FNIP1, a 130-kDa FLCN-binding protein, is evolutionarily conserved and widely expressed in human tissues. (A) Stably transfected HEK293 cells expressing doxycycline-inducible HA-FLCN were lysed, and proteins were immunoprecipitated with anti-HA antibody, subjected to SDS/PAGE, transferred to PVDF membranes, and stained with colloidal gold. FNIP1 was coimmunoprecipitated with FLCN. (B) FNIP1 and BHD(FLCN) expression patterns in human adult tissues. cDNA probes were generated by PCR and hybridized to a human 12-tissue Northern blot (Clontech). β-Actin was used as loading control. (C) Schematic structure of conserved FNIP1 domains. Amino acid alignments show five blocks of conservation between FNIP1 and its homologs (Fig.8). GenBank accession nos. for FNIP1 homologs: H. sapiens (DQ145719), X. tropicalis (ENSXETP00000036209; Ensembl), D. rerio (XM_687716), D. melanogaster (NM_140686), and C. elegans (T04C4.1a; Wormbase).

Regions of FNIP1 and FLCN necessary for binding. (A) Recombinant GST-FLCN and GST-FLCN mutants were immobilized on glutathione-Sepharose and incubated with ³⁵S in vitro-translated (IVT) FNIP1. Binding was evaluated by SDS/PAGE and autoradiography. Coomaissee Brillant blue (CBB) staining shows relative expression of GST-FLCN proteins. FLCN mutants lacking a C terminus cannot bind FNIP1. (B) Recombinant GST-FNIP1 and FNIP1 deletion mutants were bound to glutathione-Sepharose, incubated with ³⁵S IVT FLCN, and evaluated as in A. Deletion of conserved blocks 2/3 or 4/5 of FNIP1 disrupted the interaction with FLCN.

AMPK phosphorylates FNIP1 and may regulate FNIP1 expression. (A) Cell lysates from HA-FNIP1-inducible HEK293 cells grown with and without doxy were immunoprecipitated with anti-HA antibody and assayed for AMPK in vitro kinase activity with [γ-³²P]ATP and SAMS peptide with and without AMP. Radioactivity incorporated into SAMS peptide was measured by scintillation counting. Each bar represents the results of three independent experiments. Brackets, standard deviation. (B) HA-FNIP1 immunoprecipitates were incubated in AMPK kinase reaction buffer with [γ³²]ATP and various kinase inhibitors (Compound C, 4 or 40 μM; UO126, 10 μM; Wortmannin, 1 μM; and rapamycin, 20 nM). Immunoprecipitates were washed and subjected to SDS/PAGE and autoradiography. (C) HA-FNIP1-inducible 293 cells were cultured for 3 h with [³²P]orthophosphate with and without AMPK inhibitors (AraA, 2 mM and Compound C, 20 μM), and anti-HA immunoprecipitates were subjected to SDS/PAGE and autoradiography. Cold samples obtained under the same culture conditions were used for phosphoacetyl CoA carboxylase (pACC) and HA-FNIP1 Western blotting. (D and E) HA-FNIP1-inducible 293 cells (D) or 293 cells (E) were cultured with AraA (2 mM) or Compound C (Comp C) (30 μM) for 24 h. Exogenous or endogenous FNIP1 and endogenous FLCN were detected by Western blotting. WB, Western blotting.

Evaluation of mTOR activity in FLCN-null and restored cells. (A) Lack of FLCN does not affect mTOR regulation by AMPK. UOK257 (FLCN-null) and UOK257-2 (FLCN-restored) cells were cultured with serum-depleted DMEM for 48 h. To activate AMPK, cells were treated with 50 mM 2-deoxyglucose (2DG) for 2 h. AMPK was activated, and mTOR activity was suppressed by 2DG in both cell lines. (B) Lack of FLCN affects mTOR activity under certain conditions. UOK257 and UOK257-2 cells were cultured without serum for 48 h, stimulated with DMEM containing 10% serum, with or without rapamycin (Rapa) (40 nM) or amino acid-free [a.a.(−)] medium with 10% dialyzed serum for 90 min. Under serum-starved conditions, FLCN-null cells did not show complete suppression of mTOR activity, whereas FLCN-null cells were more sensitive to mTOR inhibition by amino acid deprivation.

FLCN–FNIP1 interactions. (A) HA-FLCN was cotransfected with FLAG-FNIP1 into HEK293 cells. Cell lysates were immunoprecipitated (IP) with anti-FLAG or anti-HA antibodies and blotted with both antibodies. Arrows, FLAG-FNIP1; arrowheads, HA-FLCN. (B) HEK293 lysates were immunoprecipitated with anti-FLCN and control IgG or with anti-FNIP1 and control IgG and blotted with anti-FLCN or anti-FNIP1. Arrowheads, endogenous FLCN; arrows, endogenous FNIP1; asterisks, IgG heavy-chain bands. (C) HeLa cells were cotransfected with HA-FNIP1 and FLAG-FLCN. Immunocytochemistry was performed with anti-HA and anti-FLAG antibodies. FNIP1 and FLCN colocalize with a reticular pattern in the cytoplasm. Green, FLAG-FLCN; red, HA-FNIP1.

FNIP1 interacts with AMPK in vitro and in vivo. (A) FNIP1-interacting proteins were immunoprecipitated (IP) with anti-HA antibody from doxycycline-inducible HA-FNIP1-expressing HEK293 cells, separated by SDS/PAGE, transferred to PVDF membrane, stained with colloidal gold, and analyzed by mass spectrometry. The 40-kDa protein was identified as the γ-1 subunit of AMPK. Other interacting proteins were FLCN (67 kDa) and HSP 90 (90 kDa). (B) Cell lysates from HA-FNIP1-inducible HEK 293 cells cultured with and without doxy were immunoprecipitated with anti-HA antibody and blotted with antibodies, as indicated, showing that HA-FNIP1 and AMPK subunits interact. (C) Immunoprecipitates from HA-FNIP1-inducible HEK293 cells were blotted with antibodies as indicated. Band intensities were measured with the ImageJ program. The estimated amounts of AMPKα and phospho-AMPKα(T172) binding to FNIP1 were calculated by IP/[input (4%) × 25]. Results were from three independent experiments. Brackets, standard deviation. (D) HEK293 lysates were immunoprecipitated with anti-FNIP1 or control IgG. Immunoprecipitates were blotted with anti-FNIP1, anti-FLCN mouse monoclonal antibody, and anti-AMPKβ mouse monoclonal antibody. Endogenous FNIP1 and AMPK interact. (E) HEK293 cell lysates transfected (T.F.) with HA-FLCN alone or HA-FLCN and FLAG-FNIP1,were immunoprecipitated with anti-HA antibody and blotted with antibodies as indicated. All AMPK subunits were immunoprecipitated with FLCN in a FNIP1-dependent manner. (F) UOK257 cells (FLCN null) and FLCN-restored UOK257-2 cells were immunoprecipitated with anti-FNIP1 or control IgG and blotted with anti-FNIP1, anti-FLCNmAb, and anti-AMPKβ mAb. W.B., Western blotting; Sup, supernatant.

FLCN phosphorylation is regulated by FNIP1 through mTOR signaling and AMPK. (A) FLCN phosphorylation is affected by mTOR activity. UOK257-2 FLCN-restored cells were cultured under different culture conditions: S(+), with serum; S(−), serum starvation for 48 h; or S(−) → S(+), 20% dialyzed serum stimulation for 30 min after 48-h serum starvation. Cells were pretreated for 2 h before stimulation with various inhibitors: nontreatment (N/T); U0126, 50 μM; Wortmannin (Wort), 1 μM; or rapamycin (Rapa), 20 nM. (B) AMPK inhibition suppresses FLCN phosphorylation. UOK 257–2 cells were cultured with rapamycin (20 nM), Compound C (CompC) (30 μM), or both for 24 h. Western blotting (WB) was performed for phospho-ACC (AMPK readout) and phospho-S6R (mTOR readout). Compound C treatment reduced FNIP1 expression and FLCN phosphorylation. (C) FLCN phosphorylation facilitated by FNIP1 expression is partially regulated by mTOR activity. HA-FNIP1-inducible HEK293 cells were cultured with and without doxy. For amino acid starvation, cells were cultured with Earl's balanced solution containing dialyzed serum, vitamins, pyruvate, and glucose for 16 h with and without doxycycline, followed by stimulation with 2× amino acids for 30 min. Rapamycin was added 2 h before stimulation.