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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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Nuclear accumulation of c-Met fragment is cytosolic pH-dependent. (a) Bar graph shows the intracellular pH change measured by BCECF dye as described in Materials and methods with 1 μM nigericin treatment in a time-dependent manner. Representative result from three independent experiments. pH value of each experiment is the average of data from more than 15 cells on a cover-slip. (b) HeLa cells transiently transfected with F-3 construct were treated with or without 1 μM nigericin at indicated time intervals. Western blot analysis was performed using anti-GFP antibody after cellular fractionation. Tubulin and lamin B were used as cytoplasmic and nuclear markers respectively. Bar graphs below each panel show their respective subcellular distribution of the protein in fold change. (c, d) Western blot analysis using anti-GFP antibody was performed after subcellular fractionation of F-3 (in c) or Jxtm1 (in d)-transfected HeLa cells treated with nigericin at indicated concentration for 30 min. Bar graphs below show the subcellular distribution of the protein in fold change. (e) Representative time-lapse microscopy images of either pEGFP or F-3 transfected HeLa cells treated with 1 μM nigericin at indicated time intervals. The graph displaying on right shows nucleo-cytoplasmic ratio of GFP intensity over time. Red circle; nuclear area, white circle; cytoplasmic area. Margin of the cells are indicated by white broken line, n=10. (f) Western blot analysis using indicated antibodies were performed after subcellular fractionation of F-3 (WT), histidine-to-lysine (2K), or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with or without 1 μM nigericin for 30 min. Bar graphs on right panel show the subcellular distribution of the protein in fold change. (g) Importin β binding assay (as described in Figure 3b) was performed in F-3-transfected HeLa cells after the treatment with nigericin at indicated time points. Bar graph below shows the amount of cargo-binding importin β in fold change. (h) Immunoprecipitation was performed with anti-GFP antibody with the cell lysate of F-3 (WT), histidine-to-lysine (2K) or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with nigericin for 30 min followed by immunoblotting with importin β antibody. Bar graph below shows amount of cargo-binding importin β in fold change.
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fig4: Nuclear accumulation of c-Met fragment is cytosolic pH-dependent. (a) Bar graph shows the intracellular pH change measured by BCECF dye as described in Materials and methods with 1 μM nigericin treatment in a time-dependent manner. Representative result from three independent experiments. pH value of each experiment is the average of data from more than 15 cells on a cover-slip. (b) HeLa cells transiently transfected with F-3 construct were treated with or without 1 μM nigericin at indicated time intervals. Western blot analysis was performed using anti-GFP antibody after cellular fractionation. Tubulin and lamin B were used as cytoplasmic and nuclear markers respectively. Bar graphs below each panel show their respective subcellular distribution of the protein in fold change. (c, d) Western blot analysis using anti-GFP antibody was performed after subcellular fractionation of F-3 (in c) or Jxtm1 (in d)-transfected HeLa cells treated with nigericin at indicated concentration for 30 min. Bar graphs below show the subcellular distribution of the protein in fold change. (e) Representative time-lapse microscopy images of either pEGFP or F-3 transfected HeLa cells treated with 1 μM nigericin at indicated time intervals. The graph displaying on right shows nucleo-cytoplasmic ratio of GFP intensity over time. Red circle; nuclear area, white circle; cytoplasmic area. Margin of the cells are indicated by white broken line, n=10. (f) Western blot analysis using indicated antibodies were performed after subcellular fractionation of F-3 (WT), histidine-to-lysine (2K), or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with or without 1 μM nigericin for 30 min. Bar graphs on right panel show the subcellular distribution of the protein in fold change. (g) Importin β binding assay (as described in Figure 3b) was performed in F-3-transfected HeLa cells after the treatment with nigericin at indicated time points. Bar graph below shows the amount of cargo-binding importin β in fold change. (h) Immunoprecipitation was performed with anti-GFP antibody with the cell lysate of F-3 (WT), histidine-to-lysine (2K) or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with nigericin for 30 min followed by immunoblotting with importin β antibody. Bar graph below shows amount of cargo-binding importin β in fold change.

Mentions: To explore the effect of decreased cytosolic pH on the nuclear translocation of the c-Met fragment, we used nigericin, which reduces cytosolic pH via the exchange of Na+ and H+ from vesicular organelles.41,42 We measured the actual cytosolic pH change with the pH-sensitive dye, BCECF-AM.43,44 As shown in Figure 4a, treatment of HeLa cells with 1 uM nigericin significantly decreased the cytosolic pH as early as 30 min posttreatment, and this decrease continued time-dependently thereafter. We then explored the effect of nigericin on the nuclear translocation of the c-Met fragment. As expected, decreasing the cytosolic pH significantly increased the nuclear accumulation of the fusion protein both time- and dose-dependently (Figures 4b and c). The increase in the nuclear protein level was observed as early as 15 min after nigericin treatment and reached up to 10-fold at 1 h posttreatment; the protein level in the cytosolic fraction decreased only slightly because most of the fusion proteins were localized in the cytosol (Supplementary Figure 1). We obtained essentially the same result using the Jxtm1 fragment of c-Met protein (Figure 2a), which has similar size to the~60 kDa C-terminal fragment of c-Met, ruling out the possibility that this result was an artifact due to our use of a small fragment of c-Met (Figure 4d). Since there was some discrepancy in the timings of the pH decrease and the nuclear translocation, we used time-lapse microscopy for real-time measurement of nuclear translocation (Figure 4e). When the fluorescence intensities of two circular areas inside the cytosol and nucleus, respectively, were measured from the time-lapse images (Figure 4e, right panel), we clearly saw that the nucleo-cytoplasmic ratio of the fusion protein increased significantly at ~30 min after nigericin treatment, which was consistent with the cytosolic pH change measured using BCECF-AM (Figure 4a). These data collectively indicated that the novel NLS of c-Met appears to be sensitive to changes in pH (hereinafter called ‘pH-dependent').


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)

Nuclear accumulation of c-Met fragment is cytosolic pH-dependent. (a) Bar graph shows the intracellular pH change measured by BCECF dye as described in Materials and methods with 1 μM nigericin treatment in a time-dependent manner. Representative result from three independent experiments. pH value of each experiment is the average of data from more than 15 cells on a cover-slip. (b) HeLa cells transiently transfected with F-3 construct were treated with or without 1 μM nigericin at indicated time intervals. Western blot analysis was performed using anti-GFP antibody after cellular fractionation. Tubulin and lamin B were used as cytoplasmic and nuclear markers respectively. Bar graphs below each panel show their respective subcellular distribution of the protein in fold change. (c, d) Western blot analysis using anti-GFP antibody was performed after subcellular fractionation of F-3 (in c) or Jxtm1 (in d)-transfected HeLa cells treated with nigericin at indicated concentration for 30 min. Bar graphs below show the subcellular distribution of the protein in fold change. (e) Representative time-lapse microscopy images of either pEGFP or F-3 transfected HeLa cells treated with 1 μM nigericin at indicated time intervals. The graph displaying on right shows nucleo-cytoplasmic ratio of GFP intensity over time. Red circle; nuclear area, white circle; cytoplasmic area. Margin of the cells are indicated by white broken line, n=10. (f) Western blot analysis using indicated antibodies were performed after subcellular fractionation of F-3 (WT), histidine-to-lysine (2K), or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with or without 1 μM nigericin for 30 min. Bar graphs on right panel show the subcellular distribution of the protein in fold change. (g) Importin β binding assay (as described in Figure 3b) was performed in F-3-transfected HeLa cells after the treatment with nigericin at indicated time points. Bar graph below shows the amount of cargo-binding importin β in fold change. (h) Immunoprecipitation was performed with anti-GFP antibody with the cell lysate of F-3 (WT), histidine-to-lysine (2K) or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with nigericin for 30 min followed by immunoblotting with importin β antibody. Bar graph below shows amount of cargo-binding importin β in fold change.
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fig4: Nuclear accumulation of c-Met fragment is cytosolic pH-dependent. (a) Bar graph shows the intracellular pH change measured by BCECF dye as described in Materials and methods with 1 μM nigericin treatment in a time-dependent manner. Representative result from three independent experiments. pH value of each experiment is the average of data from more than 15 cells on a cover-slip. (b) HeLa cells transiently transfected with F-3 construct were treated with or without 1 μM nigericin at indicated time intervals. Western blot analysis was performed using anti-GFP antibody after cellular fractionation. Tubulin and lamin B were used as cytoplasmic and nuclear markers respectively. Bar graphs below each panel show their respective subcellular distribution of the protein in fold change. (c, d) Western blot analysis using anti-GFP antibody was performed after subcellular fractionation of F-3 (in c) or Jxtm1 (in d)-transfected HeLa cells treated with nigericin at indicated concentration for 30 min. Bar graphs below show the subcellular distribution of the protein in fold change. (e) Representative time-lapse microscopy images of either pEGFP or F-3 transfected HeLa cells treated with 1 μM nigericin at indicated time intervals. The graph displaying on right shows nucleo-cytoplasmic ratio of GFP intensity over time. Red circle; nuclear area, white circle; cytoplasmic area. Margin of the cells are indicated by white broken line, n=10. (f) Western blot analysis using indicated antibodies were performed after subcellular fractionation of F-3 (WT), histidine-to-lysine (2K), or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with or without 1 μM nigericin for 30 min. Bar graphs on right panel show the subcellular distribution of the protein in fold change. (g) Importin β binding assay (as described in Figure 3b) was performed in F-3-transfected HeLa cells after the treatment with nigericin at indicated time points. Bar graph below shows the amount of cargo-binding importin β in fold change. (h) Immunoprecipitation was performed with anti-GFP antibody with the cell lysate of F-3 (WT), histidine-to-lysine (2K) or histidine-to-alanine (2A) mutants-transfected HeLa cells treated with nigericin for 30 min followed by immunoblotting with importin β antibody. Bar graph below shows amount of cargo-binding importin β in fold change.
Mentions: To explore the effect of decreased cytosolic pH on the nuclear translocation of the c-Met fragment, we used nigericin, which reduces cytosolic pH via the exchange of Na+ and H+ from vesicular organelles.41,42 We measured the actual cytosolic pH change with the pH-sensitive dye, BCECF-AM.43,44 As shown in Figure 4a, treatment of HeLa cells with 1 uM nigericin significantly decreased the cytosolic pH as early as 30 min posttreatment, and this decrease continued time-dependently thereafter. We then explored the effect of nigericin on the nuclear translocation of the c-Met fragment. As expected, decreasing the cytosolic pH significantly increased the nuclear accumulation of the fusion protein both time- and dose-dependently (Figures 4b and c). The increase in the nuclear protein level was observed as early as 15 min after nigericin treatment and reached up to 10-fold at 1 h posttreatment; the protein level in the cytosolic fraction decreased only slightly because most of the fusion proteins were localized in the cytosol (Supplementary Figure 1). We obtained essentially the same result using the Jxtm1 fragment of c-Met protein (Figure 2a), which has similar size to the~60 kDa C-terminal fragment of c-Met, ruling out the possibility that this result was an artifact due to our use of a small fragment of c-Met (Figure 4d). Since there was some discrepancy in the timings of the pH decrease and the nuclear translocation, we used time-lapse microscopy for real-time measurement of nuclear translocation (Figure 4e). When the fluorescence intensities of two circular areas inside the cytosol and nucleus, respectively, were measured from the time-lapse images (Figure 4e, right panel), we clearly saw that the nucleo-cytoplasmic ratio of the fusion protein increased significantly at ~30 min after nigericin treatment, which was consistent with the cytosolic pH change measured using BCECF-AM (Figure 4a). These data collectively indicated that the novel NLS of c-Met appears to be sensitive to changes in pH (hereinafter called ‘pH-dependent').

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