Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs and its potential in cancer therapy

Divya Ram Jayaram ¹ Sigal Frost ¹ Chanan Argov ² Assaf Ben-Ari ¹ Ilan Smoly ² Amitha Muraleedharan ¹ Margalit Krup ¹ Noah Isakov ¹ Deborah Toiber ³ Rose Sinay ¹ Chen Keasar ⁴ Esti Yeger-Lotem ² Etta Livneh ¹

¹The Shraga Segal department of Microbiology Immunology & Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Israel
²Department of Clinical Biochemistry & Pharmacology, Faculty of Health Sciences, Ben Gurion University of the Negev, Israel
³Life Sciences, Faculty of Life Sciences, Ben Gurion University of the Negev, Israel
⁴Department of Computer Sciences, Ben Gurion University of the Negev, Israel

Bioinformatics analysis revealed that approximately 40% of human mRNAs contain upstream open reading frames (uORFs) in their 5’ untranslated regions. Many of these uORFs, thought to impact translation or degradation of the primary ORF, were recently shown to be translated, but the function of encoded peptides remains unknown. Here we show for the first time that PKCeta, a signaling and anti-apoptotic stress kinase of PKC family, encodes for a uORF peptide with novel kinase inhibitory functions and therapeutic potential in cancer.

PKCs are involved in cell proliferation, differentiation, and apoptosis, among other processes impaired in cancer. We previously identified two uORFs upstream PKCeta that regulate its translation under normal growth conditions and upon stress. Here, we demonstrate that one of these uORFs possesses the typical pseudosubstrate motif present in all PKCs, auto-inhibiting their kinase activity. This uORF-encoded peptide (uPEP2) inhibits the kinase activity of PKCeta and other novel PKC-subfamily members, implying for network regulation in this family of kinases. Functionally, the peptide inhibits proliferation and migration of different aggressive tumors including breast cancers and leukemia cells. uPEP2 synergizes with a chemotherapeutic agent by interfering with the response to DNA damage. We show that uPEP2 interferes with DNA repair processes upstream of gH2AX phosphorylation, marking DNA double-strand breaks for repair, resulting in enhanced cell death.

Our studies introduce uORFs as new players in protein networks regulation, adding another layer of complexity to eukaryotic protein control mechanisms. Furthermore, these novel peptides may provide potential therapeutic agents for cancer treatment.