| Summary: | The Abelson protein-tyrosine kinase (c-Abl) is a non-receptor tyrosine kinase ubiquitously expressed in human tissues. c-Abl plays a critical role in many cellular functions including regulation of cell growth, survival, and differentiation, oxidative stress and DNA-damage responses, actin dynamics, and cell adhesion and migration. Its fusion protein, Bcr-Abl, has constitutive protein-tyrosine kinase activity which contributes to several disease states including chronic
myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). Though the core of Abl is similar to Src-family tyrosine kinases (SFKs) in structure, the complex regulation of both c-Abl and the Bcr-Abl fusion protein is still poorly understood. Hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the conformation and dynamics of c-Abl and understand its regulation mechanisms. Under physiological conditions, the Abl SH3 domain underwent partial unfolding which was
stabilized by ligand binding, providing a unique assay to test intramolecular and intermolecular interaction with the SH3 domain in solution. Experiments showed that Abl SH3 interacted with the SH2-kinase linker in the absence of the kinase domain. The unique conformation of the Abl SH2-kinase linker created by the insertion of few residues was further explored. Proline substitution in the linker led to high-affinity binding to the SH3 domain. All these observations implied that Abl
SH2-kinase linker sequence exhibits SH3 binding activity unique to the protein Abl. Contributions of a sequence at the N-terminus of Abl (NCap) to intramolecular interactions involving the Abl SH3 domain were studied using the same approach. HX MS results suggested that NCap stabilized SH3 domain dynamics in constructs that contain at least the SH2 domain. This NCap-induced stabilization may partially compensate for the absence of the negative regulatory C-terminal tail that is found in
the related Src family kinases. Furthermore, Bcr10 fusion of 10 residues of Bcr to Abl did not change conformation or dynamics. These results suggested that more Bcr residues may be required to sufficiently disrupt Abl downregulatory interactions. Finally, the effects of phosphorylation on Abl conformation and dynamics were assayed using HX MS. Phosphorylation of Abl SH3 prevented strong binding to high-affinity ligands. In addition, phosphorylation of NCap32L indicated disruption of
Abl intramolecular interactions. Site-directed mutagenesis implicated Tyr89 as the key residue that disrupted downregulation upon phosphorylation. These results imply that phosphorylation at Tyr89, known to occur in the leukemiogenic form Bcr-Abl, prevents engagement of the Abl SH3 domain with intramolecular binding partners and this leads to higher activity and cellular signaling by the Abl kinase.
|
|---|
