Approximately 40% of human messenger RNAs (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 the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.