Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs

Significance Bioinformatic analysis revealed that approximately 40% of human messenger RNAs contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these sequences are translated, but the function of the encoded peptides remains unknown. Our study revealed a uORF encoding for a peptide exhibiting kinase inhibitory activity. This uORF, upstream of a PKC family member, possess the typical pseudosubstrate motif, which autoinhibits the catalytic activity of all PKCs. Using mouse models and human cells, we show that this peptide inhibits cancer cell survival, tumor progression, invasion, and metastasis and synergizes with chemotherapy by interfering with DNA damage response. Together, we point to a previously unrecognized function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.
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.