Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines

Abstract

In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCN^POS and FLCN^NEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCN^NEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCN^NEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCN^NEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.

Graphical abstract

Fig. 1

Phosphoproteomic analyses of FLCN^POSand FLCN^NEGrenal epithelial cells. A, workflow of phosphoproteomic analyses of FLCN^POSversus FLCN^NEG renal epithelial cells. To gain insight into FLCN-dependent activation of specific kinases we used the INKA algorithm (15), which takes into account both phosphorylation of the kinase itself (“kinome” and “activation loop”) and phosphorylation of substrate-specific sites (PhosphoSitePlus and NetworKIN; “PSP” and “NWK,” respectively). B, numbers of identified phosphorylated sites, peptides, proteins, and kinases in both datasets. Differential phosphosites between FLCN^POS and FLCN^NEG are indicated below the total number of identified phosphosites. C, supervised hierarchical cluster analyses of FLCN differential pY peptide intensities. RPTEC hTERT (WT), RPTEC tet-on Cas9 (Cas9), and RPTEC tet-on Cas9 TP53-/- (TP53KO) were FLCN wildtype and assigned to the FLCN^POS group. Three individually isolated RPTEC tet-on Cas9 TP53-/- FLCN-/- clones (FLCNKO_C1, FLCNKO_C2, and FLCNKO_C3) were assigned to the FLCN^NEG group. D, supervised hierarchical cluster analyses of FLCN differential pSTY peptide intensities. RPTEC hTERT (WT), RPTEC tet-on Cas9 (Cas9), and RPTEC tet-on Cas9 TP53-/- (TP53KO) were FLCN wildtype and assigned to the FLCN^POS group. Three individually isolated RPTEC tet-on Cas9 TP53-/- FLCN-/- clones (FLCNKO_C1, FLCNKO_C2, and FLCNKO_C3) were assigned to the FLCN^NEG group. FLCN, folliculin; RPTEC, renal proximal tubular epithelial cell.

Fig. 2

INKA analyses of FLCN^POSversus FLCN^NEGrenal epithelial cells.A, top 10 differential kinases pY identified by INKA. Ranking is based on the difference of means of three observations per group. Kinases more active in FLCN^NEG cells are boxed yellow, and kinases more active in FLCN^POS are boxed blue. Kinase activity similarly dependent on FLCN in UOK257 cell line background are indicated by asterisks. Extended list of INKA results is attached as supplemental File S1. B, top 10 differential kinases pSTY identified by INKA. Ranking is based on the difference of means of three observations per group. Kinases more active in FLCN^NEG cells are boxed yellow, and kinases more active in FLCN^POS are boxed blue. Kinase activity similarly dependent on FLCN in UOK257 cell line background are indicated by asterisks. Extended list of INKA results is attached as supplemental File S1. FLCN, folliculin; INKA, integrative inferred kinase activity.

Fig. 3

Results of phosphosite-specific signature analyses (PTM-sigDB).A, results of phosphosite-specific signature analysis pY. Significance (p-value) of enriched signatures is indicated by color. B, results of phosphosite-specific signature analysis pSTY. Significance (p-value) of enriched signatures is indicated by color.

Fig. 4

Hyperphosphorylated tyrosine kinases in FLCN^NEGrenal epithelial cells.A, EGFR together with its substrates that were identified to be differentially phosphorylated upon FLCN loss in RPTECs. B, bar graph of normalized phosphosite intensities of EGFR and its substrates ABI1, EPS8, ERRFL1, STAT1, PTK2, and CTNND1. p-Values are indicated for each phosphosite depicted in the bar graph. C, EPHA2 together with its substrates that were identified to be differentially phosphorylated upon FLCN loss in RPTECs. D, bar graph of normalized phosphosite intensities of EPHA2 and its substrates GIT1, BCAR1, ARHGAP35, INPPL1, and VAV3. p-Values are indicated for each phosphosite depicted in the bar graph. E, MET together with its substrates that were identified to be differentially phosphorylated upon FLCN loss in RPTECs. F, bar graph of normalized phosphosite intensities of MET and its substrates PTK2, CBL, CTNND1, and CTTN. p-Values are indicated for each phosphosite depicted in the bar graph. FLCN, folliculin; RPTEC, renal proximal tubular epithelial cell.

Fig. 5

Tyrosine kinase inhibitor dose–response curves for FLCN^POSand FLCN^NEGcell lines.A, crizotinib dose–response curves (n = 4) of FLCN^POSversus FLCN^NEG RPTECs show a difference in sensitivity to MET inhibition (p = 1.64e-7). B, crizotinib dose–response curves (n = 3) of BHD renal tumor cell line UOK257 (FLCN^NEG) and the isogenic FLCN reconstituted tumor cell line UOK257-2 (FLCN^POS) show no difference in sensitivity to MET inhibition (p = 0.72). C, foretinib dose–response curves (n = 4) of FLCN^POSversus FLCN^NEG RPTECs show minor difference in sensitivity to MET inhibition (p = 0.05). D, foretinib dose–response curves (n = 4) of BHD renal tumor cell line UOK257 (FLCN^NEG) and the isogenic FLCN reconstituted tumor cell line UOK257-2 (FLCN^POS) show a difference in sensitivity to MET inhibition (p<2e-16). E, erlotinib dose–response curves (n = 3) of FLCN^POSversus FLCN^NEG RPTECs show a difference in sensitivity to EGFR inhibition (p = 4.87e-8). F. erlotinib dose–response curves (n = 3) of BHD renal tumor cell line UOK257 (FLCN^NEG) and the isogenic FLCN reconstituted tumor cell line UOK257-2 (FLCN^POS) show a difference in sensitivity to EGFR inhibition (p = 9.64e-14). G, ulixertinib dose–response curves (n = 4) of FLCN^POSversus FLCN^NEG RPTECs show a difference in sensitivity to MAPK1 inhibition (p = 0.007). H, ulixertinib dose–response curves (n = 3) of BHD renal tumor cell line UOK257 (FLCN^NEG) and the isogenic FLCN reconstituted tumor cell line UOK257-2 (FLCN^POS) show a difference in sensitivity to MAPK1 inhibition (p = 4.02e-7). BHD, Birt–Hogg–Dubé; FLCN, folliculin; RPTEC, renal proximal tubular epithelial cell.

Fig. 6

Loss of FLCN regulates MAPK signaling and TFEB phosphorylation and localization.A, MAPK together with its substrates that were identified to be differentially phosphorylated upon FLCN loss in RPTECs. B, bar graph of normalized phosphosite intensities of MAPK1/3/6/8/10 and substrates PML, NFIC, and PXN. p-values are indicated for each phosphosite depicted in the bar graph. C, PI3K/Akt/mTOR together with their substrates that were identified to be differentially phosphorylated upon FLCN loss in RPTECs. D, bar graph of normalized phosphosite intensities of PI3K/Akt/mTOR signaling, as these are known upstream regulators of ribosomal S6 kinases. p-values are indicated for each phosphosite depicted in the bar graph. E, immunofluorescence costaining of TFEB and lysosomal marker LAMP2 show enhanced nuclear TFEB upon amino acid starvation of FLCN^POS RPTECs. Upon FLCN loss, TFEB localization is nuclear independent of nutrient availability. AA, amino acids. F, quantification of TFEB-positive nuclei in FLCN^POS and FLCN^NEG RPTECs in either serum-starved (SS) medium or in medium depleted of both serum and amino acids (SS min AA). FLCN^NEG show nuclear TFEB in the presence of amino acids, while FLCN^POS show cytoplasmic TFEB as expected in a fed condition. FLCN, folliculin; RPTEC, renal proximal tubular epithelial cell; TFEB, transcription factor EB.

Fig. 7

FLCN-sensitive phosphoserines modulate nucleocytoplasmic shuttling of TFEB.A, Western blot of TFEB in FLCN^POS and FLCN^NEG RPTECs that overexpress WT, S>A, or S>D phosphomutant TFEB. FLCN protein was absent in FLCN^NEG cells and GAPDH was used as a loading control. B, representative photos of immunofluorescent stainings of FLCN^POS and FLCN^NEG RPTECs that overexpress WT, S>A, or S>D phosphomutant TFEB in serum-starved medium or in medium depleted of both serum and amino acids (minus aa). Stainings were performed with FLAG antibody to ensure that the signal of endogenous TFEB could not interfere with the results. Quantifications are shown in Figure 7C. C, quantifications of immunofluorescent stainings (shown in Fig. 7B) of FLCN^POS and FLCN^NEG RPTECs that overexpress WT, S>A, or S>D phosphomutant TFEB in serum-starved medium −/+ amino acid starvation. Nuclear:cytoplasmic ratios of mean TFEB intensities were calculated based on automated image analyses. For each condition, the total number of cells analyzed is indicated and means are shown as black bars. D, Western blot of TFEB in FLCN^POS RPTECs TFEB phosphomutants shows clear downward mobility shifts on SDS-PAGE upon amino acid (AA) starvation. In FLCN^NEG RPTECs this downward mobility shift is, even in the presence of amino acids, more abundant, which points toward additional dephosphorylation events. GAPDH was used as a loading control. E, flow cytometry histograms of fluorescent intensities of FLCN^POS and FLCN^NEG RPTECs stained for intracellular reactive oxygen species (ROS) with the CellROX reagent. As a positive control a Menadione treated (100 μM, 1 h) sample was included. Fold changes of independent experiments were calculated and are shown as a bar graph in Figure 7F. F, relative ROS levels are shown as fold change in mean fluorescent intensity (MFI) as measured by flow cytometry. FLCN^NEG cells show 2.2-fold higher ROS levels than FLCN^POS cells (n = 4). FLCN, folliculin; RPTEC, renal proximal tubular epithelial cell; TFEB, transcription factor EB.

Fig. 8

Schematic overview of differential phosphorylation pathways upon FLCN loss. A scheme portraying most dominant pathways where FLCN is involved in protein and receptor phosphorylation in our renal epithelial cell model. Dashed lines show where FLCN is involved, either directly or indirectly via (multiple) unknown other kinases as indicated by blackdiamond-shaped squares with question marks. Highlighted in pink are specific phosphosites that are differentially phosphorylated upon FLCN deficiency in both renal proximal tubular epithelial cells and UOK257 cell lines. As TFEB is dephosphorylated upon FLCN loss, we marked these specific differential phosphosites red. FLCN, folliculin; ROS, reactive oxygen species; TFEB, transcription factor EB.