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Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia.

Suzuki M, Yamagata K, Shino M, Aikawa Y, Akashi K, Watanabe T, Kitabayashi I - Cancer Sci. (2014)

Bottom Line: The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies.Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia.These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan.

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Related in: MedlinePlus

The NES region within clathrin assembly lymphoid myeloid leukemia protein (CALM) is necessary for cytoplasmic localization. (a) Schematic representations of CALM, AF10, CALM-AF10 and mutant proteins. FLAG-CALMNES4A-AF10 and FLAG-CALMNES4A were generated by alanine substitution of three leucine residues and one isoleucine residue in the putative CALM NES (red). FLAG-NES1-AF10 and FLAG-NES2-AF10 mutants were constructed by fusion of the NES sequences of CALM to the AF10 portion of CALM-AF10. (b) Diagrams of the plasmid constructs used in transfection experiments. (c) Subcellular distribution of CALM-AF10 and the NES point mutations FLAG-CALM-AF10, FLAG-CALM, FLAG-CALMNES4A-AF10, FLAG-CALMNES4A, and FLAG-NES1-AF10, FLAG-NES2-AF10 and FLAG-mAF10 in COS-7 cells. Transfected cells were stained with anti-FLAG antibody (green) and observed by fluorescence microscopy. (d) Population of cells expressing transduced genes in the nucleus and cytoplasm shown in (c). (e) Subcellular distribution of CALM-AF10 and NES mutation proteins in murine bone-marrow cells. Transduced cells were stained with anti-FLAG antibody (red). (f) Population of cells expressing transduced genes in the nucleus and the cytoplasm shown in (e). Nuclei were stained with DAPI (blue) and observed by fluorescence microscopy. The scale bar represents 20 μm in (c) and 10 μm in (e). ANTH, AP180 N-terminal homology domain binding phosphatidylinositol 4,5-bisphosphate (PIP2); DIF and DPF, motifs interacts with AP-2; NPF, a motif interacts with the EH (Eps15 homology) domain; CBS-I and -II, putative type I and II clathrin-binding sequences; NES, nuclear export signal; PHD Type1 and 2, plant homeodomain zinc finger domains; NLS, nuclear localization signal; AT-hook, DNA-binding protein motif; OMLZ, octapeptide motif-leucine zipper domain; Q-rich, glutamine-rich region.
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fig01: The NES region within clathrin assembly lymphoid myeloid leukemia protein (CALM) is necessary for cytoplasmic localization. (a) Schematic representations of CALM, AF10, CALM-AF10 and mutant proteins. FLAG-CALMNES4A-AF10 and FLAG-CALMNES4A were generated by alanine substitution of three leucine residues and one isoleucine residue in the putative CALM NES (red). FLAG-NES1-AF10 and FLAG-NES2-AF10 mutants were constructed by fusion of the NES sequences of CALM to the AF10 portion of CALM-AF10. (b) Diagrams of the plasmid constructs used in transfection experiments. (c) Subcellular distribution of CALM-AF10 and the NES point mutations FLAG-CALM-AF10, FLAG-CALM, FLAG-CALMNES4A-AF10, FLAG-CALMNES4A, and FLAG-NES1-AF10, FLAG-NES2-AF10 and FLAG-mAF10 in COS-7 cells. Transfected cells were stained with anti-FLAG antibody (green) and observed by fluorescence microscopy. (d) Population of cells expressing transduced genes in the nucleus and cytoplasm shown in (c). (e) Subcellular distribution of CALM-AF10 and NES mutation proteins in murine bone-marrow cells. Transduced cells were stained with anti-FLAG antibody (red). (f) Population of cells expressing transduced genes in the nucleus and the cytoplasm shown in (e). Nuclei were stained with DAPI (blue) and observed by fluorescence microscopy. The scale bar represents 20 μm in (c) and 10 μm in (e). ANTH, AP180 N-terminal homology domain binding phosphatidylinositol 4,5-bisphosphate (PIP2); DIF and DPF, motifs interacts with AP-2; NPF, a motif interacts with the EH (Eps15 homology) domain; CBS-I and -II, putative type I and II clathrin-binding sequences; NES, nuclear export signal; PHD Type1 and 2, plant homeodomain zinc finger domains; NLS, nuclear localization signal; AT-hook, DNA-binding protein motif; OMLZ, octapeptide motif-leucine zipper domain; Q-rich, glutamine-rich region.

Mentions: To investigate the role of subcellular localization of CALM-AF10 in leukemogenesis, we focused on the NES within the CALM portion of the fusion protein (amino acids 543–554 of CALM).9 We generated NES-deficient mutants CALMNES4A-AF10 and CALMNES4A, in which leucine-544, leucine-547, leucine-551 and isoleucine-553 in the putative NES region within CALM were substituted with alanines (NES4A) (Fig.1a). Expression vectors for FLAG-tagged CALM-AF10, CALM, CALMNES4A-AF10, CALMNES4A and mAF10 (the AF10 portion of CALM-AF10) were transiently transfected into COS-7 cells. Immunofluorescence analysis revealed that CALM and CALM-AF10 primarily localized in the cytoplasm, whereas mAF10 and the NES mutants CALMNES4A-AF10 and CALMNES4A localized in the nucleus (Fig.1b,c).


Nuclear export signal within CALM is necessary for CALM-AF10-induced leukemia.

Suzuki M, Yamagata K, Shino M, Aikawa Y, Akashi K, Watanabe T, Kitabayashi I - Cancer Sci. (2014)

The NES region within clathrin assembly lymphoid myeloid leukemia protein (CALM) is necessary for cytoplasmic localization. (a) Schematic representations of CALM, AF10, CALM-AF10 and mutant proteins. FLAG-CALMNES4A-AF10 and FLAG-CALMNES4A were generated by alanine substitution of three leucine residues and one isoleucine residue in the putative CALM NES (red). FLAG-NES1-AF10 and FLAG-NES2-AF10 mutants were constructed by fusion of the NES sequences of CALM to the AF10 portion of CALM-AF10. (b) Diagrams of the plasmid constructs used in transfection experiments. (c) Subcellular distribution of CALM-AF10 and the NES point mutations FLAG-CALM-AF10, FLAG-CALM, FLAG-CALMNES4A-AF10, FLAG-CALMNES4A, and FLAG-NES1-AF10, FLAG-NES2-AF10 and FLAG-mAF10 in COS-7 cells. Transfected cells were stained with anti-FLAG antibody (green) and observed by fluorescence microscopy. (d) Population of cells expressing transduced genes in the nucleus and cytoplasm shown in (c). (e) Subcellular distribution of CALM-AF10 and NES mutation proteins in murine bone-marrow cells. Transduced cells were stained with anti-FLAG antibody (red). (f) Population of cells expressing transduced genes in the nucleus and the cytoplasm shown in (e). Nuclei were stained with DAPI (blue) and observed by fluorescence microscopy. The scale bar represents 20 μm in (c) and 10 μm in (e). ANTH, AP180 N-terminal homology domain binding phosphatidylinositol 4,5-bisphosphate (PIP2); DIF and DPF, motifs interacts with AP-2; NPF, a motif interacts with the EH (Eps15 homology) domain; CBS-I and -II, putative type I and II clathrin-binding sequences; NES, nuclear export signal; PHD Type1 and 2, plant homeodomain zinc finger domains; NLS, nuclear localization signal; AT-hook, DNA-binding protein motif; OMLZ, octapeptide motif-leucine zipper domain; Q-rich, glutamine-rich region.
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Related In: Results  -  Collection

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fig01: The NES region within clathrin assembly lymphoid myeloid leukemia protein (CALM) is necessary for cytoplasmic localization. (a) Schematic representations of CALM, AF10, CALM-AF10 and mutant proteins. FLAG-CALMNES4A-AF10 and FLAG-CALMNES4A were generated by alanine substitution of three leucine residues and one isoleucine residue in the putative CALM NES (red). FLAG-NES1-AF10 and FLAG-NES2-AF10 mutants were constructed by fusion of the NES sequences of CALM to the AF10 portion of CALM-AF10. (b) Diagrams of the plasmid constructs used in transfection experiments. (c) Subcellular distribution of CALM-AF10 and the NES point mutations FLAG-CALM-AF10, FLAG-CALM, FLAG-CALMNES4A-AF10, FLAG-CALMNES4A, and FLAG-NES1-AF10, FLAG-NES2-AF10 and FLAG-mAF10 in COS-7 cells. Transfected cells were stained with anti-FLAG antibody (green) and observed by fluorescence microscopy. (d) Population of cells expressing transduced genes in the nucleus and cytoplasm shown in (c). (e) Subcellular distribution of CALM-AF10 and NES mutation proteins in murine bone-marrow cells. Transduced cells were stained with anti-FLAG antibody (red). (f) Population of cells expressing transduced genes in the nucleus and the cytoplasm shown in (e). Nuclei were stained with DAPI (blue) and observed by fluorescence microscopy. The scale bar represents 20 μm in (c) and 10 μm in (e). ANTH, AP180 N-terminal homology domain binding phosphatidylinositol 4,5-bisphosphate (PIP2); DIF and DPF, motifs interacts with AP-2; NPF, a motif interacts with the EH (Eps15 homology) domain; CBS-I and -II, putative type I and II clathrin-binding sequences; NES, nuclear export signal; PHD Type1 and 2, plant homeodomain zinc finger domains; NLS, nuclear localization signal; AT-hook, DNA-binding protein motif; OMLZ, octapeptide motif-leucine zipper domain; Q-rich, glutamine-rich region.
Mentions: To investigate the role of subcellular localization of CALM-AF10 in leukemogenesis, we focused on the NES within the CALM portion of the fusion protein (amino acids 543–554 of CALM).9 We generated NES-deficient mutants CALMNES4A-AF10 and CALMNES4A, in which leucine-544, leucine-547, leucine-551 and isoleucine-553 in the putative NES region within CALM were substituted with alanines (NES4A) (Fig.1a). Expression vectors for FLAG-tagged CALM-AF10, CALM, CALMNES4A-AF10, CALMNES4A and mAF10 (the AF10 portion of CALM-AF10) were transiently transfected into COS-7 cells. Immunofluorescence analysis revealed that CALM and CALM-AF10 primarily localized in the cytoplasm, whereas mAF10 and the NES mutants CALMNES4A-AF10 and CALMNES4A localized in the nucleus (Fig.1b,c).

Bottom Line: The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies.Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia.These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus