Effects of phosphorylation on the ADP-ribosylation activity of PARP16

Sammanfattning: PARP16/ARTD15 is an intracellular mono-ADP-ribosyltransferase that catalyzes auto- and heteromodification by transfer of a single ADP-ribose moiety. It belongs to the ADP-ribosyltransferase (ART) superfamily. ADP-ribosylation of amino acids on protein targets is critical in the regulation of cellular pathways in eukaryotes, especially involved in physiological functions, cell stress responses and disease. It has been discovered that PARP16 plays an important role in the Unfolded Protein Response (UPR) as the main activator of 2 endoplasmic reticulum (ER) stress sensors, PERK and IRE1α kinases. In addition, it was also found that these kinases phosphorylate PARP16. The aim of this project is to investigate the phosphorylation of PARP16 by the kinases and whether that implies alterations of its activity. To answer this question, we coexpressed Tyrosine-kinases with YopH and Serine/Threonine-kinases with Lambda from an Addgene genetic library. The kinases were purified by a 2-step elution protocol using Ni-NTA beads and imidazole. Although the samples showed signs of phosphatase contamination, we found conditions in which we could assess the phosphorylation activity of the kinases. Finally, phosphorylation and ADP-ribosylation assays were performed with PARP16 and our kinases. The results showed clear phosphorylation of PARP16 and a variation of its ADP-ribosylation activity in the presence of the modification. This discovery opens up new questions about the biological effects that the phosphorylation of PARP16 can have, especially in cancers and protein folding diseases. Therefore, these findings can be critical for therapeutic inhibition and treatment. Further investigation should address questions such as what residues from PARP16 are modified and how phosphorylation by PERK and IRE1α affect the ADP-ribosylation activity of PARP16.