cytoplasmic Abelson Tyrosine Kinase (Mus musculus)
Created by Kimberly Hoilman
Kinases perform important roles for cell signaling. Protein kinases catalyze the phosphorylation of proteins, while tyrosine kinases specifically catalyze the phosphorylation of proteins at tyrosine residues - essentially activating and inactivating a protein. There are two categories of tyrosine kinases: receptors and non-receptors. Receptor tyrosine kinases are transmembrane proteins; non-receptor tyrosine kinases are located in the nucleus or the cytoplasm of cells (1).
Cytoplasmic Abelson tyrosine kinase (c-Abl kinase) (PDB ID: 1IEP) is a non-receptor kinase of approximately 1150 residues. c-Abl functions as both a transferase and a kinase in almost every kind of cell, for which the protein performs a variety of functions. For example, c-Abl may be involved in cell differentiation, division, proliferation, and/or apoptosis (1).
c-Abl is well known for its prominent role in chronic myelogenous leukemia (CML), which comprises about 15% of adult leukemias (8). A genetic mutation (chromosome translocation) fuses the c-Abl gene with the breakpoint cluster region (bcr) gene. The hybrid chromosome resulting from chromosome translocation (the Philadelphia chromosome) contains the fused bcr-abl gene, which results in the production of a constitutively active form of c-Abl: the oncoprotein BCR-Abl (1). This mutation of c-Abl significantly elevates the level of tyrosine kinase activity of the protein that in normal cells is rather low, causing cells to develop growth factor independence due to increased phosphorylation by BCR-Abl (2). One hypothesis for the increased level of kinase activity is the deregulation/absence of autoinhibitory amino acid sequences in the c-Abl kinase domain (1).
c-Abl is found in cell nuclei and cytoplasm and exists as a dimer of bi-chain subunits in its physiological form. Its kinase domain is also composed of two subunits (chains A and B). Each chain is composed of approximately 586 residues in its crystallized form and is found in the N-terminal region of c-Abl. The domain consists of two lobes, an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The N-lobe is composed of a 5 stranded, antiparallel beta-sheet and an alpha-helix (helix alpha-C). The C-lobe is mostly alpha-helical and contains the helix alpha-l (1, 2, 3).
Within the c-Abl kinase domain, there are three key regulatory elements for autoinhibition. In its open/active conformation, the activation loop (residues 381-402) is phosphorylated at Tyr-393, the activating residue of the loop, which stabilizes the active conformation of the domain for substrate and ATP binding (2,4). A second crucial element is the Asp-381-Phe-382-Gly-383 (DFG) motif at the N-terminus of the activation loop (1,2). In c-Abl?s open/active conformation, Asp-381 is oriented towards the ATP binding site, located at the interface of the N- and C-lobes. This orientation allows Asp-381 to ligate a Mg2+ ion coordinated with the phosphate groups of bound ATP. When the kinase domain of c-Abl is inactive, the DFG motif flips, and Phe-382 projects toward the ATP binding site, preventing coordination of Mg2+ (1). The helix alpha-C is another important regulatory element. When the kinase domain is activated, the orientation of helix alpha-C allows Glu-117 to ionically bond with Lys-313 of the N-lobe, forming a salt bridge that coordinates ATP (1,3). If the helix alpha-C is rotated out of this active conformation, the salt bridge between E and K does not form (1).
Important regulatory elements lie outside the kinase domain as well. Two Src homology domains (SH3 and SH2) upstream from the c-Abl kinase domain interact with the domain, essentially clamping around it (1,2,4). Before these interactions can occur, a myristoyl group located at the extreme N-terminus (N-cap) of c-Abl must interact with the helix alpha-l of the C-lobe (1,3). When not in complex with the myristoyl group, the helix alpha-l exists in an extended form. The myristoyl group induces a bend in helix alpha-l at Phe-516, creating a new secondary structure with an almost 90° four-point turn and a new helix, alpha-l-prime. This bent conformation allows the myristoyl group to protrude far into the C-lobe. After this interaction has occurred, the docking site for the SH2 domain is appropriately oriented. The SH2 domain docks onto the distal side of the kinase domain (opposite the active site) through hydrophobic interactions and hydrogen bonding. This creates a docking site within the SH2-kinase linker region for the SH3 domain. With SH3 docking into the SH2-kinase linker region, a regulatory clamp is created that inhibits enzymatic activity of c-Abl (3).
The kinase activity of c-Abl can be activated by failure of autoinhibitory structures, dimerization, autophosphorylation or phosphorylation by other kinases (ex: c-Src, ATM) at catalytic tyrosine residues like Tyr-393, Tyr-412, and Tyr-283 (1,3,5,7). Notably, the autoinhibited conformation of the kinase domain has no phosphorylated tyrosine residues (4).
Because c-Abl is implicated in a large number of processes, consequently it has many substrates, such as DNA, p53, Hdm2, c-Crk, tyrosine phosphatase SHPTP1, etc. (1,6,7). However, c-Abl is best known for its interaction with Gleevec, a small molecule inhibitor used to treat chronic myelogenous leukemia (CML) (1,2,3,4). Chromosomal translocation of the breakpoint cluster gene (bcr) onto the Abl gene erases the N-cap/myristoyl group, and expression of the resulting mutant protein Bcr-Abl is thought to be the leading cause of CML (8).
Gleevec recognizes a unique, inactive conformation of Bcr-Abl. The activation loop is unphosphorylated, and it folds into and blocks the substrate binding site (4). The DFG motif is also in its inactive conformation. However, the Glu-Lys salt bridge remains intact, and an aspartate residue on the activation loop forms an H-bond with Tyr-393, which blocks substrate from docking in the active site (1,2). In this conformation, Gleevec settles into the ATP binding site of the Bcr-Abl kinase domain, forming H-bonds over a span of 21 residues, including to Thr-315, a critical residue for Gleevec recognition. Gleevec is hypothesized to induce this conformation of kinase domain of Bcr-Abl, and its binding to Bcr-Abl significantly reduces the mutants constitutively active kinase activity (1).
Unfortunately, mutations in the kinase domain (e.g. at Thr-315) and other regions of Bcr-Abl have led to Gleevec resistance among some CML patients, and the development of new c-Abl inhibitors is ongoing (1,2). PD173955, a novel inhibitor currently in development, has been shown to be significantly more effective in regulating Bcr-Abl kinase activity than Gleevec, due to its ability to bind active and inactive conformations of the kinase domain. In contrast, Gleevec only interacts with the inactive conformation. PD173955 and Gleevec bind to the same site of the Bcr-Abl kinase domain but with different conformations at the activation loop (2,3).
Protein BLAST and Dali server results have shown that c-Abl found in Mus musculus and Homo sapiens (PDB ID = 2HYY) have almost identical primary and tertiary structures (9). An E value of 4 e-161 with a Z score of 43.2 and a rmsd value of 0.4 Å justify comparison of c-Abl in the two species (data from Protein BLAST and Dali, respectively). There are two primary structural differences, neither of which affects the function of the c-Abl kinase. One occurs in the C-lobe of the c-Abl kinase domain, where Mus Ser-355 is mutated to Homo Asn-355. The second primary structure mutation occurs at Mus Ile-244 to Homo Val-244, located in the SH2-kinase linker region (3). Despite these mutations, the forms of c-Abl kinase in Mus musculus and Homo sapiens perform almost identical functions during cell differentiation and division, by phosphorylating substrate, and regulating DNA repair (9).