Abl holds a prominent place among the over 500 protein kinases encoded by the human genome. Abl mediates its function by participating in a number of biological processes, including actin remodeling, cell adhesion and motility, DNA damage response, and bacterial pathogen response. Although Abl is essential for cell development, it can also cause serious diseases as a result of chromosome translocations. Chronic myeloid leukemia (CML) is characterized by a translocation that fuses the Bcr and Abl genes. The Bcr-Abl fusion gene product has constitutive tyrosine kinase activity and is leukemogenic. Bcr-Abl is also involved in childhood and adult acute lymphoblastic leukemia. Although Abl provides an excellent example of how kinase inhibitors can be used for therapeutic purposes, mutations that confer resistance to drugs presents a growing problem in the treatment of CML. Thus, central to the physiological function of Abl is the proper regulation of it kinase activity. This is accomplished by the presence of a myristoylated regulatory module consisting of SH3 and SH2 signaling domains. Crystal structures of the isolated Abl kinase domain in the presence of inhibitors as well as in the assembled, autoregulated state have provided fascinating information about the overall structural organization of Abl. Nevertheless, the mechanistic basis for the regulation and activation of Abl is far from being understood. The various structural elements of the regulatory module apparently act in a synergistic fashion to yield a multi-layered regulatory mechanism that remains elusive. The intrinsic properties of the Abl kinase domain are still not known since all available structures have been solved in the presence of an inhibitor. Moreover, despite the fact that kinases are highly dynamic, very little i known about their intrinsic motions due to technical challenges in studying these systems. We propose to use primarily NMR spectroscopy to characterize the mechanisms underlying autoregulation and activation of the Abl kinase. We will obtain integrated structural, dynamic, kinetic and thermodynamic information of the activation process.
The specific aims are designed to provide atomic-resolution insight into (i) the activation process in the isolated kinase domain, (ii) the intrinsic structural and dynamic properties of the regulatory module, (iii) the activation process in the assembled, autoregulated Abl kinase, (iv) the mechanisms underlying the function of drug-resistance mutations, and (v) the binding of Crk adaptor proteins to Abl and the transactivation mechanism.
Abl fusion proteins (such as Bcr-Abl) are deregulated and cause various hematopoietic malignancies, such as chronic myeloid leukemia (CML), childhood and adult acute lymphoblastic leukemia, epithelial cell malignancies as well as invasive growth of breast cancer cells. The use of small molecules as inhibitors of Abl kinase activity holds great promise for therapeutic purposes.
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