Baba et al. 10.1073/pnas.0603781103. |
Fig. 8. FNIP1 homolog protein sequence alignments and analysis. The Gblocks method [Castresana, J. (2000) Mol Biol Evol 17:540-552] was used for selection of conserved blocks from multiple alignments of FNIP1 homologs. The sequences were aligned with ClustalX [Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997) Nucleic Acids Res 25:4876-4882] with pair-wise gap openings and gap extension penalties set at 10× and 0.1×, respectively. The following FNIP1 homologs were aligned: Homo sapiens (DQ145719), Xenopus tropicalis (ENSXETP00000036209; Ensembl), Danio rerio (XM_687716), Drosophila melanogaster (NM_140686), and Caenorhabditis elegans (T04C4.1; Wormbase). Five conserved sequence blocks were identified with similarities ³35%.
Fig. 9. C terminus of folliculin is required for FNIP1 binding in vivo. A series of FLCN-FLAG deletion mutants were expressed in doxycycline-induced HA-FNIP1-expressing HEK293 cells. Anti-HA immunoprecipitates were subjected to Western blotting with anti-FLAG or anti-HA antibody (Right). The relative expression of folliculin deletion mutants in cell lysates is shown by Western blotting with anti-FLAG antibody (Left). Lower inserts show HA-FNIP1. Folliculin mutants lacking a C terminus cannot bind to FNIP1.
Fig. 10. FNIP1 and AMPK binding in vitro. (A) Residues 602-929 of FNIP1 are the minimum region for AMPK binding. Recombinant GST-FNIP1 and GST-FNIP1 deletion mutants expressed in Sf9 cells were bound to glutathione-Sepharose beads as described in Supporting Methods. The washed beads were incubated with HEK293 cell lysates as a source of endogenous AMPK. After washing, bound proteins were eluted and subjected to SDS/PAGE, followed by Western blotting with anti-AMPK subunit-specific antibodies. Residues 602-929, located between two conserved regions, were necessary but not sufficient for AMPK binding. (B) FNIP1 and AMPK bind directly in vitro. Recombinant GST-FNIP1 and GST were bound to glutathione-Sepharose beads as described in Supporting Methods. The washed beads were incubated with 2.5 mg of purified AMPK (Upstate Biotechnology, Lake Placid, NY) in 1 ml of buffer (20 mM Tris·HCl, pH 7.5, 135 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100, 5% glycerol, and Complete Protease Inhibitor mixture). After washing, bound proteins were eluted and subjected to SDS/PAGE, followed by Western blotting with anti-AMPKa-subunit antibody. Six hundred nanograms of purified AMPK were loaded in lane 1 as positive control. The asterisk indicates the degraded GST-FNIP1 product.
Fig. 11. Folliculin is phosphorylated in vivo. HA-folliculin expression vector was transiently transfected into HeLa cells cultured for 24 h, lysed, and subjected to immunoprecipitation with anti-HA antibody. To enhance HA-folliculin phosphorylation, some cultures were treated with 100 nM Calyculin A (a serine/threonine phosphatase inhibitor) for 30 min before harvest. Immunoprecipitated HA-folliculin was incubated with phosphatases as indicated (CIAP, calf intestinal alkaline phosphatase; PP1, protein phosphatase 1 specific for phosphoserine/threonine). After phosphatase treatment, washed beads were boiled in SDS sample buffer. The eluted proteins were subjected to SDS/PAGE, followed by Western blotting with anti-HA antibody. The electrophoretic band-shift up with CalyculinA treatment and band-shift down with phosphatase treatment confirmed the presence of phosphorylated forms of folliculin, most likely involving serine or threonine residues.
Fig. 12. Folliculin is phosphorylated by AMPK in vitro. Purified recombinant GST-folliculin (11 pmol) or GST (11 pmol) was bound to glutathione-Sepharose beads and incubated with [g32P]ATP and AMPK( 10 milliunits; Upstate Biotechnology) in an AMPK reaction buffer with or without AMP (75 mM). SAMS peptide (100 mM) was added for substrate competition. After completion of the kinase reaction, glutathione-Sepharose beads were washed and boiled with 1× SDS sample buffer and subjected to SDS/PAGE and Coomassie staining, followed by autoradiography. GST-folliculin was phosphorylated by AMPK, and addition of AMP, which activates AMPK, enhanced GST-folliculin phosphorylation. GST-folliculin phosphorylation was competed by SAMS peptide, supporting AMPK as the kinase that phosphorylated GST-folliculin.
Fig. 13. Possible scheme for FNIP1-folliculin interactions with AMPK and mTOR pathways that are dysregulated in hamartoma syndromes. Yellow symbols indicate proteins defective in hamartoma syndromes. Double-headed arrows identify experimentally validated protein interactions. Dotted lines indicate functional interactions that are not yet clarified. FNIP1 is regulated by AMPK through phosphorylation. FNIP1 may serve as a scaffold regulator, which facilitates FLCN phosphorylation by mTOR and AMPK signaling.
Data Set 1. Peptide mass fingerprinting data that identified the p130 band as FNIP1 are contained in Supporting Data Set: HA-FLCN IP p130 PMF.
Data Set 2. Peptide mass fingerprinting data that identified the p40 band as AMPK g-1 subunit are contained in Supporting Data Set: HA-FNIP IP p40 PMF.
Data Set 3. Peptide mass fingerprinting data that identified the p67 band as folliculin are contained in Supporting Data Set: HA-FNIP IP p67 PMF.
Data Set 4. Peptide mass fingerprinting data that identified the p90 band as HSP90 are contained in Supporting Data Set: HA-FNIP IP p90 PMF.
Supporting Methods
Identification of Folliculin and FNIP-1-Interacting Proteins by Coimmunoprecipitation and Mass Spectrometric Analysis.
HA-folliculin or FNIP-1-HA-nducible HEK 293 cells were cultured with or without 1 mg/ml doxycycline for 36 h. For each culture, 5 ´ 107 cells were harvested and lysed in lysis buffer [20 mM Tris·HCl, pH 7.5, 135 mM NaCl, 5% glycerol, 0.1% Triton X-100, 50 mM NaF, 1 mM Na3VO4, and Complete Protease Inhibitor mixture (Roche Applied Science, Rockford, IL)]. After centrifugation, lysates were immunoprecipitated at 4°C overnight with anti-HA Affinity Matrix (Roche Applied Science). The matrix was washed seven times with lysis buffer, and proteins were eluted by HA peptide competition (2 mg/ml; Roche Applied Science) at 30°C for 15min. The eluted proteins were boiled with SDS sample buffer and subjected to SDS/PAGE, transferred to PVDF membranes (ProBlott PVDF, Applied Biosystems, Foster City, CA) that were soaked in TBST (20 mM Tris·HCl, pH 7.5, 500 mM NaCl, and 0.3% Tween 20) and stained with Colloidal Gold Total Protein stain (Bio-Rad, Hercules, CA). The specific bands that were observed in doxycycline-induced lanes were excised. The immobilized proteins were reduced, S-carboxymethylated, and digested in situ with Achromobacter protease I [Iwamatsu, A. (1992) Electrophoresis 13:142-147]. Molecular mass analyses of Lys-C fragments were performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using an Applied Biosystems Voyager-DE/STR [Jensen, O.N., Podtelejnikov, A. & Mann, M. (1996) Rapid Commun Mass Spectrom 10:1371-1378.] Identification of proteins was carried out by peptide mass fingerprinting and searching the human NCBI nonredundant protein database [Jensen, O. N., Podtelejnikov, A. & Mann, M. (1996) Rapid Commun Mass Spectrom 10:1371-1378].Cloning and Characterization of the Human FNIP1 cDNA (DQ145719).
The 130-kDa protein that coimmunoprecipitated with folliculin, designated FNIP1, was identified by mass spectrometric analysis (17 peptide matches) as the protein product of KIAA1961 (AB075841). The KIAA1961 mRNA encodes an ORF of 943 aa, followed by a poly (A) tail, but lacks an ATG start codon (BAB85547). BLAST analysis of the publicly available databases (UCSC Human Genome Browser, human EST track; NCBI, human EST) identified several overlapping human ESTs corresponding to KIAA1961. The clone BC001956 encodes a 508-aa protein with an ATG start codon (AAH01956) that shares 284 aa from its C terminus with the N terminus of BAB85547. We performed a genomic BLAST analysis and confirmed that these two cDNA clones were located at the same genomic locus, 5q23.3, and shared coding sequences. Further evaluation of the mass spectrometric data for FNIP1 revealed one peptide fragment that matched the AAH01956 protein. The full-length FNIP1 transcript was amplified from a pooled tissue cDNA library (Clontech, Mountain View, CA) by PCR with primers designed from 5'-end sequences of BC001956 cDNA (5' UTR of FNIP1) and 3'-end sequences of AB075841 cDNA (3' UTR of FNIP1): 5'-GCCTAGCAAGCGCCCAGCG-3' (forward primer) and 5'-CTCCATAAATGCATGTTGTGTCTGC-3' (reverse primer). Amplified cDNAs were TOPO-cloned into pCR II cloning vectors (Invitrogen, Carlsbad, CA). Double-stranded sequencing reactions using Big Dye Terminators (Applied Biosystems) were purified by using Performa plates (Edge BioSystems, Gaithersburg, MD) and electrophoresed on an ABI 3700 genetic analyzer. Sequencing reactions provided >6-fold coverage of all clones. Sequence alignment was performed with Lasergene (DNAStar, Madison, WI) software. The consensus sequence was determined from the majority of clones and was identical to the assembled overlapping sequences of BC001956 and AB075841 cDNAs. Sequencing results confirmed that the majority of cDNA clones contained the full-length 3,626-nt FNIP1 sequence. Several alternative transcripts lacking 1 or more of the 18 coding exons were identified and are currently being verified.Generation of Gateway
Entry Clones for Full-Length FNIP1. A full-length FNIP1 cDNA clone was amplified from overlapping AB075841 (KIAA1961, obtained from Kazusa DNA Research Institute, Chiba, Japan) and BC001956 cDNAs for cloning into Gateway Entry vectors (Invitrogen) as follows. The N-terminal fragment was amplified with the following primers, where initiator codon, ATG, is underlined: 5'-ATGGCCCCTACGCTGTTCC-3' (forward primer) and 5'-GATGAGTCTTTGCCAACATGTCC-3' (reverse primer), KOD Hot Start DNA polymerase (Novagen, San Diego, CA) and BC001956 cDNA as template. The C-terminal fragment was amplified with the following primers: 5'-AGAAAAGCATTCCTCTCAGAGTGTG-3' (forward primer) and 5'-TTAAAGGAGTATTTGTGCAACATATGGA-3' (reverse primer), KOD Hot Start DNA polymerase and AB075841 cDNA as template. Then both PCR products were gel purified, mixed, and used as templates for a final PCR with the following primers: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGCCCCTACGCTGTTCC-3' (forward primer) and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAAGGAGTATTTGTGCAACATATGGAGA-3' (reverse primer) that contain attB sequences for recombination of full-length FNIP1 cDNA into Entry vectors using the Gateway Protein Expression System (Invitrogen). The FNIP1 PCR product containing attB recombination sites was recombined into the Entry vector pDONR223 (Invitrogen) by using BP Clonase (Invitrogen), and the resulting FNIP1 Entry clone was sequence-verified. The Entry clones were used for Destination vector production.Plasmids, Transfection, Recombinant Protein Expression and Purification.
To produce a variety of mammalian expression vectors, we used the Gateway Protein Expression System (Invitrogen). The full-length and deletion-mutant series cDNAs encoding folliculin and FNIP1 were generated by PCR with specific primers containing attB sequences and then inserted into pDONR223 (modified pDONR221 in which the kanamycin-resistance gene was replaced by the spectinomycin-resistance gene) with BP Clonase and sequence-verified (Entry clones). The sequence-verified Entry clones were recombined, by using LR Clonase and manufacturer's protocols, into a series of Destination vectors to produce HA, FLAG, and GST fusion expression clones. Expression clones containing GST full-length folliculin, GST-folliculin deletion mutants, GST full-length FNIP1 and GST-FNIP1 deletion mutants were transformed into Escherichia coli DH10Bac (Invitrogen), and transposed bacmid DNA was selected by using media containing gentamycin, kanamycin, and tetracycline according to manufacturer's protocols. Colonies were picked, and bacmid DNA was prepared and verified by PCR amplification of the bacmid junction regions. Verified bacmid DNA was transfected into insect Sf9 cells and cultured for 96 h at 27°C in HyClone SFX insect cell culture medium. The recombinant baculovirus-containing supernatant was harvested, and 2 ml of virus was used to infect a 200-ml culture of Sf9 cells. The infected cells were collected after 72 h of incubation, washed with ice-cold PBS and snap-frozen in an alcohol/dry ice bath. Fusion proteins were released by sonication of cell pellets and used in in vitro binding assays (see below). Transfections of HEK293 cells were performed by the lipofection method using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. For recombinant GST-folliculin purification, GST-folliculin fusion protein was expressed in Sf9 insect cells infected with bacmid DNA from a GST-folliculin-expressing baculovirus Destination vector. The protein was purified on a GST-Trap column (Amersham Pharmacia, Piscataway, NJ), eluted with a reduced glutathione gradient, and further purified by ion-exchange chromatography.In Vitro
Binding Assay. Bacmid DNA generated from a variety of baculovirus Destination vectors expressing full-length GST-folliculin, GST-folliculin deletion mutants, GST-FNIP1, and GST-FNIP1 deletion mutants were transfected into Sf9 insect cells for expression of GST fusion proteins (details above). The culture supernatants were clarified by centrifugation. Cell pellets were washed twice with PBS, resuspended in 8 ml of sonication buffer per 200 ml of cell pellet (500 mM NaCl, 50 mM Tris·HCl, pH 7.5, 5% glycerol, 1 mM b-mercaptoethanol, 5 mM MgCl2, and Complete Protease Inhibitor mixture), and lysed by sonication. Benzonase solution (1 unit/ml; Novagen) was added to the cell lysate and incubated on ice for 20 min. Lysates were centrifuged at 11,000 ´g for 30 min at 4°C, and supernatant was recovered and stored at -80°C until further analysis. For affinity purification, 10 ml of equilibrated glutathione-Sepharose 4B beads (Amersham Pharmacia Biosciences) were preincubated in 0.3% BSA TBS-T for 1 h at 4°C and then mixed with cell extracts diluted with binding buffer (25 mM Tris·HCl, pH 7.5, 135 mM NaCl, 5 mM DTT, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100 and Complete Protease Inhibitor mixture) for 1 h at 4°C. The beads were washed four times with binding buffer. T7 promoter-driven Destination vector plasmids encoding wild-type FNIP1 or wild-type folliculin were used for in vitro transcription/translation (IVT) with the rabbit reticulocyte TNT T7 Quick kit (Promega, Madison, WI) and [35S]methionine (Amersham Pharmacia) according to the manufacturer's protocols. The reactions were incubated for 90 min at 30°C. The IVT reactions containing 35S-labeled folliculin or FNIP1 were diluted with binding buffer, mixed with GST fusion protein immobilized on glutathione-Sepharose beads, and incubated at 4°C for 1 h. After washing four times with binding buffer, beads were boiled with SDS sample buffer and subjected to SDS/PAGE, followed by Coomaisse Brilliant blue (CBB) staining and exposure to x-ray film.For recombinant FNIP1 and AMPK binding studies, GST-FNIP1 and GST were immobilized on glutathione-Sepharose beads as above. The beads were mixed with 293 cell lysate lysed in lysis buffer [20 mM Tris·HCl, pH 7.5, 135 mM NaCl, 5% glycerol, 0.1% Triton X-100, 50 mM NaF, and 1 mM Na3VO4] and Complete Protease Inhibitor mixture (Roche Applied Science)] or purified AMPK (Upstate Biotechnology, Lake Placid, NY), and incubated at 4°C overnight. After washing five times with lysis buffer, beads were boiled with SDS sample buffer and subjected to SDS/PAGE, followed by Western blotting and CBB staining.
Immunofluorescence Microscopy.
HeLa cells transfected with FNIP1-HA and FLAG-folliculin expression plasmids were cultured on Chamber Slides (Nalgene Nunc International, Roskilde, Denmark) for 12 h. Cells were washed with ice-cold PBS and fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were then permeabilized with 0.1% (vol/vol) Triton X-100 for 10 min and blocked with 3% goat serum and 10% calf serum in PBS for 1 h at room temperature. Anti-HA antibody (3F10; Roche Applied Science) and anti-FLAG antibody (Sigma) incubations were performed at room temperature for 1 h in buffer containing 10 mM Tris·HCl, p H7.5, 150 mM NaCl, 0.01% (vol/vol) Tween 20, 1.5% goat serum, and 0.1% (wt/vol) BSA. The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR) and Cy3-conjugated goat anti-mouse antibody (Amersham Pharmacia Biosciences). TO-PRO-3 iodide (Molecular Probes) was used to stain nuclei. The samples were mounted with a Slow Fade Antifade kit (Molecular Probes) and were viewed with a confocal microscope system (LSM 510; Zeiss, Thornwood, NY).Immunoprecipitation and Western Blotting.
Cells were lysed in lysis buffer (20 mM Tris·HCl, pH 7.5, 135 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100, 5% glycerol, and Complete Protease Inhibitor mixture) or RIPA buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1.0% Triton X-100, 0.5% deoxycholate, 0.1% SDS, and Complete Protease Inhibitor mixture), and lysates containing equal amounts of protein were immunoprecipitated at 4°C overnight with various antibodies bound to protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ). The resin was washed five times with lysis buffer, proteins were eluted with SDS sample buffer, followed by boiling, and Western blotting was performed as described [Baba, M., Hirai, S., Yamada-Okabe, H., Hamada, K., Tabuchi, H., Kobayashi, K., Kondo, K., Yoshida, M., Tahashita, A., Kishida, T., et al. (2003) Oncogene 22:2728-2738.]