Insights into Aurora-A Kinase Activation UsingUnnatural Amino Acids Incorporated by Chemical Modification

摘要

Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated andsupported substrate phosphorylation. Aurora-A activity is enhancedby phosphorylation at position 287 alone but is suppressed when position288 is also phosphorylated. This is rationalized by competition betweenphosphorylated T287 and T288 for a binding site composed of arginines,based on a structure of Aurora-A in which phospho-T287 occupies thissite. This is, to our knowledge, the first example of a Ser/Thr kinasewhose activity is controlled by the phosphorylation state of adjacentresidues in its activation loop. Overall we demonstrate an approachthat combines mutagenesis and selective chemical modification of selectedcysteine residues to investigate otherwise impenetrable aspects ofkinase regulation.