Abstract
Kinases are responsible for protein phosphorylation, which regulates a myriad of cellular activities pertaining to cell proliferation, growth, and communication. The unregulated activity of kinases leads to uncontrolled cell growth and, ultimately, cancer; therefore, This has caused kinases to be critical drug targets and has elicited extensive research aimed at elucidating the details of their regulation. Src and Abl are two non–receptor tyrosine kinases that share structural similarities in their auto-inhibited states, but have distinct mechanisms by which they achieve those states. Published crystal structures have revealed essential phosphorylation sites and allosteric players that regulate activity of these enzymes. However, specifics on the quantitative effects of these regulators remain elusive. \r Here, we set out to provide detailed kinetic schemes for the regulation of Src and Abl and provide insight into the evolution of their regulation. As a starting point, we determined the quantitative effect of modifying the essential tyrosine residues and later proceeded to investigate the effects of the allosteric regulators on kinase activity. Our results demonstrate that Src and Abl are not regulated by simple ‘on’ and ‘off’ switches where they exist only in two states; instead, they exist in equilibria between various states with different activities. \r In order to gain a better understanding of the evolution of Src and Abl regulation, we evaluated the effects of various regulatory players on the activity of common ancestors of Src and Abl. Interestingly, we observed a clear pattern: the effect of phosphorylation and other regulatory elements was greater in modern enzymes and gradually decreased for older ancestors. \r Our kinetic experiments provided detailed quantitative information about the effect of different players on Src and Abl activity. With this information in hand, we are now proceeding to developing a single-molecule FRET assay. The main objective of this experiment is to couple the observed kinetic effects with structural information. This will shed light on the interplay between different conformational ensembles of Src and Abl, affording us a better understanding of their regulation.