Englerin A (EA) is a small-molecule natural product with
selective
cytotoxicity against renal cancer cells. EA has been shown to induce
apoptosis and cell death through cell-cycle arrest and/or insulin
signaling pathways. However, its biological mode of action or targets
in renal cancer remains enigmatic. In this study, we employed advanced
mass spectrometry-based phosphoproteomics approaches to identify EA’s
functional roles in renal cancer. We identified 10,940 phosphorylation
sites, of which 706 sites exhibited EA-dependent phosphorylation changes.
Integrated analysis of motifs and interaction networks suggested activation
of stress-activated kinases including p38 upon EA treatment. Of note,
a downstream target of p38, Hsp27, was found to be hyperphosphorylated
on multiple sites upon EA treatment. Among these, a novel site Ser65
on Hsp27, which was further validated by targeted proteomics, was
shown to be crucial for EA-induced cytotoxicity in renal cancer cells.
Taken together, these data reveal the complex signaling cascade that
is induced upon EA treatment and importantly provide insights into
its effects on downstream molecular signaling.