TY - JOUR
T1 - Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines
AU - Glykofridis, Iris E.
AU - Henneman, Alex A.
AU - Balk, Jesper A.
AU - Haas, Richard Goeij-De
AU - Westland, Denise
AU - Piersma, Sander R.
AU - Knol, Jaco C.
AU - Pham, Thang V.
AU - Boekhout, Michiel
AU - Zwartkruis, Fried J. T.
AU - Wolthuis, Rob M. F.
AU - Jimenez, Connie R.
N1 - Funding Information: Acknowledgments—We thank the members of the Onco-Proteomics and Oncogenetics laboratories for fruitful discussions, Tim Schelfhorst for technical assistance, Andrea Vallés Martí and Henk Dekker for sharing reagents, Govind Pai for help with flow cytometry measurements, and Jürgen Claesen for statistical analyses of drug sensitivity assays. BHD renal tumor cell lines UOK257 and UOK257-2 were kindly provided by Laura Schmidt, and we thank Rosa Puertollano for sharing TFEB expression constructs. This work was funded by the VUmc-Cancer Center Amsterdam (grant CCA2018-5-51 and Core Funding Mass Spectrometry Infrastructure) and the Netherlands Organisation for Scientific Research (NWO-Mid-delgroot project number 91116017). Funding Information: We thank the members of the Onco-Proteomics and Oncogenetics laboratories for fruitful discussions, Tim Schelfhorst for technical assistance, Andrea Vallés Martí and Henk Dekker for sharing reagents, Govind Pai for help with flow cytometry measurements, and Jürgen Claesen for statistical analyses of drug sensitivity assays. BHD renal tumor cell lines UOK257 and UOK257-2 were kindly provided by Laura Schmidt, and we thank Rosa Puertollano for sharing TFEB expression constructs. This work was funded by the VUmc-Cancer Center Amsterdam (grant CCA2018-5-51 and Core Funding Mass Spectrometry Infrastructure) and the Netherlands Organisation for Scientific Research (NWO-Middelgroot project number 91116017). Publisher Copyright: © 2022 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2022/9/1
Y1 - 2022/9/1
N2 - 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 FLCNPOS and FLCNNEG 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 FLCNNEG 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 FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.
AB - 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 FLCNPOS and FLCNNEG 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 FLCNNEG 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 FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.
UR - http://www.scopus.com/inward/record.url?scp=85138016230&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.mcpro.2022.100263
DO - https://doi.org/10.1016/j.mcpro.2022.100263
M3 - Article
C2 - 35863698
SN - 1535-9476
VL - 21
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 9
M1 - 100263
ER -