody and the presence of HA- or Flag-FLCN in the precipitates was analyzed by western blotting. B. Purified 6xHis tagged recombinant FLCN was analyzed by native gel electrophoresis followed by coomassie blue staining. C. Flag-tagged FLCN was co-expressed with HA-tagged full length (Full) or various truncated FLCN mutant proteins in HEK293 cells. The association between Flag-FLCN and HA-tagged FLCN proteins was analyzed by immunoprecipitation with anti-HA antibody. The IgG control was done by precipitating extracts from cells expressing full length Flag-FLCN and HA-FLCN proteins using a mouse IgG. The levels of Flag-FLCN and HA-FLCN mutant proteins in the precipitates (IgG and α-HA IP) and extracts (extract) were determined by western blotting. D. Schematic presentation of the HA-tagged FLCN mutant proteins used in C. The association between the expressed proteins was quantified based on the levels of co-purified Flag-FLCN. Data from three independent experiments were analyzed.

To determine the regions in FLCN that mediate its dimerization, we co-expressed various Flag-tagged truncation mutants of FLCN with HA-tagged full length of FLCN in HEK293 cells and examined the ability of these mutants to bind with the full length FLCN using co-immunoprecipitation. We found that the binding ability was limited to a region comprising of the N-terminal 314 amino acids of FLCN (Fig. 1C). Interestingly, this N-terminal region bound to HA-FLCN much stronger than its full length counterpart (Fig. 1D). In contrast, the C-terminal half (315-579 amino acids) of the protein was unable to interact with HA-FLCN. When the N-terminal region was further divided into two fragments comprising of the first 115 amino acids or the 116-314 amino acids, both fragments retained a strong binding with HA-FLCN (Fig. 1C and 1D). These observations indicate that the entire N-terminal half of FLCN, which contains the Longin domain, is involved in its dimerization.

The loop region of FLCN mediates its interaction with kinesin-2

To determine the regions in FLCN that mediate its interaction with kinesin-2, we co-expressed various HA-tagged truncation mutants of FLCN together with Flag-tagged KIF3A and assessed the association of the expressed proteins by co-immunoprecipitation. We found that all truncation mutants of FLCN capable of binding with Flag-KIF3A contained a fragment of 19 amino acids corresponding to the region of 327-345 amino acids in FLCN (Fig. 2A and 2B). To determine whether the 19-amino acid fragment was sufficient for binding with Flag-KIF3A, we generated a GST fusion construct that contained a GST extended at its C-terminal with the 19 amino acids and tested its ability for binding with KIF3A in vitro. We found that the fusion protein, but not GST alone, was able to interact with KIF3A (Fig. 2C), suggesting that the 19-amino acid sequence of FLCN is sufficient for KIF3A binding. When this sequence was fused to GFP and expressed in HEK293 cells, the resulting fusion protein but not GFP alone was able to associate with both endogenous KIF3A and KIF3B (Fig. 2D). However, we failed to detect the expressed fusion protein in primary cilia by confocal imaging (data not shown), suggesting that the domain alone is not sufficient to target FLCN into the cilia or that the level of the fusion protein accumulated in the cilia was below the threshold for detection. Similarly, we were unable to detect ciliary localization of fusion constructs containing the kinesin-2 binding domain with the N-terminal half or the C-terminal half of FLCN (data not shown). Collectively, the above results demonstrate that the region between 327-345th residues of FLCN comprises of its kinesin-2 binding domain and that binding with kinesin-2 is not sufficient for the ciliary localization of FLCN.

Figure 2. Identification of the kinesin-2 binding domain in FLCN.

Figure 2.

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A. Flag-tagged KIF3A was co-expressed with various HA-tagged truncated mutants of FLCN in HEK293 cells. The association between Flag-KIF3A and the HA-FLCN mutants was assayed by co-immunoprecipitation with anti-HA antibody. The IgG control was done by precipitating extracts from cells expressing Flag-tagged KIF3A and HA tagged wild type (WT) FLCN using a mouse IgG. The levels of Flag-KIF3A and HA-FLCN proteins in the precipitates (IgG and α-HA IP) and extracts (extract) were determined by western blotting. B. Schematic presentation of FLCN mutants and their relative binding affinity toward KIF3A in the co-immunoprecipitation shown in A. The association between the expressed proteins was quantified based on the levels of co-purified KIF3A. Data from four independent experiments were analyzed. C. Purified GST and a GST fusion protein containing the 19 amino acids of the kinesin-2 binding domain (327-345 amino acids) were incubated with purified 6xHis tagged C-terminal cargo binding domain (601-701 amino acids) of KIF3A. The amounts of His-tagged KIF3A pulled down with GST and the GST fusion protein were analyzed by western blotting. D. GFP and a GFP fusion protein containing the kinesin-2 binding domain of FLCN (GFP-BD) were expressed in ciliated HEK293 cells. The association of the expressed GFP and GFP-BD with endogenous KIF3A and KIF3B was assayed by co-immunoprecipitation with anti-GFP antibody.

Binding with kinesin-2 is essential for ciliary entry of FLCN

A sequence comparison revealed many invariant amino acids within the kinesin-2 binding domain of FLCN among different species. We selected the five residues in the middle of the 19-amino acid sequence and examined their role in the binding (Fig. 3A). Replacing any of the residues with alanine yielded a mutant FLCN protein that when ectopically expressed in HEK293 cells, failed to bind KIF3A (Fig. 3B). One exception was the Q339A substitution, which retained a residual binding activity. A double replacement at positions 338 and 339 (W338A Q339A) completely abolished the binding (Fig. 3B). Utilizing these point mutants, we then examined whether the binding with kinesin-2 is essential for targeting FLCN to primary cilia. We found that when expressed in the FLCN null cells of UOK257 line, none of the kinesin-2 binding deficient mutants of FLCN localized to primary cilia, whereas, wild type FLCN did (Fig. 4). These observations suggest that the binding with kinesin-2 is essential for the ciliary localization of FLCN.

Figure 3. Point mutations within the kinesin-2 binding domain of FLCN abolish its binding with KIF3A.

Figure 3.

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A. Five residues (red letters) between amino acid positions 335 and 339 within the kinesin-2 binding domain of FLCN were mutated by single or double alanine substitution. The invariant amino acids within the binding domain are marked with *. B. HA-tagged FLCN mutants with a single or double point mutation shown in A were co-expressed with Flag-tagged KIF3A in HEK293 cells. The association of the mutant proteins with Flag-KIF3A was assayed by co-immunoprecipitation with anti-HA antibody (α-HA IP). The IgG control was done by precipitating extracts from cells expressing Flag-KIF3A and HA-tagged wild type FLCN using a mouse IgG. The levels of Flag-KIF3A and HA-FLCN in the precipitates (IP) and extracts (Ext) were determined by western blotting.

Figure 4. Ciliary localization of FLCN mutants defective for kinesin-2 binding.

Figure 4.

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A. HA-tagged wild type and mutant FLCN proteins with point mutations within the kinesin-2 binding domain were expressed in UOK257 cells. Ciliary localization of the expressed proteins was analyzed by confocal imaging. Expressed FLCN proteins were marked with anti-HA antibody (red) and cilia by anti-acetylated-tubulin antibody (green). B. Percentages of ciliated cells showing ciliary localization of FLCN were quantified for cells expressing control (Ctrl), wild type and mutants of FLCN. Data were from three independent experiments and ~200 ciliated cells were analyzed for each experiment. Data were presented as means ± SD. The differences in ciliation between control cells and cells expressing wild type and mutant FLCN proteins were analyzed by one-way ANOVA, * p > 0.05, ** p < 0.05.

Ciliary localization of FLCN is imperative for its function in mTOR regulation

We next investigated the role of ciliary localized FLCN in regulation of mTOR signaling and tumorigenesis potential. We found that transient overexpression of FLCN or its kinesin-2 binding deficient mutants in HEK293 cells did not impact mTORC1 and mTORC2 signaling activities (data not shown), which is consistent with our previous finding that transiently modulating FLCN expression level in cultured cells has no obvious effect on mTORC1 and mTORC2 signaling activities under normal growth conditions [16]. We thus established lines stably expressing wild type and the mutants of FLCN in FLCN null UOK257 cells. UOK257 cells were derived from renal tumors of a BPH patient [22]. The cells exhibit highly elevated mTORC1 and mTORC2 signaling activities that can be suppressed by restoration of FLCN expression [23]. Consistent with the previous finding, we found that in the UOK257 cells expressing wild type FLCN, activity of mTORC1 signaling, as assessed by mTORC1-dependent phosphorylation of S6 ribosomal protein, and that of mTORC2 signaling, as measured based on the phosphorylation of AKT at S473, were diminished (Fig. 5A). In contrast, in cells expressing the kinesin-2 binding deficient FLCN mutants, the abnormal mTORC1 and mTORC2 signaling activities sustained (Fig. 5A). Since FLCN has been shown to regulate mTORC1 through its association with FNIP1 and FNIP2 [8,10,11], we examined if the failure of the mutants in rescuing mTORC1 and mTORC2 activities was due to a defect in their association with FNIP1 and FNIP2. Accordingly, we assessed the association of the FLCN mutants with endogenous FNIP1 and FNIP2 by co-immunoprecipitation with FLCN antibody. We found that like the wild type, the mutants were co-precipitated with FNIP1 and FNIP2. This finding suggests that the mutants are as capable of binding with FNIP1 and FNIP2 as is the wild type FLCN (Fig. 5B). As FLCN functions as a GTPase activating protein (GAP) for the RagC/D small GTPases that localize to the lysosomes [25], we further examined the association of the FLCN mutants with RagC by co-immunoprecipitation with anti-RagC antibody. We found that the FLCN mutants, together with FNIP2, were co-precipitated with RagC as was the wild type FLCN (Fig. 5C). These findings indicate that the kinesin-2 binding deficient mutants retain the lysosomal function of FLCN and that their inability in restoring mTOR signaling is unrelated to their function in regulating the RagC/D small GTPases. Collective, these observations demonstrate that the interaction of FLCN with kinesin-2 is required for its role in mTOR regulation in ciliated cells.

To assess whether the FLCN mutants retain their tumor suppressor potential, we examined their ability to inhibit anchorage independent growth of UOK257 cells in a soft agar assay. We found that similar to a previous report [23], expressing wild type FLCN prevented UOK257 cells from forming colonies in soft agar. In contrast, the mutants defective for ciliary localization all failed to suppress the anchorage independent growth (Fig. 6). This finding suggests that the ability of FLCN to inhibit colony formation depends on its ciliary localization. Interestingly, we found that UOK257 cells expressing one of the kinesin-2 binding deficient FLCN mutants (G336A) displayed a significantly higher number of colonies in the anchorage independent growth. This observation indicates that the cells may have a higher survival rate than the control UOK257 cells.

Figure 6. FLCN mutants defective for ciliary localization are unable to suppress anchorage-independent growth of UOK257 cells.

Figure 6.

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A. UOK257 cells stably expressing wild type and mutant FLCN genes were grown in soft agar and colony formation was visualized. B. Quantitative presentation of the colony formation shown in A. Data were from four independent experiments and presented as means ± SD. The differences in colony numbers between control cells and cells expressing wild type and mutant FLCN proteins were analyzed by one-way ANOVA, * p > 0.05, ** p < 0.05.

Discussion

We have previously shown that FLCN localizes to primary cilia and binds directly to kinesin-2 motor complex in quiescent non-cycling cells [16]. In the present study we show that binding with kinesin-2 is essential for FLCN to localize to cilia, suggesting that FLCN is a cargo of the anterograde IFT. We identify a sequence motif of 19 amino acids in FLCN that mediates its binding with kinesin-2, which lies in a loop region that links the N-terminal Longin domain and C-terminal DENN domain (Fig. 2B). Previous structural analyses indicate that this linker region is highly flexible [18,21]. Hence, the interaction with kinesin-2 is unlikely to impact the two functional domains of FLCN, which are involved in binding with FNIP1 and FNIP2 [8,21]. The 19-amino acid binding motif is able to confer a foreign protein the ability for interacting with kinesin-2. However, it is not sufficient to target the protein to primary cilia, indicating that additional regions of FLCN are needed for its ciliary localization. We also find that FLCN is able to form a homodimer (Fig. 1A and 1B). The dimerization of FLCN is mediated through its N-terminal half that contains the Longin domain. The domain has been previously shown to be involved in binding with FNIP2 [21], indicating that the dimerization and binding with FNIP2 may be mutually exclusive. However, the Longin domain of FLCN is not required for its binding with FNIP1, which is mediated by its C-terminal DENN domain [8]. The role of the dimerization in FLCN function remains unclear. The dimerization is not required for kinesin-2 binding. It is possible that the dimerization promotes the ciliary entry of the FLCN-FNIP1 complex. Additional study is needed to verify this notion.

Within the 19-amino acid kinesin-2 binding domain, several residues are found to be essential for the binding. Replacing any of them individually with alanine results in a mutant protein incapable of binding with kinesin-2. None of the binding deficient mutants is able to localize to primary cilia, suggesting the binding with kinesin-2 is essential, although not sufficient, for ciliary localization of FLCN. Using these binding deficient mutants, we show that the ciliary localization of FLCN is required for its function in mTOR regulation (Fig. 5) and its potential as a tumor suppressor (Fig. 6). While it remains possible that the point mutations in the kinesin-2 binding domain affect other activities of FLCN, we consider the possibility unlikely, given the fact that binding domain lies within a highly flexible linker region where the alanine substitution minimalizes potential structural disturbance. In support of this view, we find that the kinesin-2 binding deficient mutants of FLCN retain their ability to interact with RagC, FNIP1 and FNIP2, the mediators of FLCN for its activity in the lysosome-dependent regulation of mTORC1 (Fig. 5B and 5C). Collectively, our observations demonstrate a critical role of ciliary localization in FLCN function.

FLCN has been found to associate with AMPK indirectly through their mutual binding with FNIP1 [8]. The association appears to be a key link for the effect of FLCN in many cellular processes [26–30]. In quiescent cells the association of FLCN with AMPK is cilium-dependent [16], which explains why loss of ciliary localization abolishes the ability of FLCN to control mTORC1 signaling activity. However, how the ciliary function of FLCN is connected to mTORC2 is unclear. A previous study has shown that CCDC28B, a cilium-associated protein, is able to interact with mTORC2 and regulates its activity [31]. It is possible that ciliary function of FLCN acts through CCDC28B to regulate mTORC2. Alternatively, the effect of FLCN on mTORC2 may be an indirect consequence of reduced proliferation of UOK257 cells when FLCN function is restored. Additional studies are needed to address molecular basis underlying the ciliary function of FLCN in mTORC2 regulation.

Acknowledgements:

We thank Laura Schmidt and Marston Linehan at NIH for providing the FLCN plasmid, UOK257 and UOK257-2 cell lines and appreciate other laboratory members for comments and discussion during the course of the study. This study is supported in part by NIH grant GM132137 to YJ. YZ is supported by the Shanghai Science and Technology Commission grant 19410741800.

Footnotes

Conflict of Interest: The authors declare that they have no conflict of interest.

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