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A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met.

Chaudhary SC, Cho MG, Nguyen TT, Park KS, Kwon MH, Lee JH - Exp. Mol. Med. (2014)

Bottom Line: This substitution also decreased the association of c-Met fragment with importin β.The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS.Indeed, decreasing the cytosolic pH either with nigericin, an Na(+)/H(+) exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low pH itself enhanced nuclear translocation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea [2] Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, Korea.

ABSTRACT
The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin β. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na(+)/H(+) exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.

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The C-terminal fragment of c-Met localizes in the nuclei of HeLa cells. (a) Sub-confluent HeLa cells were fixed and stained with C-terminal antibody for c-Met (C-28, green) and DAPI for nucleus (blue). Representative images show the nuclear patches of c-Met. Size bar,10 μm. (b) Cell lysates from the indicated cells were fractionated as described in Materials and methods, and then subjected to western blot analysis by using antibody against C terminus of c-Met (25H2). Notice the c-Met RTK (145 kDa) in cytosolic fraction and a 60 kDa fragment of c-Met (triangle) in nuclear fraction only in HeLa cells. Purity of cellular fractionation was checked by Lamin B (nuclear marker) and tubulin (cytoplasmic marker). (c) Cell lysates from HeLa cells transfected with indicated siRNAs were fractionated and subjected to western blot analysis as in (b), triangle; 60kDa nuclear c-Met fragment. (d) HeLa cells were transfected with control or c-Met siRNAs at the indicated concentrations and fixed at 24 h after transfection. Immunocytochemistry was performed using C-28 antibody, and DAPI staining was used to visualize nuclei. Representative images show disappearance of nuclear c-Met signal in the presence of c-Met siRNA. Met (green) and nuclei (blue). Size bar; 10 μm. Each experiment was performed in triplicate with similar results.
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fig1: The C-terminal fragment of c-Met localizes in the nuclei of HeLa cells. (a) Sub-confluent HeLa cells were fixed and stained with C-terminal antibody for c-Met (C-28, green) and DAPI for nucleus (blue). Representative images show the nuclear patches of c-Met. Size bar,10 μm. (b) Cell lysates from the indicated cells were fractionated as described in Materials and methods, and then subjected to western blot analysis by using antibody against C terminus of c-Met (25H2). Notice the c-Met RTK (145 kDa) in cytosolic fraction and a 60 kDa fragment of c-Met (triangle) in nuclear fraction only in HeLa cells. Purity of cellular fractionation was checked by Lamin B (nuclear marker) and tubulin (cytoplasmic marker). (c) Cell lysates from HeLa cells transfected with indicated siRNAs were fractionated and subjected to western blot analysis as in (b), triangle; 60kDa nuclear c-Met fragment. (d) HeLa cells were transfected with control or c-Met siRNAs at the indicated concentrations and fixed at 24 h after transfection. Immunocytochemistry was performed using C-28 antibody, and DAPI staining was used to visualize nuclei. Representative images show disappearance of nuclear c-Met signal in the presence of c-Met siRNA. Met (green) and nuclei (blue). Size bar; 10 μm. Each experiment was performed in triplicate with similar results.

Mentions: To search for the NLS of c-Met, we first needed to identify a suitable cell line that contained all the molecular machineries required for nuclear translocation of the c-Met fragment. To this end, we investigated the presence of the c-Met fragment in the nuclei of HeLa cells. Cells were stained with an antibody specific for the C terminus of c-Met (C-28) and observed by confocal microscopy, which revealed a fine and distinct granular pattern of staining in the nuclei (Figure 1a). We next performed cellular fractionation to confirm the immunocytochemical data. Immunoblotting using another antibody specific to the C terminus of c-Met (25H2) clearly detected the ~60 kDa C-terminal fragment of c-Met in the nuclear fraction. This is consistent with previous reports on the nuclear C-terminal fragment of c-Met in various cells.18,19 In contrast, the nuclear fraction from Chang liver cells did not show the nuclear c-Met band despite the presence of wild-type c-Met in the non-nuclear fraction (Figure 1b), indicating that the nuclear translocation of the c-Met fragment is cell-type-specific. The observed expression of the c-Met fragment in the nuclear fraction of HeLa cells was clearly abolished by the c-Met-small interfering RNA (siRNA) (Figure 1c), confirming the presence of the nuclear c-Met fragment in the nuclei of HeLa cells. To confirm the specificity of this signal, we carried out RNAi-mediated gene knockdown experiments, and found that the distinct punctate pattern was significantly and dose-dependently abrogated by a c-Met-specific siRNA (Figure 1d) supporting the specificity of antibody. Based on these findings, we used the HeLa cell line as the model for our investigations.


A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met.

Chaudhary SC, Cho MG, Nguyen TT, Park KS, Kwon MH, Lee JH - Exp. Mol. Med. (2014)

The C-terminal fragment of c-Met localizes in the nuclei of HeLa cells. (a) Sub-confluent HeLa cells were fixed and stained with C-terminal antibody for c-Met (C-28, green) and DAPI for nucleus (blue). Representative images show the nuclear patches of c-Met. Size bar,10 μm. (b) Cell lysates from the indicated cells were fractionated as described in Materials and methods, and then subjected to western blot analysis by using antibody against C terminus of c-Met (25H2). Notice the c-Met RTK (145 kDa) in cytosolic fraction and a 60 kDa fragment of c-Met (triangle) in nuclear fraction only in HeLa cells. Purity of cellular fractionation was checked by Lamin B (nuclear marker) and tubulin (cytoplasmic marker). (c) Cell lysates from HeLa cells transfected with indicated siRNAs were fractionated and subjected to western blot analysis as in (b), triangle; 60kDa nuclear c-Met fragment. (d) HeLa cells were transfected with control or c-Met siRNAs at the indicated concentrations and fixed at 24 h after transfection. Immunocytochemistry was performed using C-28 antibody, and DAPI staining was used to visualize nuclei. Representative images show disappearance of nuclear c-Met signal in the presence of c-Met siRNA. Met (green) and nuclei (blue). Size bar; 10 μm. Each experiment was performed in triplicate with similar results.
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Related In: Results  -  Collection

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fig1: The C-terminal fragment of c-Met localizes in the nuclei of HeLa cells. (a) Sub-confluent HeLa cells were fixed and stained with C-terminal antibody for c-Met (C-28, green) and DAPI for nucleus (blue). Representative images show the nuclear patches of c-Met. Size bar,10 μm. (b) Cell lysates from the indicated cells were fractionated as described in Materials and methods, and then subjected to western blot analysis by using antibody against C terminus of c-Met (25H2). Notice the c-Met RTK (145 kDa) in cytosolic fraction and a 60 kDa fragment of c-Met (triangle) in nuclear fraction only in HeLa cells. Purity of cellular fractionation was checked by Lamin B (nuclear marker) and tubulin (cytoplasmic marker). (c) Cell lysates from HeLa cells transfected with indicated siRNAs were fractionated and subjected to western blot analysis as in (b), triangle; 60kDa nuclear c-Met fragment. (d) HeLa cells were transfected with control or c-Met siRNAs at the indicated concentrations and fixed at 24 h after transfection. Immunocytochemistry was performed using C-28 antibody, and DAPI staining was used to visualize nuclei. Representative images show disappearance of nuclear c-Met signal in the presence of c-Met siRNA. Met (green) and nuclei (blue). Size bar; 10 μm. Each experiment was performed in triplicate with similar results.
Mentions: To search for the NLS of c-Met, we first needed to identify a suitable cell line that contained all the molecular machineries required for nuclear translocation of the c-Met fragment. To this end, we investigated the presence of the c-Met fragment in the nuclei of HeLa cells. Cells were stained with an antibody specific for the C terminus of c-Met (C-28) and observed by confocal microscopy, which revealed a fine and distinct granular pattern of staining in the nuclei (Figure 1a). We next performed cellular fractionation to confirm the immunocytochemical data. Immunoblotting using another antibody specific to the C terminus of c-Met (25H2) clearly detected the ~60 kDa C-terminal fragment of c-Met in the nuclear fraction. This is consistent with previous reports on the nuclear C-terminal fragment of c-Met in various cells.18,19 In contrast, the nuclear fraction from Chang liver cells did not show the nuclear c-Met band despite the presence of wild-type c-Met in the non-nuclear fraction (Figure 1b), indicating that the nuclear translocation of the c-Met fragment is cell-type-specific. The observed expression of the c-Met fragment in the nuclear fraction of HeLa cells was clearly abolished by the c-Met-small interfering RNA (siRNA) (Figure 1c), confirming the presence of the nuclear c-Met fragment in the nuclei of HeLa cells. To confirm the specificity of this signal, we carried out RNAi-mediated gene knockdown experiments, and found that the distinct punctate pattern was significantly and dose-dependently abrogated by a c-Met-specific siRNA (Figure 1d) supporting the specificity of antibody. Based on these findings, we used the HeLa cell line as the model for our investigations.

Bottom Line: This substitution also decreased the association of c-Met fragment with importin β.The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS.Indeed, decreasing the cytosolic pH either with nigericin, an Na(+)/H(+) exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low pH itself enhanced nuclear translocation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea [2] Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, Korea.

ABSTRACT
The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068-H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin β. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible 'pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na(+)/H(+) exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a 'pH-dependent' NLS.

Show MeSH