Abstract
Abl is a member of the non-receptor tyrosine kinase family, a group of enzymes responsible for propagation of cell signals, including those responsible for cell division. BCR-Abl, a deregulated fusion protein, is the cause of Chronic Myeloid Leukemia. This has made Abl the focus of a wide breadth of successful research efforts, yielding a fairly comprehensive understanding the structural and functional changes induced by each of its regulatory elements: Abl’s regulatory SH2 and SH3 domains, activation by binding of peptides to these domains or via phosphorylation, and inhibition by myristoylation. However, relatively little is known about how these elements work together to shape the enzyme’s regulation. Typically, myristoylated or phosphorylated states are obtained by purifying Abl from mammalian or insect cells, where these reactions occur naturally. However, the resulting Abl forms are often mixtures of different states, which can be difficult to differentiate and purify. By myristoylating and phosphorylating Abl in vitro, we have been able to obtain enzyme samples with only our desired post-translational modifications and build up these interactions from the unmodified protein. From this myristoylated Abl, we determined that a myristoylated peptide added in trans, while sufficient for crystallography, does not bind to activated states well enough to accurately mimic cis myristoylation. In addition, activation by phosphorylation is able to override most of the inhibition from myristoylation. These insights into the interplay between Abl’s different regulatory mechanisms will hopefully pave the way for more effective novel cancer treatments.