Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.     

Structure of the carboxyl-terminal Src kinase, Csk

The carboxyl-terminal Src kinase (Csk) is an indispensable negative regulator for the Src family tyrosine kinases (SFKs) that play pivotal roles in various cell signalings. To understand the molecular basis of the Csk-mediated regulation of SFKs, we elucidated the crystal structure of full-length Csk. The Csk crystal consists of six molecules classified as active or inactive states according to the coordinations of catalytic residues. Csk assembles the SH2 and SH3 domains differently from inactive SFKs, and their binding pockets are oriented outward enabling the intermolecular interaction. In active molecules, the SH2-kinase and SH2-SH3 linkers are tightly stuck to the N-lobe of the kinase domain to stabilize the active conformation, and there is a direct linkage between the SH2 and the kinase domains. In inactive molecules, the SH2 domains are rotated destroying the linkage to the kinase domain. Cross-correlation matrices for the active molecules reveal that the SH2 domain and the N-lobe of the kinase domain move as a unit. These observations suggest that Csk can be regulated through coupling of the SH2 and kinase domains and that Csk provides a novel built-in activation mechanism for cytoplasmic tyrosine kinases.     

Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase

The catalytic activity of protein tyrosine kinases is commonly regulated by domain-domain interactions. The C-terminal Src kinase (Csk) contains a catalytic domain and the regulatory SH3 and SH2 domains. Both the presence of the regulatory domains and binding of specific phosphotyrosine-containing proteins to the SH2 domain activate Csk. The structural basis for both modes of activation is investigated here. First, the SH3-SH2 linker is crucial for Csk activation. Mutagenic and kinetic studies demonstrate that this activation is mediated by a cation-pi interaction between Arg68 and Trp188. Second, Ala scanning and kinetic analyses on residues in the SH2-catalytic domain interface identify three functionally distinct types of residues in mediating the communication between the SH2 and the catalytic domains. Type I residues are important in mediating a ligand-triggered activation of Csk because their mutation severely reduces Csk activation by the SH2 domain ligand. Type II residues are involved in suppressing Csk activity, and their mutation activates Csk, but makes Csk less sensitive to activation by the SH2 ligand. Both type I and type II residues are likely involved in mediating SH2 ligand-triggered activation of Csk. Type III residues are those located in the SH2 domain whose mutation severely decreases Csk catalytic activity without affecting the SH2 ligand-triggered activation. These residues likely mediate SH2 activation of Csk regardless of SH2-ligand interaction. These studies lead us to propose a domain-domain communication model that provides functional insights into the topology of Csk family of protein tyrosine kinases.     

Distal Loop Flexibility of a Regulatory Domain Modulates Dynamics and Activity of C-Terminal Src Kinase (Csk)

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Csk and SFKs share a modular design with the kinase domain downstream of the N-terminal SH2 and SH3 domains that regulate catalytic function and membrane localization. While the function of interfacial segments in these multidomain kinases are well-investigated, little is known about how surface sites and long-range, allosteric coupling control protein dynamics and catalytic function. The SH2 domain of Csk is an essential component for the down-regulation of all SFKs. A unique feature of the SH2 domain of Csk is the tight turn in place of the canonical CD loop in a surface site far removed from kinase domain interactions. In this study, we used a combination of experimental and computational methods to probe the importance of this difference by constructing a Csk variant with a longer SH2 CD loop to mimic the flexibility found in homologous kinase SH2 domains. Our results indicate that while the fold and function of the isolated domain and the full-length kinase are not affected by loop elongation, native protein dynamics that are essential for efficient catalysis are perturbed. We also identify key motifs and routes through which the distal SH2 site might influence catalysis at the active site. This study underscores the sensitivity of intramolecular signaling and catalysis to native protein dynamics that arise from modest changes in allosteric regions while providing a potential strategy to alter intrinsic activity and signaling modulation.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

Domain interactions in protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.     

Csk suppression of Src involves movement of Csk to sites of Src activity.

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.     

Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk

Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.     

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.     

Theoretical Insights Reveal Novel Motions in Csk’s SH3 Domain That Control Kinase Activation

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk’s activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk.     

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.     

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.     

Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.     

Regulation of the Src Family Kinases by Csk

The non-receptor tyrosine kinase Csk serves as an indispensable negative regulator of the Src family tyrosine kinases (SFKs) by specifically phosphorylating the negative regulatory site of SFKs, thereby suppressing their oncogenic potential. Csk is primarily regulated through its SH2 domain, which is required for membrane translocation of Csk via binding to scaffold proteins such as Cbp/PAG1. The binding of scaffolds to the SH2 domain can also upregulate Csk kinase activity. These regulatory features have been elucidated by analyses of Csk structure at the atomic levels. Although Csk itself may not be mutated in human cancers, perturbation of the regulatory system consisting of Csk, Cbp/PAG1, or other scaffolds, and certain tyrosine phosphatases may explain the upregulation of SFKs frequently observed in human cancers. This review focuses on the molecular bases for the function, structure, and regulation of Csk as a unique regulatory tyrosine kinase for SFKs.     

Mutations in the N-terminal regulatory region reduce the catalytic activity of Csk, but do not affect its recognition of Src.

In addition to the C-terminal catalytic domain, Csk is a protein tyrosine kinase that has an N-terminal regulatory region that contains SH3 and SH2 domains. The role this region plays relative to the function of the catalytic domain is not clear. To study its role, we introduced either deletion or site-specific mutations within this region and analyzed the effect of such mutations on the catalytic activity of Csk and its ability to phosphorylate/inactivate Src protein tyrosine kinase, its physiological substrate in the cell. Deletion of the SH3 domain and the SH2 domain resulted in reductions of kinase activity by 70 and 96%, respectively. Mutations within the SH2 domain that abolished its ability to bind phosphotyrosine did not result in a significant loss of kinase activity. Mutation of Ser78 to Asp, located between the SH3 and the SH2 domains, resulted in a reduction of over 90% of the catalytic activity. The reduction in specific activity is not the result of any apparent physical instability of the mutants. Kinetic analyses indicate that the mutations did not affect the Km values for ATP-Mg or the polypeptide substrate. The ability of the mutants to phosphorylate and inactivate Src is directly correlated to their kinase activity. These results indicate that the regulatory region is important in optimizing the kinase activity of the catalytic domain, but apparently plays no direct or specific role in substrate recognition.     

Controlling the activity of the Tec kinase Itk by mutation of the phenylalanine gatekeeper residue

The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2-kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.     

Activation of the Src family kinase Hck without SH3-linker release

Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein (human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.     

Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.     

Autoinhibition of Bcr-Abl through its SH3 domain

Bcr-Abl is a dysregulated tyrosine kinase whose mechanism of activation is unclear. Here, we demonstrate that, like c-Abl, Bcr-Abl is negatively regulated through its SH3 domain. Kinase activity, transformation, and leukemogenesis by Bcr-Abl are greatly impaired by mutations of the Bcr coiled-coil domain that disrupt oligomerization, but restored by an SH3 point mutation that blocks ligand binding or a complementary mutation at the intramolecular SH3 binding site defined in c-Abl. Phosphorylation of tyrosines in the activation loop of the catalytic domain and the linker between the SH2 and catalytic domains (SH2-CD linker) is dependent on oligomerization and required for leukemogenesis. These results suggest that Bcr-Abl has a monomeric, unphosphorylated state with the SH3 domain engaged intramolecularly to Pro1124 in the SH2-CD linker, the form that is sensitive to the inhibitor imatinib (STI-571). The sole function of the coiled-coil domain is to disrupt the autoinhibited conformation through oligomerization and intermolecular autophosphorylation.