STRUCTURAL AND FUNCTIONAL INSIGHTS INTO ALPHA-KINASES AND THEIR REGULATION

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Protein phosphorylation represents one of the most fundamental processes in living organisms, with ~30% of all human proteins undergoing phosphorylation at some point during their lifetime. Of the protein kinases present in the human genome, >90% belong to a superfamily of conventional eukaryotic protein kinases (CPKs). The alpha-kinase family, lacking sequence homology with CPKs, is an intriguing class of atypical kinases which were discovered for their phosphorylation of alpha-helical substrates. While our long-term objective is to deepen our understanding of the function, mechanism and regulation of protein phosphorylation, we will focus on the alpha-kinase family in this proposal. The alpha-kinases play critical roles in cell survival, proliferation and motility, but they are still poorly understood. Currently, the structures of two alpha-kinases have been determined: TRMP7 (a channel kinase involved in Mg2+ sensing) and MHCK (a Thr-specific kinase which acts on myosin II), the latter of which was determined by our group. Our structure revealed that an indispensable Asp residue near the active site of MHCK is phosphorylated, suggesting that the phosphate is transferred from ATP to the Asp and then to substrate, in contrast to the direct phosphotransfer from ATP in CPKs. We will investigate this unprecedented two-step mechanism using 31P-NMR and mass spectrometry. Further, we will elucidate a novel regulatory feature, discovered in our lab, involving phospho-ligand binding to an allosteric pocket for kinase activation. For eukaryotic elongation factor 2 kinase (eEF2K), which is arguably the most biologically important alpha-kinase, multiple regulatory factors exist. This is not surprising as it is a “master regulator” of protein synthesis. One of the most important, but poorly understood, regulatory elements is eEF2K's dependence on calmodulin (CaM) for activation. The main challenge has been the expression and purification of eEF2K which we have recently conquered. Based on this breakthrough, we propose to determine an eEF2K-CaM complex structure to reveal the structural basis of its activation. Moreover, the structural basis for recognition of an alpha-helical substrate is unknown due to a lack of alpha-kinase-substrate complex structures. We plan to use a peptide substrate conjugated to ATP as a high-affinity “bisubstrate” to evaluate substrate recognition and binding. Based on our discovery that a conserved N/D loop binds Mg2+, we will dissect the role of Mg2+ sensing of TRMP7 and its implication in cellular function using patch-clamp in combination with various N/D mutants. Taken together, we have proposed a comprehensive research program which is largely based on our own discoveries and progress. As such, we have a competitive edge in making significant contributions to the under-characterized alpha-kinases which are important for potential therapeutic applications.