Ile164 Is a Critical Residue for the Integrity of the Active Conformation of Bcr-Abl
The SH2 domain is thought to act as an intramolecular allosteric activator of the tyrosine kinase domain in the protooncogenic kinases Fes and c-Abl (Filippakopoulos et al., 2008, 2009
). Recently, a mutation of threonine 231 (Thr231) in the SH2 domain of the Bcr-Abl protein was identified in imatinib-treated patients and implicated in causing imatinib resistance (Sherbenou et al., 2010
). The T231R mutation may stabilize the SH2-kinase domain interface by the formation of an additional ionic interaction with Glu294 in the N lobe of the kinase domain (A and Sherbenou et al., 2010
). In contrast, mutation of isoleucine 164 (Ile164), located in the αA-βB-loop of the Abl SH2 domain, to glutamate (I164E) may nullify the allosteric activation of the Abl kinase by the SH2 domain by disrupting the SH2-kinase domain interface (Filippakopoulos et al., 2008; Nagar et al., 2006
and A). These two observations may suggest a possible role of the Bcr-Abl SH2-kinase domain interface for the activity of the oncoprotein and even in CML pathobiology.
Structure-Function Analysis of the SH2-Kinase Domain Interface in Bcr-Abl
We introduced the I164E and T231R single mutations and the I164E/T231R double mutation into Bcr-Abl. Upon expression in HEK293 cells, global tyrosine phosphorylation, Bcr-Abl autophosphorylation (on Tyr412 in the activation loop and on Tyr245 in the SH2-kinase linker), and in vitro tyrosine kinase activity were reduced in the I164E but increased in the T231R mutant. The I164E mutation was dominant over the T231R mutation, consistent with the proposed disrupting effect of the I164E mutation versus the stabilizing role of the T231R mutation (B and 1C). In contrast, levels of phosphorylated Tyr177 in the Bcr part of Bcr-Abl, a site thought to be phosphorylated by Src kinases, were slightly higher in the T231R mutant. This might suggest higher Src activity in the presence of the T231R mutation (B).
To study the structural role of Ile164 in more detail, we mutated Ile164 to different polar/charged amino acids (Glu, Gln, Thr, Asp, or Lys) or to Ala. All mutations led to a strong reduction of Abl autophosphorylation and in vitro kinase activity. Somewhat weaker effects were observed by mutating Ile164 to structurally related hydrophobic amino acids (Val or Leu) ( available online).
Effects of Mutations of Ile164 and/or Thr231 in the SH2-Kinase Interface on Abl Kinase Activity and Phosphorylation of the Abl Substrate Paxillin, Related to
In enzyme-kinetic experiments, the Bcr-Abl I164E mutant protein displayed a >3-fold reduction in vmax compared to Bcr-Abl wild-type (WT) and a modest increase in Michaelis-Menten constant (KM) when assayed in vitro using an optimal Abl substrate peptide containing one single tyrosine (A).
The SH2-Kinase Domain Interface in Bcr-Abl Is Both Necessary and Sufficient for High Catalytic Activity of the Enzyme
Relationship between Phosphotyrosine Binding and Kinase Domain Binding of the Abl SH2 Domain
SH2 domain-containing tyrosine kinases have been proposed to use their SH2 domains to bind primed substrates to facilitate multisite (processive) phosphorylation of substrates with multiple tyrosine phosphorylation sites (Mayer et al., 1995
). Upon cotransfection of the Bcr-Abl substrate paxillin with Bcr-Abl I164E, we observed a strong reduction in multisite phosphorylation of paxillin, whereas the T231R mutation had the opposite effect (B and ). These data indicate that disruption of the SH2-kinase domain interface also influences the cellular activity of Bcr-Abl.
As the canonical function of the SH2 domain is phosphotyrosine binding, we analyzed whether the I164E mutation would interfere with phosphopeptide-SH2 interactions (Pawson et al., 2001
). Recombinant WT and I164E-mutated Abl SH2 domains displayed indistinguishable low micromolar binding constants for an optimal Abl SH2-domain phoshopeptide ligand (). In contrast, Abl SH2 domains bearing the FLVR
mutants R171L or S173N were unable to bind tyrosine-phosphorylated peptides ( and data not shown). Together these data show that the I164E mutant does not compromise phosphotyrosine binding or structural integrity of the SH2 domain. Nonetheless, in the context of full-length Abl kinase, the I164E mutant was as defective as the FLVRES
mutant S173N in multisite phosphorylation of paxillin (). This shows that aside from the intrinsic reduction in in vitro and cellular kinase activity, disruption of the SH2-kinase interface also interferes with multisite phosphorylation of Abl substrates, even though the phosphotyrosine peptide-binding properties of the SH2 domain are retained in Abl I164E. In addition, this indicates that the correct positioning of the SH2 domain may just be as important as the ability of the SH2 domain to bind primed tyrosine-phosphorylated substrates for further rounds of phosphorylation.
The I164E Mutation Does Not Interfere with the Phosphotyrosine-Binding Capability of the Abl SH2 Domain, Related to
Induced Tethering of the SH2 Domain to the Kinase Domain Activates Abl and Depends on Ile164
In order to assess its potential for pharmacological exploitation, we further investigated the molecular mechanism of the positive effect of the SH2 domain on kinase activity. We made use of the FKBP-FRB fusion protein system, in which the SH2 and kinase domains of Abl are expressed separately but can be induced to physically associate upon addition of rapamycin (Belshaw et al., 1996
) (C). Induced tethering of the Abl SH2 domain to the Abl kinase domain led to an unexpectedly strong increase in phosphorylation of cotransfected paxillin and in total cellular tyrosine phosphorylation (C). The effect was reliant on the presence of both protein components and on the dimerization-inducing drug. Importantly, this effect was entirely dependent on the SH2-kinase interface, as mutation of Ile164 abolished the stimulatory effect. This provided conclusive evidence that docking of the SH2 domain is sufficient for the observed positive allosteric effect on kinase activity and could thus represent a targetable interaction.
The Bcr-Abl SH2-Kinase Interface Is Critical for the Transformation of Primary Murine Hematopoietic Cells and Leukemia Formation In Vivo
We next determined whether mutation of the SH2-kinase domain interface had an effect on oncogenic transformation and leukemogenicity. Primary murine bone marrow hematopoietic cells transduced with Bcr-Abl WT were able to form colonies in the absence of cytokines in semisolid media (A). In contrast, this transforming capability was dramatically reduced in cells expressing Bcr-Abl I164E (A).
Bcr-Abl I164E Is Not Leukemogenic in a Bcr-Abl Mouse Bone Marrow Transplantation Model
We then tested whether this reduction was also manifest in an in vivo model of Bcr-Abl-induced leukemia (Daley et al., 1990
). Equal numbers of Bcr-Abl WT- and Bcr-Abl I164E-transduced hematopoietic stem cells were injected into lethally irradiated recipient mice. Mice transplanted with Bcr-Abl-expressing cells developed an aggressive myeloproliferative disorder, leading to death of all animals within 3 weeks (B). These mice displayed massively infiltrated spleen and liver and loss of normal organ architecture (C). In contrast, all mice transplanted with Bcr-Abl I164E-expressing cells remained alive for the 120 days of the study (B). After this period, no obvious pathological alterations in the liver or spleen were detected despite confirmation of the presence of Bcr-Abl I164E-transduced cells in all lineages in peripheral blood, bone marrow, and spleen (C and 3D and data not shown). On one hand, this indicates a lack of oncogenic properties of Bcr-Abl I164E that are necessary to induce a fatal leukemia, but it rules out a possible defect in engraftment and/or hematopoietic differentiation caused by the I164E mutation. Together, our results indicate a crucial role for the SH2-kinase domain interface in Bcr-Abl-mediated leukemic transformation.
Bcr-Abl I164E Confers Growth-Factor Independence to Ba/F3 and UT-7 Cells despite Reduced Kinase Activity
To assess cellular effects and downstream signaling of the Bcr-Abl I164E mutation, we engineered the murine IL-3-dependent cell line Ba/F3 and the human GM-CSF-dependent cell line UT-7 to express Bcr-Abl WT and Bcr-Abl I164E. In both cell lines, we consistently observed higher expression levels of Bcr-Abl I164E as compared to Bcr-Abl WT, despite equal virus titers used (A and data not shown). Nevertheless, global tyrosine phosphorylation was still lower in cells expressing Bcr-Abl I164E (A). Single-cell clone pairs, initially selected for equal expression levels of Bcr-Abl WT and I164E, quickly showed increased Bcr-Abl I164E expression within a few passages (data not shown).
Bcr-Abl I164E Renders Ba/F3 or UT-7 Cell Lines Factor Independent and Differentially Impacts Cellular Signaling Pathways
A widely used read-out of Bcr-Abl activity is the ability to confer cytokine-independent growth to Ba/F3 or UT-7 cells. Despite the complete lack of leukemogenic activity of Bcr-Abl I164E (see ), we observed that Bcr-Abl I164E was equally potent in transforming Ba/F3 or UT-7 cell lines to cytokine independence (B). A possible explanation is that the reduced kinase activity of the I164E mutant may be compensated by upregulation of the Bcr-Abl mutant protein levels in order to allow cytokine-independent proliferation of hematopoietic cell lines.
Analysis of Downstream Signaling Pathways
It is surprising that a single point mutation located outside of the tyrosine kinase domain and not affecting phosphotyrosine binding of the SH2 domain could have such a dramatic effect on the oncogenic activity of Bcr-Abl. Given this crucial function, we investigated the downstream signaling events that may be involved. We tested some major phosphorylation events in Ba/F3 cells, as well as in human U937 cells. As already observed in HEK293 cells, mutation of Ile164 led to a strong reduction of global tyrosine phosphorylation and phosphorylation of Tyr412 in the activation loop of Bcr-Abl (A and 4D). Important signaling mediators downstream of Bcr-Abl include the phosphorylation and activation of STAT5, Akt, and Erk1/2 (Ren, 2005
). We observed a strong reduction in tyrosine phosphorylation of STAT5 and CrkL in cells expressing Bcr-Abl I164E as compared to Bcr-Abl WT (C, 4D, and ). As activation of STAT5 is required for the induction and maintenance of CML by Bcr-Abl (Nieborowska-Skorska et al., 1999; Hoelbl et al., 2010
), it is tempting to speculate that the inability of Bcr-Abl I164E to induce CML may mainly be caused by its inability to activate STAT5.
Bcr-Abl I164E-Expressing Ba/F3 Cells Show Reduced Tyrosine Phosphorylation of Stat5 and CrkL, Related to
Surprisingly, despite the strong effect on Bcr-Abl activity, the I164E mutation had no effect on Erk1/2 and Akt phosphorylation or other MAPK or PI3K pathway members (C and 4D and data not shown). This is in line with comparable levels of phosphorylated Tyr177 in Bcr-Abl, which was shown to be critical for PI3K and MAPK pathway activation (B) (Sattler et al., 2002
). Thus, interference with the SH2-kinase domain interface appears to result in a complex rewiring of the signaling network with specific downregulated events rather than general attenuation of signaling, suggesting that uncoupling of the SH2-kinase module may generate a not only quantitatively but also qualitatively impaired abnormal Bcr-Abl output. Furthermore, these data are consistent with different thresholds in Bcr-Abl activity that are required for full signaling output, with STAT5 activation being a critical event that is highly sensitive to disruption of the SH2-kinase domain interface.
Disruption of the SH2-Kinase Domain Interface Sensitizes Bcr-Abl WT and Drug-Resistant Forms to TKI Inhibition
Pharmacological interference with the SH2-kinase domain interaction surface would be particularly attractive if it could alter the sensitivity of Bcr-Abl to existing TKIs. We tested the sensitivity of Bcr-Abl I164E toward the CML TKIs imatinib and dasatinib in in vitro kinase assays (A and 5B). Whereas imatinib and nilotinib exclusively bind Bcr-Abl when its activation loop is not phosphorylated, binding of dasatinib requires an active conformation (Schindler et al., 2000; Vajpai et al., 2008
). Therefore, different sensitivities to the two classes have been used by others and us to monitor different conformational states of the target enzymes (De Keersmaecker et al., 2008
). We observed a 4-fold increase in sensitivity of Bcr-Abl I164E for imatinib, whereas no differences were observed in response to dasatinib (A and 5B). In line with this, the sensitivity of Ba/F3 cells expressing Bcr-Abl I164E for nilotinib was increased 3-fold (C). We next addressed whether disruption of the SH2-kinase domain interface also sensitized TKI-resistant mutants to nilotinib inhibition. Introduction of the I164E mutation in the imatinib-resistant Bcr-Abl clones H396R or E255K also increased the nilotinib sensitivity of these mutants 3-fold (D and data not shown).
Bcr-Abl I164E Sensitized WT and Imatinib-Resistant Bcr-Abl Forms to TKI Inhibition
The T315I mutation in Bcr-Abl is the only mutation that is resistant to imatinib, nilotinib, and dasatinib. At concentrations that only marginally inhibited Bcr-Abl T315I, nilotinib caused a dramatic reduction in the in vitro tyrosine kinase activity in the Bcr-Abl T315I/I164E double mutant (E). Finally, the I164E mutation also increased the sensitivity of Bcr-Abl T315I-expressing Ba/F3 cells toward the allosteric myristate-binding pocket inhibitor GNF-2 (Zhang et al., 2010
and F). These results extend the recently proposed cooperativity between the myristate pocket and the ATP-binding site (Zhang et al., 2010
) to the SH2-kinase domain interface and indicate that disruption of the SH2-kinase domain interface not only sensitizes Bcr-Abl WT to imatinib/nilotinib but could also be used to enhance inhibition of TKI-resistant Bcr-Abl mutants.
Targeting of the Bcr-Abl SH2-Kinase Interface using Monobodies
To test whether the SH2-kinase interface can be targeted using an inhibitor in trans
, we generated single-domain binding proteins based on the fibronectin type III domain (FN3), termed monobodies, that target the Abl SH2 domain (Koide and Koide, 2007; Wojcik et al., 2010
). We have previously described the Abl SH2-binding monobody HA4, which acts as a competitive inhibitor of phosphotyrosine binding (Wojcik et al., 2010
). Using HA4 as a competitor in phage-disply library sorting, we identified an Abl SH2 monobody, designated 7c12, that bound to a distinct site. 7c12 binds the Abl SH2 domain with a dissociation constant (KD
) of ~50 nM ( and data not shown). In in vitro kinase assays, 7c12 inhibited kinase activity of Bcr-Abl WT and T315I, but not of Bcr-Abl I164E or Bcr-Abl T315I/I164E (A and ). Although the effect was mild, it was statistically significant and did not occur with a recombinant control protein, which does not bind the Abl SH2 domain. This suggested a possible involvement of the Bcr-Abl SH2-kinase domain interface in 7c12 binding. In fact, the I164E mutation reduced binding of 7c12 by a factor of ~400 (B).
Binding Parameters, Inhibition of Active Forms of Abl, Binding Properties, and Sequence of the SH2 Domain Monobody 7c12, Related to
Targeting the SH2-Kinase Domain Interface with the Engineered Monobody Protein 7c12 Leads to Bcr-Abl Inhibition
Crystal Structure of the Abl SH2 Domain-7c12 Monobody Complex
To elucidate the molecular details of the 7c12-Abl SH2 interaction, we determined the crystal structure of the complex at 2.1 Å resolution (PDB ID: 3T04; Table S1
; C and ). The asymmetric unit consists of a single monobody-SH2 domain complex (C and A). The 7c12 monobody and the Abl SH2 domain contribute nearly equally to the ~1340 Å2
of surface area buried in the complex. The diversified loops contribute nearly 75% of the binding interface with each of the three loops making substantial contributions (91, 178, and 234 Å2
for the BC, DE, and FG loops, respectively). The D strand of the monobody scaffold also makes substantial contribution to the interface. It packs against the C-terminal tail of the SH2 domain through an intermolecular β sheet (A, red box and B). Outside of these backbone-mediated interactions, the interface is dominated by hydrophobic contacts between monobody loop residues and two surface concavities on either side of β strand B of the SH2 domain. At the end of the monobody D strand, the DE loop forms a hairpin that packs against one of these concave surfaces that is formed by the βB strand and the αA helix of the Abl SH2 domain (C). Pro60 and Tyr62 make most of the DE loop contacts, together burying more than 150 Å2
of surface area.
Crystal Structure of the 7c12sm-Abl SH2 Domain Complex and Comparison to the Abl SH2-Kinase Domain Interface, Related to
The second surface concavity is bounded on one side by β strand B of the Abl SH2 domain, on the other by α helix B, and from above and below by the N-terminal and C-terminal tails of the Abl SH2 domain (A, cyan box). Phe87 of the 7c12 FG loop penetrates deep into the center of this pocket, burying 133 Å2 of surface area on its own. Other residues in the FG loop (Phe88, Pro89), Val38, and the aliphatic portion of Lys39 of the BC loop form a ring of hydrophobic contacts at the periphery of this surface pocket (D). Together, these residues contribute nearly 300 Å2 to total monobody surface area burial.
The interface observed in the crystal structure is also consistent with the strong negative effect of the I164E mutant on 7c12-SH2 interaction (B). Ile164 is located at the edge of the 7c12-binding interface and contributes 29 Å2 of surface to the interface. It makes hydrophobic interactions with Tyr62 of the DE loop (C). These features rationalize the ~400-fold reduction in binding affinity by the I164E mutation (B).
In order to further confirm the authenticity of the interaction interface observed in the crystal structure, we used nuclear magnetic resonance (NMR) spectroscopy to conduct epitope mapping. The Abl SH2 backbone amide resonances most greatly affected by the presence of 7c12, as measured in 15N HSQC experiments, are in good agreement with portions of the SH2 domain that are contacted by 7c12 in the crystal structure (F), suggesting that the interface observed in the crystal structure corresponds to the actual solution-phase interaction. To further validate the crystal structure using an independent experimental approach, we mutated each of the loop regions individually back to the template sequence and measured the binding affinity of these mutants. In each case, the mutant-Abl SH2 domain interaction was at least 100 times weaker than the wild-type 7c12-Abl SH2 interaction (data not shown). These results are consistent with the crystal structure, in which each loop makes a significant contribution to the 7c12-Abl SH2 interaction. Together, these studies provide significant validation of the observed crystal structure interface.
Comparison of the 7c12-Abl SH2 Interface to the Abl SH2-Kinase Domain Interface
We next compared the Abl SH2-kinase domain interface with the 7c12-Abl SH2 interface (C and ). The size of the SH2-kinase domain interface is ~1030 Å2, ~75% of that of the 7c12-Abl SH2 interface. More than half of the total surface area of the SH2-kinase interface overlaps with the 7c12-SH2 domain interface centered on β strand B of the Abl SH2 domain (G and S5H). This significant overlap in the 7c12-Abl SH2 and Abl SH2-kinase interfaces strongly suggests that the binding of the SH2 domain to the kinase domain and to 7c12 are mutually exclusive (C, G, and S5H). The incompatibility of simultaneous binding of the SH2 to both the kinase domain and 7c12, therefore, likely explains the observed inhibition of Bcr-Abl kinase activity, as full 7c12 binding requires the disruption of the SH2-kinase domain interface. Given the theoretical difficulty of competing intermolecularly with an ~1100 Å2 intramolecular protein-protein interaction and the partial occlusion of the 7c12-binding site on the SH2 domain by the kinase domain, we consider the degree of inhibition that is achieved by the 7c12 monobody to be symptomatic and strongly indicative of a specific vulnerability.
Improved Targeting of the SH2 Interface with a Tandem Fusion of the HA4 and 7c12 Monobodies
Despite the clear demonstration of the general feasibility of targeting the SH2-kinase interface using 7c12, we aimed at improving its biological potency. Superposition of the structure of the previously characterized HA4 monobody bound to the Abl SH2 domain (Wojcik et al., 2010
) with the 7c12-Abl SH2 structure reveals that the two monobodies bind on opposite faces of the SH2 domain with no overlap (D and E). This arrangement is consistent with the selection scheme employed for the generation of 7c12, in which HA4 was used as a competitor, and with sandwich-ELISA data indicating that HA4 and 7c12 can bind simultaneously to the same Abl SH2 domain molecule (data not shown). HA4 binds with low nanomolar affinity to the phosphotyrosine-binding pocket of the Abl SH2 domain, which is not part of an intramolecular interaction in Bcr-Abl and is able to effectively outcompete phosphotyrosine ligand binding (Wojcik et al., 2010
). For these reasons, we attempted to link HA4 and 7c12 in tandem, which should increase the local concentration of 7c12 in proximity to the SH2-kinase domain interface and further enhance the specificity toward the Abl SH2 domain. A 19 amino acid Gly-Ser linker was used to bridge the C terminus of HA4 to the N terminus of 7c12, based on structural modeling (D). As negative controls, we introduced point mutations in both parts of the HA4-7c12 tandem monobody. For HA4, we used the Y88A (Y87A in the original numbering scheme) mutation that was previously shown to reduce binding to the Abl SH2 domain >1000-fold (Wojcik et al., 2010
). We also designed a double point mutation (Y172E/F179K; Y62E/F87K in the original numbering scheme, ) for the 7c12 part, which is located at structurally central positions of the 7c12-Abl SH2 interface and showed a strong reduction in binding in pull-down experiments (data not shown). We tested the HA4-7c12 tandem monobody in Bcr-Abl in vitro kinase assays. We observed a strong inhibition of Bcr-Abl activity with low micromolar concentrations of the HA4-7c12 tandem monobody, whereas the nonbinding Y88A/Y172E/F179K mutant failed to inhibit Bcr-Abl activity (E). Importantly, the degree of inhibition of Bcr-Abl activity achieved by HA4-7c12 was comparable to that upon introduction of the I164E mutant (Figures C and A). HA4-7c12 was also able to inhibit Bcr-Abl T231R, although to a lesser degree than Bcr-Abl WT (). This indicates that the tandem monobody may lead to a complete disruption of the Bcr-Abl kinase domain interface.
The T231R Mutation in the SH2-Kinase Interface Partially Blocks Inhibition of Bcr-Abl Activity by the HA4-7c12 Tandem Monobody, Related to
The HA4-7c12 Tandem Monobody Strongly Inhibits Cellular Bcr-Abl Activity and Induces Apoptosis in CML Cells
Finally, we tested whether the HA4-7c12 tandem monobody would also inhibit Bcr-Abl activity in CML cells. Transient expression of a HA4-7c12-GFP fusion protein in the Bcr-Abl-positive CML cell line K562 led to a strong reduction of activation loop (Tyr412) phosphorylation of Bcr-Abl (A). This effect was dependent on both HA4 and 7c12 moieties of the tandem monobody, as expression of point mutants that abolish binding of either HA4 (Y88A) or 7c12 (Y172E/F197K) or both to the Abl SH2 domain did not show this effect (A).
The Tandem Monobody HA4-7c12 Strongly Inhibits Cellular Bcr-Abl Kinase Activity and Induces Apoptosis in CML Cell Lines and Primary Cells
We next tested whether the HA4-7c12-induced decrease in Bcr-Abl activity with HA4-7c12 would result in cell death. Indeed, K562 cells expressing the HA4-7c12 tandem monobody showed high amounts of apoptosis, as measured by TUNEL and cleaved caspase 3 staining (B and A). Again, this effect was not evident when the nonbinding mutant was used.
The Tandem Monobody HA4-7c12 Induces Apoptosis in CML Cell Lines and Primary Cells from CML Patients, Related to
Next, we asked whether HA4-7c12 would inhibit Bcr-Abl-induced transformation of primary murine bone marrow cells. In contrast to the nonbinding mutants, bicistronic expression of Bcr-Abl and GFP-HA4-7c12 WT severely impaired cytokine-independent colony formation in semisolid media (C).
Finally, we tested whether HA4-7c12 would induce apoptosis in primary bone marrow or peripheral blood cells from CML patients at different stages of the disease (Table S2
). Lentiviral expression of HA4-7c12 induced an ~65% increase in apoptotic cells over the nonbinding mutant HA4 Y88A-7c12-Y172E/F197K in cells from patients in chronic phase (D, left panel). This increase was comparable to the induction of apoptosis upon treatment of the same cultures with 2 μM nilotinib for 5 days (41%). Similar results were obtained when cells from patients in the accelerated phase of the disease were used (D, right panel). Here, nilotinib treatment led to a 40% increase whereas expression of HA4-7c12 induced a 49% increase in apoptosis.
Together, these data show that Bcr-Abl activity can be inhibited by targeting the SH2-kinase interface using FN3-based monobodies in CML cell lines, as well as in primary cells from CML patients.