Hugues de Thé Collège de France Oncologie cellulaire et moléculaire Année 2023-2024 Exploring the World of Protein Post-translational Modifications Conférence - Tony Hunter : Noncanonical Protein Phosphorylation – Is Histidine Phosphorylation Used as a Reversible Regulatory Mechanism? Tony Hunter Salk Institute, La Jolla, California, USA Résumé Protein phosphorylation is not restricted to serine, threonine and tyrosine, but can occur on six other amino acids, including histidine. In this lecture, I will discuss what is known about non-canonical protein phosphorylation, focusing on our recent work developing new tools to study histidine phosphorylation in cells and tissues, and their use to investigate the possible roles of histidine phosphorylation in liver cancer, pediatric neuroblastoma, breast cancer and pancreatic cancer. Histidine phosphorylation of proteins was discovered in 1962, before tyrosine phosphorylation, but much less is known about this PTM because phosphohistidine is chemically very labile and therefore hard to study. We set out to investigate histidine phosphorylation over thirty years ago but were stymied by the lack of key reagents. Because anti-phosphotyrosine antibodies have been so useful in studying tyrosine phosphorylation, we set out to develop anti-pHis antibodies, using chemically stable pHis analogues as antigens. We were successful in generating rabbit monoclonal antibodies selective for the 1-pHis and 3-pHis isoforms of phosphohistidine (pHis), using synthetic peptides containing the 1-Tza and 3-pTza pHis analogues as antigens, respectively. We have carried out structural studies of these mAbs bound to their cognate pTza peptide, which reveal how these mAbs achieve isoform specificity, and we have recently used these structures to engineer a higher affinity anti-3-pHis mAb. We have also exploited these mAbs to survey the pHis phosphoproteome, using mass spectrometry to identify novel sites of histidine phosphorylation and new pHis-containing proteins, which we are exploring. I will go on to describe how we have utilized these mAbs to investigate roles for histidine phosphorylation in cancer, showing that elevated histidine phosphorylation is a potential driver of hepatocellular carcinoma and pediatric neuroblastoma. Finally, I will discuss ongoing work investigating a role for histidine phosphorylation in triple negative breast cancer and pancreatic cancer, and, in particular, a possible role for histidine phosphorylation in the formation and function of invadopodia structures in neuroblastoma cell invasiveness.