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Induction of apoptosis by directing oncogenic Bcr-Abl into the nucleus.

Huang ZL, Gao M, Li QY, Tao K, Xiao Q, Cao WX, Feng WL - Oncotarget (2013)

Bottom Line: We show that a rapalog nuclear transport system (RNTS) containing six nuclear localization signals directs Bcr-Abl into the nucleus and that nuclear entrapped Bcr-Abl induces apoptosis and inhibits proliferation of CML cells by activating p73 and shutting down cytoplasmic oncogenic signals mediated by Bcr-Abl.Coupling cytoplasmic depletion with nuclear entrapment of Bcr-Abl synergistically enhances the inhibitory effect of nuclear Bcr-Abl on its oncogenicity in mice.These results provide evidence that direction of cytoplasmic Bcr-Abl to the nucleus offers an alternative CML therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, Chongqing, People's Republic of China.

ABSTRACT
The chimeric Bcr-Abl oncoprotein, which causes chronic myeloid leukemia, mainly localizes in the cytoplasm, and loses its ability to transform cells after moving into the nucleus. Here we report a new strategy to convert Bcr-Abl to be an apoptotic inducer by altering its subcellular localization. We show that a rapalog nuclear transport system (RNTS) containing six nuclear localization signals directs Bcr-Abl into the nucleus and that nuclear entrapped Bcr-Abl induces apoptosis and inhibits proliferation of CML cells by activating p73 and shutting down cytoplasmic oncogenic signals mediated by Bcr-Abl. Coupling cytoplasmic depletion with nuclear entrapment of Bcr-Abl synergistically enhances the inhibitory effect of nuclear Bcr-Abl on its oncogenicity in mice. These results provide evidence that direction of cytoplasmic Bcr-Abl to the nucleus offers an alternative CML therapy.

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RNTS directs Bcr-Abl from cytoplasm into the nucleus
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Figure 1: RNTS directs Bcr-Abl from cytoplasm into the nucleus

Mentions: Three NLSs were fused to FRBT2098L with FLAG tag incorporated to the N-terminus of the protein, which is termed F3NF. Two FKBP domains were in tandem and fused to ABD (Abl binding domain of RIN1) with HA tag fused to the N-terminus, which is termed H2FA (Figure 1A). The ABD of RIN1 interacts with both the SH3 and SH2 domains of Bcr-Abl specifically with high affinity. Because all three potential tyrosine phosphorylation sites Y36, Y121, and Y148 at ABD N-terminus are critical for ABD binding to Bcr-Abl, all three sites were mutated to phenylalanine as a triple mutant, which is termed ABD™[23]. Heterodimerization was induced between F3NF and H2FA upon AP21967. Nuclear location signal was transferred from F3NF to H2FA, then to Bcr-Abl (Figure 1B). This strategy is called rapalog nuclear transport system (RNTS). As expected, recombinant F3NF localized mainly in the nucleus, and both H2FA and H2FA™ localized in the cytoplasm (Supplemental Figure 1). In K562 cells, Bcr-Abl located mostly in the cytoplasm. When RNTS was introduced, Bcr-Abl was transported into the nucleus significantly. To retain Bcr-Abl in the nucleus, LMB was added, because LMB is a potent and specific nuclear export inhibitor which functions by inhibiting CRM1/exportin 1, a protein required for nuclear export of NES-containing proteins[24]. When both RNTS and LMB were used, Bcr-Abl was mostly transported into the nucleus (Figure 1C). The amount of Bcr-Abl in the nucleus and cytoplasm was quantified by western blot separately. We found that the level of nuclear Bcr-Abl increased with decreased Bcr-Abl in the cytoplasm (Figure 1D). These results demonstrate that RNTS can direct Bcr-Abl from the cytoplasm and into the nucleus.


Induction of apoptosis by directing oncogenic Bcr-Abl into the nucleus.

Huang ZL, Gao M, Li QY, Tao K, Xiao Q, Cao WX, Feng WL - Oncotarget (2013)

RNTS directs Bcr-Abl from cytoplasm into the nucleus
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926824&req=5

Figure 1: RNTS directs Bcr-Abl from cytoplasm into the nucleus
Mentions: Three NLSs were fused to FRBT2098L with FLAG tag incorporated to the N-terminus of the protein, which is termed F3NF. Two FKBP domains were in tandem and fused to ABD (Abl binding domain of RIN1) with HA tag fused to the N-terminus, which is termed H2FA (Figure 1A). The ABD of RIN1 interacts with both the SH3 and SH2 domains of Bcr-Abl specifically with high affinity. Because all three potential tyrosine phosphorylation sites Y36, Y121, and Y148 at ABD N-terminus are critical for ABD binding to Bcr-Abl, all three sites were mutated to phenylalanine as a triple mutant, which is termed ABD™[23]. Heterodimerization was induced between F3NF and H2FA upon AP21967. Nuclear location signal was transferred from F3NF to H2FA, then to Bcr-Abl (Figure 1B). This strategy is called rapalog nuclear transport system (RNTS). As expected, recombinant F3NF localized mainly in the nucleus, and both H2FA and H2FA™ localized in the cytoplasm (Supplemental Figure 1). In K562 cells, Bcr-Abl located mostly in the cytoplasm. When RNTS was introduced, Bcr-Abl was transported into the nucleus significantly. To retain Bcr-Abl in the nucleus, LMB was added, because LMB is a potent and specific nuclear export inhibitor which functions by inhibiting CRM1/exportin 1, a protein required for nuclear export of NES-containing proteins[24]. When both RNTS and LMB were used, Bcr-Abl was mostly transported into the nucleus (Figure 1C). The amount of Bcr-Abl in the nucleus and cytoplasm was quantified by western blot separately. We found that the level of nuclear Bcr-Abl increased with decreased Bcr-Abl in the cytoplasm (Figure 1D). These results demonstrate that RNTS can direct Bcr-Abl from the cytoplasm and into the nucleus.

Bottom Line: We show that a rapalog nuclear transport system (RNTS) containing six nuclear localization signals directs Bcr-Abl into the nucleus and that nuclear entrapped Bcr-Abl induces apoptosis and inhibits proliferation of CML cells by activating p73 and shutting down cytoplasmic oncogenic signals mediated by Bcr-Abl.Coupling cytoplasmic depletion with nuclear entrapment of Bcr-Abl synergistically enhances the inhibitory effect of nuclear Bcr-Abl on its oncogenicity in mice.These results provide evidence that direction of cytoplasmic Bcr-Abl to the nucleus offers an alternative CML therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, Chongqing, People's Republic of China.

ABSTRACT
The chimeric Bcr-Abl oncoprotein, which causes chronic myeloid leukemia, mainly localizes in the cytoplasm, and loses its ability to transform cells after moving into the nucleus. Here we report a new strategy to convert Bcr-Abl to be an apoptotic inducer by altering its subcellular localization. We show that a rapalog nuclear transport system (RNTS) containing six nuclear localization signals directs Bcr-Abl into the nucleus and that nuclear entrapped Bcr-Abl induces apoptosis and inhibits proliferation of CML cells by activating p73 and shutting down cytoplasmic oncogenic signals mediated by Bcr-Abl. Coupling cytoplasmic depletion with nuclear entrapment of Bcr-Abl synergistically enhances the inhibitory effect of nuclear Bcr-Abl on its oncogenicity in mice. These results provide evidence that direction of cytoplasmic Bcr-Abl to the nucleus offers an alternative CML therapy.

Show MeSH
Related in: MedlinePlus