A new layer of protein kinase regulation by uORF-encoded small peptides

About 40% of human mRNAs contain upstream open reading frames (uORFs) in their 5’-untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of most encoded peptides remains unknown. Our recent study was first to reveal a uORF-encoded peptide exhibiting kinase inhibitory activity (Jayaram DR, et al. PNAS (2021)). This uORF (uORF2), upstream of the main ORF of a protein kinase C (PKC) family member (PKCeta), encodes for a peptide (uPEP2) containing the typical PKC pseudosubstrate (PS) motif present in all PKCs that auto-inhibits their kinase activity. We demonstrate that this PS motif underlies the direct binding, and kinase inhibition of four kinases consisting of the novel sub-group of PKCs (but not of the classical or atypical PKCs). Thus, uORF2 acts in cis on its own ORF and in trans on other novel PKCs. Moreover, we show that uPEP2 has therapeutic potential; it reduces breast cancer cells survival, their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, uPEP2 suppressed tumor progression, invasion and metastasis of MDA-MB-231 triple-negative breast cancer xenografts in mice models. CRISPR/Cas9 deletions of uORF2 and its downstream main ORF suggest that it acts as a growth suppressor. Notably, we have recently identified an additional uORF with a PS motif upstream of another PKC. Together, we revealed a new layer of protein regulation by uORFs, opening new views into eukaryotic protein regulation and cancer biology.