Role of phosphorylation in regulation of redox-mediated events in apoptotic cell death

The blue-green algal toxins: nodularin and microcystin, induce apoptotic cell death in an ultrarapid manner (Fladmark et al., 1999). These toxins inhibit the general protein phosphatases (PP1 and PP2A) thereby resulting in Ca2+/calmodulin-dependent protei n kinase (CaMKII) hyperphosphorylation of a number of proteins (Fladmark et al., 2002). We believe that by identifying the phosphorylated target proteins in our apoptotic model, new clues for understading the regulation of cell death may be found. But, as most phosphoproteins exist in very low amounts in the cell, identification of phosphorylated proteins is a difficult task. We have recently identified a number of phosphorylated proteins in nodularin-induced apoptotic cells. Among these were two antioxid ant proteins, namely Cu/Zn superoxide dismutase (SOD1) and antioxidant protein 2 (AOP2). Additionally, proteins known to be involved in the regulation of reactive oxygen species (ROS): PARK7 and GAMT were are shown to be phosphorylated. None of our above mentioned proteins have previously shown to be regulated by phosphorylation. We have also identified interaction partners of some of the identified proteins, interacting exclusively during the apoptotic process. In the applied project will characterize th e specific phosphorylation sites of the identified proteins and find the kinase(s) responsible. We will search for more interaction partners and study the effect of phosphorylation upon these interactions. The effect of removing the specific phosphorylat ion sites, phosphoproteins or interaction partners will be studied in respect to ROS release apoptosis development. Establishment of methods for identification and quantitation of phosphoprotein will be done in collaboration Centre of for Element and Iso top Analysis, UiB (LA-ICP-MS), National Proteomics Platform (LC-MS/MS, phosphoprotein/peptide up-concentration) and world-renowed international partners in this field.