Identification of Novel Protein Kinases Dependent on Phosphocreatine Rather than ATP

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Abstract Post-translational modifications of proteins are a major mechanism for the modulation of protein activity. Among these, the most common is protein phosphorylation by a set of enzymes called protein kinases. Protein kinases (PKs) are involved in almost every pathway of biological significance in eukaryotes. Metabolic regulation requires protein kinase function in every system, including insulin and glucagon action, glycogenolysis, adipogenesis and adaptive thermogenesis. Excessive AKT protein kinase activity downstream of insulin action is also suspected of being a key link between obesity and cancer. The mammalian kinome includes some 560 known enzymes, about 0.2% of the entire coding genome. As far as it is known, every one of these enzymes utilizes ATP as a high-energy phosphate donor, transferring the γ-phosphate of ATP onto (mainly) serine, threonine or tyrosine residues. Nature has developed another high-energy phosphate containing molecule that is often more abundant than ATP: phosphocreatine (CrP). Because ATP is an inhibitor of ATP synthase, cells can't store ATP. Instead, the γ-phosphate of ATP can be transferred to creatine (Cr), regenerating ADP and allowing the electron transport chain to continue functioning in the “forward” direction. Our recent work has demonstrated a non-canonical function of Cr and CrP in thermogenic adipose cells, which run a futile cycle of creatine phosphorylation and de-phosphorylation. This futile cycle expends energy without doing work and hence, results in the generation of heat. This work demonstrating a broader function of creatine than “just” energy storage caused us to ask an unusual question: are there protein kinases that preferentially use CrP? In fact, we have demonstrated here, using high-resolution protein Mass Spectrometry, that brown fat cell extracts can utilize CrP to phosphorylate certain peptide sites (at both S/T and Y residues) that are not modified when ATP is used as a substrate. Our key goals moving forward are to (1) demonstrate that these phosphorylation events are direct phosphate transfer reaction from CrP to target proteins (2) to demonstrate that these phosphorylations are dependent on CrP in vivo, using murine models of Cr and CrP-deficient animals (3) purify and characterize the CrP-dependent PKs. These may be new members of the kinome or known PKs that alter peptide target specificity when they use CrP as a substrate (4) perform biochemical and biophysical studies to characterize the enzymatic reactions and identify the CrP binding sites on the PKs. (5) investigate the physiological importance of the CrP PKs, by mutating specific target sites in protein targets and ablating the CrP-dependent PKs themselves. This project will open up a potentially important new area in biochemistry and physiology, and represents a “high-risk, high-reward type of project for which the Catalyst Award is intended.