ABSTRACT
The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS–RAF–MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: L.T., P.N., R.J.C., J.F.H.; Methodology: L.T., K.-J.C., W.E.K., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Software: L.T., K.-J.C., W.E.K., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Validation: L.T., K.-J.C., W.E.K., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Formal analysis: L.T., K.-J.C., W.E.K., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Investigation: L.T., K.-J.C., W.E.K., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Resources: L.T., K.-J.C., C.M.G., Y.Z., P.N., R.J.C., J.F.H.; Data curation: L.T., K.-J.C., Y.Z., J.F.H.; Writing - original draft: L.T., J.F.H.; Writing - review & editing: L.T., K.-J.C., W.E.K., C.M.G., P.N., R.J.C., J.F.H.; Visualization: L.T., Y.Z., P.N., R.J.C., J.F.H.; Supervision: L.T., K.-J.C., Y.Z., R.J.C., J.F.H.; Project administration: L.T., J.F.H.; Funding acquisition: J.F.H.
Funding
This work was supported by grants from the Cancer Prevention and Research Institute of Texas (CPRIT; RP170233 to J.F.H.) and the National Cancer Institute (R00CA188593 to K.-J.C.). Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.232132.supplemental
- Received March 19, 2019.
- Accepted June 18, 2019.
- © 2019. Published by The Company of Biologists Ltd
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