Project Summary Protein kinases are an important class of enzymes that transfer a molecule of phosphate from ATP to protein substrates, a process known as phosphorylation. Virtually every cellular process is regulated by protein kinases, and abnormal phosphorylation has been linked to numerous diseases. More than 500 human protein kinases have been identified and assembled into an evolutionary tree known as the human “kinome”. Interestingly, approximately 10% of human kinases are predicted to be inactive and named pseudokinases because they lack one or more active site residues that are thought to be necessary for catalytic activity. We have discovered that the predicted inactive pseudokinase selenoprotein O (SelO) adopts an atypical protein kinase fold, yet has a novel enzymatic activity. SelO uses ATP as a co-substrate to transfer AMP, instead of phosphate, to proteins (AMPylation). Our results illustrate the catalytic versatility of the kinase fold and suggest that predicted pseudokinases should be analyzed for alternative transferase activities. Therefore, the goal of this proposal is to determine whether other protein pseudokinases can AMPylate protein substrates or perform entirely new chemistry. We propose that some pseudokinases may be catalytically active and have developed an innovative approach that combines the rational and targeted identification of candidate pseudokinases with a novel strategy to screen these candidates for alternative catalytic activities. Importantly, inactive pseudoenzymes are widespread in all major enzyme families throughout life and our strategy is applicable to other pseudoenzyme families as well. Thus, we anticipate that the results obtained in this proposal will have the potential to define new paradigms of cellular regulation and signal transduction.