Interplay between kinase domain autophosphorylation and F-actin binding domain in regulating imatinib sensitivity and nuclear import of BCR-ABL

Abstract

Background: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood.

Methodology/principal findings: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib.

Conclusions/significance: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML.

Figure 1. Kinase activity of BCR-ABL inhibits its nuclear import.

A: Domain structure of ABL, the BCR-ABL p210 and p185 fusion proteins, and the minimal BCR₆₃-ABL used in this study. All numbering used herein refers to amino acid positions in the human ABL-1a isoform. The kinase-defective (KD) constructs bear a lysine-to-histidine substitution (K271H) in the ATP-binding site, which renders the kinase catalytically inactive. Abbreviations used are: SH3, src-homology 3; SH2 src-homology 2; FABD, F-actin binding domain; NLS, nuclear localization signal; NES nuclear export signal; cc, coiled-coil oligomerization domain; KD, kinase-defective. B and C: COS cells ectopically expressing active BCR₆₃-ABL (B) or the kinase-defective mutant (BCR₆₃-ABL^KD) (C) were treated with the CRM1-inhibitor LMB (10 nM) for either 1 or 6 hours, which leads to accumulation in the nuclei of cells only if the protein is imported. The presence of nuclear staining in LMB-treated cells demonstrates that the protein is imported. Cells displaying notable nuclear staining (resulting from nuclear import) of BCR-ABL are marked with white arrows. The BCR-ABL kinase activity was also blocked by treatment with the kinase inhibitors imatinib (10 µM) or PD166326 (10 nM) for 16 hours to enable nuclear import. BCR-ABL localization was determined by immunofluorescence staining with an anti-ABL antibody (8E9, shown in red). The endogenous ABL was not observed under the experimental conditions, which were designed to detect only the ectopically expressed proteins that were present at a much higher abundance than the endogenous ABL protein. DNA is counterstained in blue with Hoechst dye.

Figure 2. Trans-phosphorylation of kinase-defective BCR-ABL blocks its nuclear import.

A: Scheme of experimental design. Kinase-defective BCR₆₃-ABL constructs were co-transfected with kinase active p185-BCR-ABL to induce tyrosine phosphorylation of the kinase-defective protein. B: BCR₆₃-ABL^KD constructs were immunoprecipitated with an anti-HA antibody from COS cells that were co-transfected with the indicated plasmids. Immunoblots from HA-pulldowns (top) and total cell lysates (bottom) were probed with the indicated antibodies to detect the tyrosine phosphorylation of BCR₆₃-ABL^KD. The previously described β53-BCR₆₃-ABL^KD has a beta-turn inserted at position 53, which disables the coiled-coil oligomerization domain . C: COS cells were transfected with the indicated HA-tagged, kinase-defective BCR₆₃-ABL^KD constructs either alone or in co-transfection with a kinase-active p185-BCR-ABL. The localization of the kinase-defective BCR₆₃-ABL proteins was detected by immunostaining with an anti-HA antibody (red).

Figure 3. Mutation of nine tyrosines in BCR₆₃-ABL does not restore nuclear import.

A: In the BCR₆₃-ABL^9Y/F protein, nine autophosphorylation sites are mutated to phenylalanines. The position and amino acid number (according to that of ABL-1a) of the Tyr/Phe (Y/F) substitutions are indicated in the schematic drawing. B: The BCR₆₃-ABL^9Y/F protein and the BCR₆₃-ABL protein were immunoprecipitated from transfected cells. The levels of phosphotyrosine and the BCR₆₃-ABL protein were detected by immunoblotting from immunoprecipitates (top) and whole cell lysates (bottom) using monoclonal antibodies 4G10 (for phosphotyrosine) and 8E9 (for ABL). C: HA-tagged kinase-defective BCR₆₃-ABL or a corresponding 9Y/F-mutant were co-transfected with kinase-active p185-BCR-ABL to allow for trans-phosphorylation. The kinase-defective proteins were immunoprecipitated using an anti-HA antibody, and immunoblotted as in (B). D: The phosphorylation site mutant BCR₆₃-ABL^9Y/F was transfected in COS cells and its localization determined by immunofluorescence after treatment with 10 nM LMB for 6 hours, or 10 µM imatinib and LMB. Nuclear localization was only observed after treatment with imatinib and LMB, as indicated by the solid white arrows.

Figure 4. Mutation of Tyr232, Tyr253, and Tyr257 to phenylalanine blocks nuclear import of kinase-defective BCR₆₃-ABL.

A: Schematic representation of BCR₆₃-ABL^9Y/F-KD and BCR₆₃-ABL^3Y/F-KD constructs. The constructs encode catalytically inactive proteins due to the KD mutation (Lys271His) in the kinase domain. The position and amino acid number of the 9Y/F and the 3Y/F substitutions are indicated in the scheme according to the ABL-1a amino acid numbering. B: The kinase-defective BCR₆₃-ABL^9Y/F-KD construct was transfected into COS cells and the localization of the protein determined by immunofluorescence. Nuclear localization of the kinase-defective protein was only observed in cells treated with 10 µM imatinib and 10 nM LMB, as indicated by the white arrows. C: The gatekeeper mutation (T315I), which prevents imatinib from binding to the ATP binding pocket of BCR-ABL, was introduced into the BCR₆₃-ABL^9Y/F-KD backbone to test whether imatinib enables the nuclear import of the kinase-defective protein through direct binding to its kinase domain. The respective construct (BCR₆₃-ABL^{9Y/F-KD-T315I}) was transfected into COS cells and the subcellular localization of the ectopically expressed protein examined by immunofluorescence. No nuclear localization of the protein was detected in the absence or presence of imatinib and LMB. D: The BCR₆₃-ABL^3Y/F-KD protein, in which the three tyrosines Y232, Y253, and Y257 are mutated to phenylalanines, was expressed in COS cells. The subcellular localization was again determined by immunofluorescence in untreated cells, as well as after the treatment with imatinib and LMB. Dashed arrows indicate minimal nuclear staining in the absence of imatinib, white arrows point to cells showing predominately nuclear localization in the presence of imatinib.

Figure 5. Phosphomimetic mutation of either Tyr232, Tyr253, or Tyr257 blocks nuclear import of kinase-defective BCR₆₃-ABL.

A: Position of tyrosines 232, 253, and 257 as seen in a crystal structure of the ABL kinase domain bound to imatinib (, PDB code 1IEP). The Tyr253 and Tyr257 are in the P-loop (yellow), and are engaged in interactions with other P-loop side chains (Gln252 and Glu255) in this structure. Tyr232 is located in the SH2-kinase linker region and situated right above the kinase N-lobe in this structure. B: BCR₆₃-ABL^KD, in which either Tyr232, 253 or 257 was mutated to glutamic acid, was transiently expressed in COS cells. The localization of each of these three Y/E-mutant proteins after the indicated treatments with LMB and imatinib was determined by immunofluorescence. Merged images of BCR-ABL staining (red) and DNA (blue) are shown. Cells displaying nuclear staining are marked by the white arrows.

Figure 6. The FABD but not binding to actin filaments is required to block import of BCR₆₃-ABL.

A: Schematic representation of the constructs used. BCR₆₃-ABL was either truncated by introducing a stop codon at the indicated amino acid positions or a point mutation (F1081E) within helix-3 of the actin-binding domain (FABD) to abolish binding to filamentous actin. B: NMR structure of the FABD of ABL (, PDB code 1ZZP). The C-terminal residues that are deleted in the truncation mutants Δ1080, Δ1121 and Δ1127 are highlighted in yellow, orange, and red, respectively. The phenyl side-chain of F1081 in helix-3 (αIII), which was mutated to glutamic acid, is shown in white. C, D and E: COS cells expressing the indicated BCR₆₃-ABL mutants were left untreated or treated with LMB as indicated. Merged images of anti-ABL staining show the respective mutant BCR₆₃-ABL in red and DNA in blue. Cells showing nuclear BCR₆₃-ABL staining are marked by the white arrows. F: COS cells were transfected with BCR₆₃-ABL or the indicated mutants and processed for immunofluorescence. Images of anti-ABL staining (red) and F-actin counterstained with Alexa-488-conjugated phalloidin (green) are shown individually, and merged with DNA (blue) images. Co-localization of BCR₆₃-ABL with actin fibers results in yellow color in the merged images.

Figure 7. Kinase activity and imatinib-sensitivity of BCR₆₃-ABL mutants.

A and B: COS cells were transfected with BCR₆₃-ABL or the indicated mutant constructs. The cells were left untreated or treated with 10 µM imatinib (A) or different doses of imatinib (B) for 16 hours to inhibit BCR-ABL kinase activity. Immunoblotting of whole cell lysates with an antibody (4G10) against phophostyrosine (pTyr) was used to indicate the levels of the BCR-ABL tyrosine kinase activity. The levels of the BCR₆₃-ABL protein were determined by immunoblotting with an anti-ABL antibody (8E9). The levels of tubulin were shown as a loading control. The positions of the molecular weight markers (in kilodalton) are indicated at the left of the blot.

Figure 8. A model for the regulation of BCR-ABL nuclear import through conformational interplay between the kinase domain, the FABD and the NLS region.

(i) Tyrosine phosphorylation at Y232, Y253 or Y257 causes the kinase domain to adopt a conformation that affects the folding of the C-terminal region and leading to the inhibition of the NLS function (indicated by the red color of the three nuclear localization signals depicted as small circles embedded in a proline-rich linker between the kinase domain and the F-actin binding domain, FABD). The kinase domain autophosphorylation-induced occlusion of the NLS also requires the C-terminal region beyond the third NLS (NLS-3) and including an intact helix-3 of the FABD. Binding of imatinib reverts the kinase domain back to the “DFG-Asp out” N-lobe conformation that alters the folding of the C-terminal region to un-mask the NLS (indicated by the green color of the three nuclear localization signals). (ii) Deletion of C-terminal sequences beyond the NLS-3 unmasks the NLS despite the kinase domain autophosphorylation. (iii) Mutation of Y232, Y253 or Y257 to glutamic acid (E) also alters the kinase domain conformation to trigger the inhibition of the NLS function. The NLS-inhibitory effect of the tyrosine to glutamic acid substitutions can be observed in a kinase-defective BCR₆₃-ABL. Binding of imatinib induces the “DFG-Asp out” conformation of the kinase domain , and this imatinib-bound conformation can override the effect of the glutamic acid substitution to re-activate the NLS function.