Unraveling the hidden function of small uORF-encoded peptides as novel kinase inhibitors of PKCs

Introduction. Translational regulatory elements function in cells to rapidly change protein landscape in response to internal or external stimuli. Among these elements are upstream open reading frames (uORFs) located at the 5`UTR of mRNAs. Bioinformatics studies revealed the presence of uORFs in about 40% of human mRNAs. The current notion is that uORFs regulate translation of their downstream main ORFs, mostly by attenuating scanning ribosomes. However, whether peptides encoded by uORFs have biological functions beyond translational control remains unknown. In few cases, uORF-encoded peptides were shown to have a phenotypic impact, though their modes-of-action remained elusive. Here we show for the first time a novel uORF-encoded peptide with kinase inhibitory functions and cancer therapeutic potential.

Materials and Methods. Bioinformatic analysis was used to identify uORFs in PKCs of different species and their conservation. The translational probability was determined by ribosome profiling data analysis and by generating uORF-luciferase fusion plasmids with point mutations in their respective initiation and stop codons. The ability of the peptides (w/o mutations) to inhibit PKCs kinase activity was investigated using in vitro kinase assays. Effects on cell viability and synergy with chemotherapy were studied as well as effects on cell migration.

Results and Discussion. We previously identified two uORFs upstream of protein kinase C eta (PKCeta) that regulate its translation under normal growth conditions and upon stress. PKCeta is a signaling and anti-apoptotic stress kinase that belongs to the PKC family of serine/threonine protein kinases involved in cell proliferation, differentiation, and apoptosis, among other processes involved cancer. Here we show that one of these uORFs encodes a peptide having functional features of a PKC inhibitor, possessing the typical PKC kinase inhibitory motif, the pseudosubstrate sequence (PS). In all PKCs the PS binds to the kinase domain, acting as an internal inhibitor. We show that the uORF-encoded peptide is an efficient inhibitor of the kinase activity of PKCeta itself, and can also inhibit other PKCs of the novel PKC-subfamily. Importantly, the peptide suppresses cell proliferation/survival and migration of breast cancer cells and synergizes with the cell death–promoting effect of chemotherapeutic agents.

Conclusion. Our studies show that a peptide encoded in a translatable uORF is an inhibitor of the kinases encoded by the primary ORF. Thus, we introduce uORFs as new players in protein networks regulation, adding another layer of complexity to eukaryotic protein control mechanisms, providing basis for novel agents with potential therapeutic impact in cancer.