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

Preyer M, Vigneri P, Wang JY - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology and Moores Cancer Center, Department of Medicine, University of California San Diego School of Medicine, La Jolla, California, United States of America.

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.

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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-ABLKD 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-ABLKD. The previously described β53-BCR63-ABLKD has a beta-turn inserted at position 53, which disables the coiled-coil oligomerization domain [6]. C: COS cells were transfected with the indicated HA-tagged, kinase-defective BCR63-ABLKD 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).
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pone-0017020-g002: 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-ABLKD 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-ABLKD. The previously described β53-BCR63-ABLKD has a beta-turn inserted at position 53, which disables the coiled-coil oligomerization domain [6]. C: COS cells were transfected with the indicated HA-tagged, kinase-defective BCR63-ABLKD 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).

Mentions: The kinase-defective BCR63-ABLKD, which is catalytically inactive through Lys271His (Lys290 in ABL-1b numbering) substitution in the kinase domain [32], was predominantly cytoplasmic in COS cells (Figure 1C), but became partially nuclear after 1 hour LMB treatment (Figure 1C) and mostly nuclear after 6 hours LMB exposure (Figure 1C and 2C). This demonstrates that BCR63-ABLKD, similar to BCR-ABLKD [22](supplementary Figure S1), can undergo nucleo-cytoplasmic shutting, and the continuous nuclear import allows its nuclear accumulation when export is blocked by LMB.


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

Preyer M, Vigneri P, Wang JY - PLoS ONE (2011)

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-ABLKD 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-ABLKD. The previously described β53-BCR63-ABLKD has a beta-turn inserted at position 53, which disables the coiled-coil oligomerization domain [6]. C: COS cells were transfected with the indicated HA-tagged, kinase-defective BCR63-ABLKD 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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037956&req=5

pone-0017020-g002: 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-ABLKD 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-ABLKD. The previously described β53-BCR63-ABLKD has a beta-turn inserted at position 53, which disables the coiled-coil oligomerization domain [6]. C: COS cells were transfected with the indicated HA-tagged, kinase-defective BCR63-ABLKD 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).
Mentions: The kinase-defective BCR63-ABLKD, which is catalytically inactive through Lys271His (Lys290 in ABL-1b numbering) substitution in the kinase domain [32], was predominantly cytoplasmic in COS cells (Figure 1C), but became partially nuclear after 1 hour LMB treatment (Figure 1C) and mostly nuclear after 6 hours LMB exposure (Figure 1C and 2C). This demonstrates that BCR63-ABLKD, similar to BCR-ABLKD [22](supplementary Figure S1), can undergo nucleo-cytoplasmic shutting, and the continuous nuclear import allows its nuclear accumulation when export is blocked by LMB.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology-Oncology and Moores Cancer Center, Department of Medicine, University of California San Diego School of Medicine, La Jolla, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus