A Src-like inactive conformation in the abl tyrosine kinase domain

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.     

The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation

The regulation of the activity of Abl and Src family tyrosine kinases is mediated by intramolecular interactions between the SH3, SH2, and kinase (SH1) domains. We have determined the crystal structure of an unphosphorylated form of c-Src in which the SH2 domain is not bound to the C-terminal tail. This results in an open structure where the kinase domain adopts an active conformation and the C terminus binds within a hydrophobic pocket in the C-terminal lobe. NMR binding studies support the hypothesis that an N-terminal myristate could bind in this pocket, as observed for Abl, suggesting that c-Src may also be regulated by myristate binding. In addition, the structure contains a des-methyl analog of the antileukemia drug imatinib (STI571; Gleevec). This structure reveals why the drug shows a low affinity for active kinase conformations, contributing to its excellent kinase selectivity profile.     

Structural basis for the autoinhibition of c-Abl tyrosine kinase

c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks a phosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571) to inhibit the catalytic activity of Abl, but not that of c-Src.     

Crystal structures of active SRC kinase domain complexes

c-Src was the first proto-oncoprotein to be identified, and has become the focus of many drug discovery programs. Src structures of a major inactive form have shown how the protein kinase is rigidified by several interdomain interactions; active configurations of Src are generated by release from this "assembled" or "bundled" form. Despite the importance of Src as a drug target, there is relatively little structural information available regarding the presumably more flexible active forms. Here we report three crystal structures of a dimeric active c-Src kinase domain, in an apo and two ligand complexed forms, with resolutions ranging from 2.9A to 1.95A. The structures show how the kinase domain, in the absence of the rigidifying interdomain interactions of the inactivation state, adopts a more open and flexible conformation. The ATP site inhibitor CGP77675 binds to the protein kinase with canonical hinge hydrogen bonds and also to the c-Src specific threonine 340. In contrast to purvalanol B binding in CDK2, purvalanol A binds in c-Src with a conformational change in a more open ATP pocket.     

Steered Molecular Dynamics Simulations Reveal the Likelier Dissociation Pathway of Imatinib from Its Targeting Kinases c-Kit and Abl

Development of small molecular kinase inhibitors has recently been the central focus in drug discovery. And type II kinase inhibitors that target inactive conformation of kinases have attracted particular attention since their potency and selectivity are thought to be easier to achieve compared with their counterpart type I inhibitors that target active conformation of kinases. Although mechanisms underlying the interactions between type II inhibitors and their targeting kinases have been widely studied, there are still some challenging problems, for example, how type II inhibitors associate with or dissociate from their targeting kinases. In this investigation, steered molecular dynamics simulations have been carried out to explore the possible dissociation pathways of typical type II inhibitor imatinib from its targeting protein kinases c-Kit and Abl. The simulation results indicate that the most favorable pathway for imatinib dissociation corresponds to the ATP-channel rather than the relatively wider allosteric-pocket-channel, which is mainly due to the different van der Waals interaction that the ligand suffers during dissociation. Nevertheless, the direct reason comes from the fact that the residues composing the ATP-channel are more flexible than that forming the allosteric-pocket-channel. The present investigation suggests that a bulky hydrophobic head is unfavorable, but a large polar tail is allowed for a potent type II inhibitor. The information obtained here can be used to direct the discovery of type II kinase inhibitors.     

Molecular dynamics simulations provide insights into the origin of gleevec’s selectivity toward human tyrosine kinases

Protein kinases are critical drug targets against cancer. Since the discovery of Gleevec, a specific inhibitor of Abl kinase, the capability of this drug to distinguish between Abl and other tyrosine kinases, such as Src, has been intensely investigated but the origin of Gleevec’s selectivity to Abl against Src is less studied. Here, we performed molecular dynamics (MD) simulations, dynamical cross-correlation matrices (DCCM), dynamical network analysis, and binding free energy calculations to explore Gleevec’s selectivity based on the crystal structures of Abl, Src, and their common ancestors (ANC-AS) and the two constructed mutation systems (AS→Abl and AS→Src). MD simulations revealed that the conformation of the phosphate-binding loop (P-loop) was altered significantly in the AS→Abl system. DCCM results unraveled that mutations increased anticorrelated motions in the AS→Abl system. Community network analysis suggested that the P-loop established special contacts in the AS→Abl system that are devoid in the AS→Src system. The binding free energy calculations unveiled that the affinity of Gleevec to AS→Abl increased to near the Abl level, whereas its affinity to AS→Src decreased to near the Src level. Analysis of individual residue contributions showed that the differences were located mainly at the P-loop. This study is valuable for understanding the sensitivity of Gleevec to human tyrosine kinases.

Communicated by Ramaswamy H. Sarma     

High yield bacterial expression of active c-Abl and c-Src tyrosine kinases

The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.     

Crystal structures of c-Src reveal features of its autoinhibitory mechanism.

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.     

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.     

STI-571: an anticancer protein-tyrosine kinase inhibitor

STI-571 (imatinib, Gleevec, Glivec, CGP 57148) is an inhibitor of the Abl group of protein-tyrosine kinases. One of these enzymes, the Bcr-Abl oncoprotein, results from the fusion of the BCR and ABL genes that result from the reciprocal chromosomal translocation that forms the Philadelphia chromosome. The Philadelphia chromosome occurs in 95% of people with chronic myeloid leukemia. ABL is the cellular homologue of the oncogene found in murine Abelson leukemia virus, and BCR refers to breakpoint cluster region. The Bcr-Abl oncoprotein exhibits elevated protein-tyrosine kinase activity, which is strongly implicated in the mechanism of development of chronic myeloid leukemia. STI-571 is effective in the treatment of the stable phase of chronic myeloid leukemia. The c-Abl protein kinase domain exists in an active and inactive conformation. STI-571 binds only to the inactive state of the enzyme as shown by X-ray crystallography. The drug binds to a portion of the ATP-binding site and extends from there into adjacent hydrophobic regions. STI-571 is a competitive inhibitor of Abl kinase with respect to ATP. Resistance to STI-571 is often the result of mutations in residues of the Bcr-Abl kinase that ordinarily bind to the drug. Inhibition of target protein kinases represents an emerging therapeutic strategy for the treatment of cancer.     

A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP site inhibitor STI-571

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) alpha- and beta-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit is, however, not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the beta5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGFbeta-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGFbeta-receptor mutant is inhibited by STI-571. Docking of STI-571 into computer models of the PDGFbeta-receptor and Flt3 kinase domains and comparison with the crystal structure of the STI-571 Abl kinase complex indicated very similar binding sites among the three nonphosphorylated kinases, suggesting corresponding courses of their Asp-Phe-Gly motifs and activation loops. Accordingly, we observed reduced sensitivity of preactivated compared with nonactivated PDGFR-beta for the inhibition by STI-571. Courses of the activation loop that collide with STI-571 binding explain its inactivity at other kinases as the insulin receptor. The binding site models of PDGFR-beta and Flt3 were applied to predict structural approaches for more selective PDGFbeta-receptor inhibitors.     

Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.     

The D816V Mutation of c-Kit Circumvents a Requirement for Src Family Kinases in c-Kit Signal Transduction

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis, and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia, gastrointestinal stromal tumors, and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study, we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition, we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensable for c-Kit/D816V cell survival, proliferation, and colony formation. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.The receptor for stem cell factor (SCF),² c-Kit, is a type III receptor tyrosine kinase that belongs to the same subfamily as the platelet-derived growth factor receptors, the Flt3 receptor, and the macrophage colony-stimulating factor receptor (1). The c-Kit gene is identical to the white spotting locus (W) in the mouse. c-Kit is expressed in the hematopoietic system, in the gastrointestinal system, in melanocytes, and in germ cells, and therefore loss-of-function mutations in c-Kit lead to defects in hematopoiesis, melanogenesis, and gametogenesis. Stimulation of the c-Kit receptor with its ligand, SCF, leads to receptor dimerization and activation of its intrinsic tyrosine kinase activity. Specific tyrosine residues are autophosphorylated, which results in the activation of downstream signaling pathways, including the Ras/Erk pathway and the PI3-kinase pathway (for review, see Ref. 2).One of the crucial steps in oncogenic transformation of cells is the gain of independence of external growth stimuli. This can be achieved in several different ways, including mutations that render receptor tyrosine kinases constitutively active in the absence of ligand stimulation. In the case of c-Kit, these mutations most commonly occur either in exon 11 (encoding the juxtamembrane region) and are found predominantly in gastrointestinal stromal tumors or in exon 17 (encoding the activation loop of the kinase domain). A frequently occurring type of mutation in exon 17 in c-Kit is at codon 816. This type of mutation has been found in several human malignancies including acute myeloid leukemia, mastocytosis, germ cell tumors of the seminoma or dysgerminoma types, sinonasal natural killer/T-cell lymphomas, and in intracranial teratomas (3–10). These mutations at codon 816 lead to conversion of an aspartic acid residue to a valine, a tyrosine, a phenylalanine, an asparagine, or a histidine residue. The recently elucidated crystal structure of the c-Kit kinase domain has helped define the mechanism of activation by this type of mutation (11). In the unstimulated wild-type c-Kit, the juxtamembrane region inserts directly into the clefts between the amino- and carboxyl-terminal lobes of the kinase domain, disrupting the c-Kit control helix, and physically blocking the conserved kinase DFG motif from attaining a productive conformation. The activation loop folds back over the substrate binding groove and interacts with the active center of the kinase as a pseudosubstrate. It is not fully known how mutation of aspartic acid 816 leads to activation of c-Kit. It has been suggested either that the mutation inverts the conformation of the protein backbone so that the side chain of arginine 815 is being flipped from its position in the autoinhibited or that the effect of aspartic acid 816 mutations may be derived from its ability to stabilize the small positively charge α-helical dipole through its negative charge of its side chain. Asp-816 mutations in c-Kit promote receptor autophosphorylation and thereby constitutively activate downstream signaling pathways independent of SCF binding and therefore contribute to cell transformation (12). Imatinib (Gleevec) is a well known inhibitor of c-Kit juxtamembrane mutations and has been used in the treatment of gastrointestinal stromal tumors with activating mutations in the juxtamembrane region of c-Kit. In contrast, cells expressing c-Kit/D816V are resistant to Imatinib, whereas the Abl/Src dual inhibitor Dasatinib also inhibits the D816V mutant of c-Kit (13).Negative regulation of c-Kit signaling has been shown to occur mainly through ubiquitin-mediated internalization and degradation of the receptor (14, 15). Ubiquitination is mediated by ubiquitin E3 ligases that attach ubiquitin to their target proteins, resulting in either monoubiquitination or polyubiquitination. Key components in this machinery are the Cbl family of ubiquitin E3 ligases, represented by Cbl, Cbl-b, and Cbl-c (16). Signaling through receptor tyrosine kinases must be tightly regulated, and inhibition of Cbl activation and receptor ubiquitination can lead to cell transformation (17). It has been shown that both direct and indirect binding of Cbl to wild-type c-Kit can induce Cbl activation and receptor ubiquitination followed by receptor internalization and degradation (15, 18). In contrast, the mechanisms behind negative regulation of the oncogenic c-Kit/D816V are so far unknown.Extracellular signal-regulated kinase (Erk) proteins are the evolutionary conserved products of the two genes, Erk1 and Erk2, and are central proteins in the Ras/Erk signaling pathway. Erk1 and Erk2 are activated by dual phosphorylation on their regulatory tyrosine and threonine residues (19). The serine/threonine kinase Akt, also known as protein kinase B, is activated downstream of PI3-kinase and plays a central role in signaling induced by growth factors, cytokines, and other cellular stimuli (20). The Ras/Erk pathway and the PI3-kinase pathway are key signaling pathways involved in the regulation of cell proliferation, survival, and differentiation induced by c-Kit. A key player in the relay of signals from c-Kit into the cells is Src. Binding of Src to Tyr-568 in c-Kit leads to its activation and subsequent phosphorylation of Shc and activation of the Ras/Erk pathway (21). PI3-kinase is activated by c-Kit through two alternate routes, either through direct binding to Tyr-721 in c-Kit (22) or through indirect binding to the scaffolding protein Gab2, which associates to c-Kit via the adapter protein Grb2. Activation of PI3-kinase is dependent on Src-mediated phosphorylation of Gab2 (23). Other investigators have shown that neither Erk nor Akt is constitutively activated in cells expressing c-Kit/D816V (24).In this report, we demonstrate that in c-Kit/D816V-expressing Ba/F3 cells, a low constitutive activation of both Erk and Akt exists and that this activation can be further augmented by SCF stimulation. We also present data showing that c-Kit/D816V evades the need of Src family kinases for receptor ubiquitination and Erk activation by having an altered substrate specificity resembling Src family kinases. We conclude that Src family kinases play different roles in wild-type c-Kit and c-Kit/D816V-induced cell survival and growth.     

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.     

Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and SRC family tyrosine kinases

Vaccinia virus (VacV) enters mammalian cells, replicates extranuclearly, and produces virions that move to the cell surface along microtubules, fuse with the plasma membrane, and move from infected cells toward apposing cells on actin-filled membranous protrusions or actin tails. To form actin tails, cell-associated enveloped virions (CEV) require Abl and Src family tyrosine kinases. Furthermore, release of CEV from the cell requires Abl but not Src family tyrosine kinases and is blocked by imatinib mesylate (STI-571; Gleevec), an Abl family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Here we demonstrate that the Poxviridae family members monkeypox virus (MPX) and variola virus (VarV) use conserved mechanisms for actin motility and extracellular enveloped virion (EEV) release. Furthermore, we show that imatinib mesylate is effective in a mouse model of infection with VacV, whether delivered prophylactically or postinfection, and restricts spread of virions from the site of inoculation. While inhibitors of both Src and Abl family kinases, such as dasatinib (BMS-354825; Sprycel), are effective in limiting dissemination of VacV, VarV, and MPX in vitro, members of this class of drugs appear to have immunosuppressive effects in vivo that preclude their use as anti-infectives. Together, these data suggest a possible utility for imatinib mesylate in treating smallpox or MPX infections or complications associated with vaccination.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

Structural basis for the recognition of c-Src by its inactivator Csk

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.     

Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib

Current structural understanding of kinases is largely based on x-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. ABL kinase is an important drug target in the treatment of chronic myelogenous leukemia. Here, we present the first characterization of ABL kinase in complex with three clinical inhibitors (imatinib, nilotinib, and dasatinib) by modern solution NMR techniques. Structural and dynamical results were derived from complete backbone resonance assignments, experimental residual dipolar couplings, and (15)N relaxation data. Residual dipolar coupling data on the imatinib and nilotinib complexes show that the activation loop adopts the inactive conformation, whereas the dasatinib complex preserves the active conformation, which does not support contrary predictions based upon molecular modeling. Nanosecond as well as microsecond dynamics can be detected for certain residues in the activation loop in the inactive and active conformation complexes.     

c-Abl is an effector of Src for growth factor-induced c-myc expression and DNA synthesis

The mechanism by which the ubiquitously expressed Src family kinases regulate mitogenesis is not well understood. Here we report that cytoplasmic tyrosine kinase c-Abl is an important effector of c-Src for PDGF- and serum-induced DNA synthesis. Inactivation of cytoplasmic c-Abl by the kinase-inactive Abl-PP-K(-) (AblP242E/P249E/K290M) or by microinjection of Abl neutralizing antibodies inhibited mitogenesis. The kinase-inactive SrcK295M induced a G(1) block that was overcome by the constitutively active Abl-PP (AblP242E/P249E). Conversely, the inhibitory effect of Abl-PP-K(-) was not compensated by Src. c-Src-induced c-Abl activation involves phosphorylation of Y245 and Y412, two residues required for c-Abl mitogenic function. Finally, we found that p53 inactivation and c-myc expression, two cell cycle events regulated by Src during mitogenesis, also implied c-Abl: c-Abl function was dispensable in cells deficient in active p53 and inhibition of c-Abl reduced mitogen-induced c-myc expression. These data identify a novel function of cytoplasmic c-Abl in the signalling pathways regulating growth factor-induced c-myc expression and we propose the existence of a tyrosine kinase signalling cascade (PDGFR/c-Src/c-Abl) important for mitogenesis.     

Evaluation of residue variability in a conformation‐specific context and during evolutionary sequence reconstruction narrows drug resistance selection in Abl1 tyrosine kinase

Diseases with readily available therapies may eventually prevail against the specific treatment by the acquisition of resistance. The constitutively active Abl1 tyrosine kinase known to cause chronic myeloid leukemia is an example, where patients may experience relapse after small inhibitor drug treatment. Mutations in the Abl1 tyrosine kinase domain (Abl1‐KD) are a critical source of resistance and their emergence depends on the conformational states that have been observed experimentally: the inactive state, the active state, and the intermediate inactive state that resembles Src kinase. Understanding how resistant positions and amino acid identities are determined by selection pressure during drug treatment is necessary to improve future drug development or treatment decisions. We carry out in silico site‐saturation mutagenesis over the Abl1‐KD structure in a conformational context to evaluate the in situ and conformational stability energy upon mutation. Out of the 11 studied resistant positions, we determined that 7 of the resistant mutations favored the active conformation of Abl1‐KD with respect to the inactive state. When, instead, the sequence optimization was modeled simultaneously at resistant positions, we recovered five known resistant mutations in the active conformation. These results suggested that the Abl1 resistance mechanism targeted substitutions that favored the active conformation. Further sequence variability, explored by ancestral reconstruction in Abl1‐KD, showed that neutral genetic drift, with respect to amino acid variability, was specifically diminished in the resistant positions. Since resistant mutations are susceptible to chance with a certain probability of fixation, combining methodologies outlined here may narrow and limit the available sequence space for resistance to emerge, resulting in more robust therapeutic treatments over time.