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Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
* To whom correspondence should be addressed. E-mail: bhushan.nagar{at}mcgill.ca.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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-phosphate of ATP onto serine, threonine, or tyrosine residues in proteins. Kinases permeate almost all cell signaling networks and coordinate basic cellular functions such as metabolism, gene expression, cell growth, motility, differentiation, and cell division. Through the efforts of the human genome project, it has been determined that there are 
500 different types of protein kinases in human cells comprising 2% of the genome, making it one of the largest gene families in eukaryotes (1). Protein kinases can essentially be thought of as molecular switches that toggle between on and off states. The transition between active and inactive kinase states is in general tightly regulated. Improper regulation of kinases can lead to cell transformation, and mutations that disrupt the normal regulation of kinases are a hallmark of many cancers. These findings are most notable for tyrosine kinases, because their evolution coincided with metazoans and are therefore often involved in intercellular communication (2).
Of the 500 protein kinases, 90 are tyrosine kinases, which are classified into 2 broad families: the receptor tyrosine kinases (RTK)5 and the nonreceptor tyrosine kinases (NRTK) (3). RTK are transmembrane proteins whose cytoplasmic region contains a tyrosine kinase domain. The extracellular regions are more varied, reflecting the different functions of various receptors. NRTK can be found in both the nucleus and the cytoplasm and are often associated with membranes through lipid modifications. NRTK generally consist of a single tyrosine kinase domain interspersed in various other modular protein domains, including Src homology 2 domain (SH2) and SH3 domains, which interact with phosphotyrosine and polyproline-containing peptides, respectively.
Protein kinase domains are bilobal in structure and have been structurally conserved through evolution (4). The N-terminal lobe (N-lobe) contains a 5-stranded ß-sheet and a single 
-helix referred to as helix C (Fig. 1). The C-terminal lobe (C-lobe) is largely -helical and contains the peptide substrate binding site and the catalytic loop. The binding site for ATP is situated between the 2 lobes in proximity to the peptide substrate binding site. Present within the kinase domain are several intrinsic regulatory elements (5). The first is a centrally located segment termed the activation loop, whose phosphorylation state determines whether a kinase is active. In tyrosine kinases, the site of activating phosphorylation is generally a single tyrosine residue located in the middle of the loop that once phosphorylated, can interact electrostatically with a neighboring arginine residue, resulting in the stabilization of an extended and open conformation of the loop. This phosphorylated conformation of the loop permits access to the peptide substrate binding site. When the activation loop is not phosphorylated, it can be disordered or assume various stable conformations, often blocking the peptide substrate binding site in the process. A second important regulatory feature of kinases is the conformation of a highly conserved aspartate-phenylalanine-glycine (DFG) motif located at the N-terminal end of the activation loop. In active kinases, the conformation of the aspartate residue of the DFG motif is such that it is oriented toward bound ATP and is capable of ligating a critical magnesium ion bound to the phosphate groups of ATP. Conversely, the phenylalanine residue points in the opposite direction, wedged between the C-lobe and helix C (6). In inactive kinases, the conformation of the DFG motif is often flipped such that the aspartate residue can no longer properly interact with the magnesium ion (7). Another important regulatory component is the orientation of helix C. Helix C presents a glutamate residue that forms a salt bridge with a lysine residue from the N-lobe and this pairing coordinates the phosphate groups of ATP. In inactive kinases, helix C is sometimes found to be rotated out of position, preventing the formation of the salt bridge (8). The conformation of a flexible glycine-rich loop in the N-lobe and the relative disposition of the N- and C-lobe are also important determinants of kinase activity.
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In addition to the intrinsic regulatory elements present within the kinase domain, other levels of regulatory control are often present in protein kinases, usually by means of accessory domains. One of the best understood kinases in terms of regulation by accessory domains is the Src family kinases (10). c-Src was originally discovered as a viral oncogenic form (v-Src) from an avian sarcoma. Src kinases are 550 residue tyrosine kinases consisting of a unique N-terminal region, followed by an SH3 domain, an SH2 domain, and a kinase domain that contains at its C terminus a critical regulatory tyrosine residue (Fig. 2). Phosphorylation of this tyrosine residue inactivates the kinase and its absence results in oncogenesis. The crystal structures of the Src family kinases c-Src and Hck revealed that they are autoinhibited by 2 key intramolecular interactions (8,11). The first is between the C-terminal phosphotyrosine and the SH2 domain. Additionally, the interaction of the SH2 domain with the internal phosphotyrosine ligand allows the linker region leading to the kinase domain to assume a left-handed polyproline helix that forms an internal binding for the SH3 domain. Together, these intramolecular interactions stabilize a conformation of the kinase domain in which the N-lobe is rotated, causing helix C to swing out and break the critical salt bridge between lysine and glutamate.
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The focus of this article is the mechanisms that govern the regulation of the tyrosine kinase c-Abl and how these mechanisms allow c-Abl to be specifically recognized by the cancer drug imatinib. Like c-Src, c-Abl was first discovered as the normal cellular counterpart to a virally encoded gene, v-Abl, found in a murine leukemia virus. c-Abl is a large protein of 1150 residues (13). The N-terminal half of c-Abl resembles closely the Src family tyrosine kinases, both in terms of the overall sequence as well as the order and spacing of the SH3, SH2, and kinase domains (Fig. 2). The C-terminal half of c-Abl, encoded by a single exon, contains domains of unknown structure and includes binding elements for SH3 domains, nuclear localization and export signals, a DNA-binding functionality, and an actin binding domain (14). Human cells express 2 alternative splice variants of c-Abl, Abl 1a and Abl 1b, which differ only at the very N-terminal region; Abl 1b is myristoylated, whereas Abl 1a is not known to be. c-Abl is expressed in almost all types of cells and carries out functions in both the nucleus and cytoplasm. Some of its normal cellular functions in the nucleus include mediating cell differentiation, cell division, apoptosis, and the stress response to DNA damage. In the cytoplasm, it is thought to mediate integrin binding. However, c-Abl is perhaps best known for its role in the pathogenesis of chronic myelogenous leukemia (CML), where its tyrosine kinase activity is deregulated.
CML is a disease that arises from a chromosomal translocation that takes place in hematopoietic stem cells (15). The exchange of DNA between the c-Abl gene and the breakpoint cluster region (bcr) gene, whose normal function is not known, results in a hybrid chromosome termed the Philadelphia chromosome, which carries on it a chimeric bcr-abl gene. This gene gives rise to a fusion protein, Bcr-Abl, which displays abnormally high levels of tyrosine kinase activity (Fig. 2). The expression of Bcr-Abl has important consequences on signal transduction and can result in enhanced proliferation, morphological transformation, or abrogation of growth factor dependence.
In 1996 a search for inhibitors of the tyrosine kinase platelet-derived growth factor receptor culminated in the discovery of a small-molecule compound, STI-571 (imatinib), a remarkably specific inhibitor that targeted only a handful of kinases, including platelet-derived growth factor receptor, c-Kit, and Bcr-Abl (16) (Fig. 3A). Imatinib is an ATP-competitive inhibitor and therefore binds to the cleft between the N- and C-lobes of the kinase domain of Bcr-Abl. The differences between Bcr-Abl and c-Abl occur upstream of the SH3 domain and, thus, imatinib would be expected to inhibit c-Abl as well, because it targets the kinase domain. Imatinib is now approved for use in patients afflicted with CML and is especially effective in the chronic phase of the disease (17).
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Imatinib appears to have either induced or recognized an inactive and unphosphorylated conformation of the kinase, because the DFG motif has been flipped and the activation loop is folded in toward the protein (Fig. 3B). However, the salt bridge between glutamate from helix C and lysine from the N-lobe is intact. The inverted conformation of the DFG prevents the aspartate residue from ligating magnesium. The activation loop is in a conformation such that the tyrosine residue that becomes phosphorylated upon activation forms a hydrogen bond with the catalytic aspartate on the catalytic loop, essentially mimicking bound substrate and blocking exogenous substrate from docking.
The active phosphorylated conformation of the activation loop is incompatible with binding imatinib as is the inactive conformation of the activation loop seen in the Src family kinases. Thus, imatinib appears to achieve its specificity for Abl by selecting a particular inactive conformation of the kinase that only Abl seems capable of attaining. But the question still remains as to why Src family kinases are unable to achieve this particular inactive conformation given the high degree of sequence conservation between the kinase domains of the 2 proteins. To address this question, an analysis of how this particular inactive conformation of the c-Abl kinase domain is achieved in the context of its accessory domains is required.
Abl regulation has been a long-standing problem and it has been shown that there are similarities between Src and Abl, most notably the activation of Abl through SH3 domain ligands (21). The similarity between Src and Abl is evident in the N-terminal region encompassing the SH3, SH2, and kinase domains, with one notable exception: the absence in c-Abl of the critical site of inactivating tyrosine phosphorylation at the C-terminus of c-Src (Fig. 1) (22). In v-Src, this site is also missing and it is an oncogene, yet Abl is regulated properly in normal cells. Because of the lack of a site of inactivating phosphorylation, it was postulated that c-Abl may be fundamentally different from the Src kinases and its regulation may involve interactions with other proteins in the cell (23). A subsequent biochemical analysis of c-Abl, however, showed that exogenous inhibitor proteins are not required (24). Thus, like the Src kinases, c-Abl is autoinhibited, but it achieves this regulation without a C-terminal tail phosphotyrosine. Moreover, deletion of the C-terminal half of the protein following the kinase domain does not deregulate the protein. In c-Abl, it is the N-terminal 80 residues that are critical for regulation and their removal causes the protein to become deregulated (24). Conversely, in the Src kinases, the N-terminal region is dispensable for proper regulation and its main function appears to be to tether Src to the cell membrane via the N-terminal myristoyl modification (25). Thus, the Src-like SH3, SH2, and kinase domains of c-Abl were autoinhibited properly only in the presence of the N-terminal region, which led to the hypothesis that this N-terminal region (N-cap) somehow "caps" the protein and stabilizes the autoinhibited state (24).
To ascertain how this regulation of c-Abl is achieved, the crystal structure of the Src-like portion of c-Abl1b, the major splice form, was determined (26–28). The structure shows that Abl1b adopts an assembled state that in general terms resembles c-Src (Fig. 4A). The SH3 and SH2 domains docked onto the back surface of the kinase domain that is distal to the active site. As in c-Src, docking of the SH2 domain allows the linker connecting the SH2 domain to the kinase domain to form an internal binding site for the SH3 domain. Together, the SH3-SH2 unit stabilizes an inactive conformation of the kinase domain in which the DFG motif and activation loop are distorted. The structure of the SH3-SH2 unit is known to form a rigid clamp-like structure in Src kinases when bound to the kinase domain (12) and molecular dynamics simulations show that this feature is also conserved in Abl (26). The N-cap, which was shown to be important for regulation, makes several interactions with the SH3-SH2 substructure and essentially forms a clasp that further stabilizes it. The most astounding aspect of the structure is that the very N terminus of the N-cap, which is modified by a myristoyl group, is bound stably to the base of the kinase domain. The myristoyl group penetrates deeply (10 Å) into the base of the C-lobe of the kinase domain, burying 500 Å2 of surface area.
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Superposition of the SH3-SH2 substructures of Src and Abl reveal a good structural conservation with a root mean square deviation of 1.2 Å. However, because of the differing mechanisms of SH2 domain docking, the consequence of the conservation of the SH3-SH2 substructure leads to differences in the conformations of the kinase domains of Src and Abl (Fig. 4D). In Abl, the C-lobe of the kinase domain is rotated by 20° relative to that in c-Src, so that it approaches the SH2 domain more closely, resulting in a considerably tighter SH2-kinase interface. This results in a more open conformation of the Abl kinase domain in which the DFG motif is flipped, a conformation that has been shown to be required for imatinib binding (9). Thus, the differing mechanisms of autoinhibition may explain why imatinib preferentially binds to Abl over Src. Although imatinib targets only the kinase domain, having evolved in the context of the rigid SH3-SH2 substructure and the myristoyl-dependent inactivation mechanism, the isolated kinase domain of c-Abl would be expected to display the open inactive conformation preferred by imatinib more easily than the kinase domain of c-Src. This is especially relevant in CML, where imatinib targets Bcr-Abl in which the kinase domain would be expected to be more "isolated" relative to that in wild-type c-Abl, because the myristoyl group has been removed.
In summary, by grafting small changes (N-terminal myristoyl group vs. C-terminal phosphotyrosine) onto a framework of autoinhibition controlled by the SH3-SH2 unit, nature has been able to retain a robust mechanism of regulation in 2 related, but different, tyrosine kinases. The conservation of the structure of the SH3-SH2 unit has led to conformational differences in the kinase domains because of the distinct mechanisms used to achieve autoinhibition. The resultant differences in the kinase domains of Src and Abl underlies the ability of imatinib to preferentially inhibit Abl. Although the drug imatinib has been an effective therapy for patients afflicted with CML, it is becoming vulnerable to resistance mutations, especially at later stages in the disease (20,29). These mutations are scattered throughout the protein, many of which coincide with segments that are important for mediating autoinhibition (30) and likely disrupt imatinib binding via allosteric mechanisms. Understanding the structural basis for resistance to anticancer therapeutics such as imatinib will be a key to designing new and more powerful drugs.
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FOOTNOTES |
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2 Supported by a fellowship from the Human Frontier's Science Program.
3 Author disclosure: Expenses to travel to the meeting were paid by Ajinomoto Company, Inc.
4 Color versions of Figures 1–3 are available with the online posting of this paper at jn.nutrition.org.
5 Abbreviations used: BCR, breakpoint cluster region; C-lobe, C-terminal lobe; CML, chronic myelogenous leukemia; DFG, aspartate-phenylalanine-glycine; N-lobe, N-terminal lobe; SH2/3, Src homology 2/3 domain; RTK, receptor tyrosine kinase; N-cap, N-terminal cap region; NRTK, nonreceptor tyrosine kinase.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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