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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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Two histidine residues are crucial for the nuclear translocation of c-Met fragment. (a) HeLa cells were transfected with wild-type F-3, indicated histidine-to-alanine mutant constructs or pEGFP as a vector control. Immunocytochemistry by using anti-GFP antibody was performed as in Figure 2. Representative images were taken from fluorescence microscopy at × 40 objective. Bar graph on right panel shows the percentage of subcellular distribution of cells (n=120–225). ***P<0.001 by Student's t-test. (b) Cell lysates from HeLa cells transfected with F-3(WT) or H1068A_H1079A mutant were subjected to immunoprecipitation by using anti-GFP antibody, which was followed by immunoblotting with importin β antibody. Bar graph in right shows the relative binding tendency of importin β with cargo protein compared to F-3(WT). (c) HeLa cells were transfected with wild-type F-3 or indicated histidine-to-lysine mutant constructs, and subjected to immunocytochemistry by using anti-GFP antibodies. Bar graph displaying on right upper panel shows the percentage of subcellular distribution of cells (n=102–156) and on the right lower panel shows the relative nucleo-cytoplasmic ratio of green fluorescence intensity. Middle panel shows the intensity changes revealed by line scan analysis performed following the arrow. Error bars; standard deviations of three independent experiments. *P<0.05, **P<0.01 by Student's t-test, n=25.
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fig3: Two histidine residues are crucial for the nuclear translocation of c-Met fragment. (a) HeLa cells were transfected with wild-type F-3, indicated histidine-to-alanine mutant constructs or pEGFP as a vector control. Immunocytochemistry by using anti-GFP antibody was performed as in Figure 2. Representative images were taken from fluorescence microscopy at × 40 objective. Bar graph on right panel shows the percentage of subcellular distribution of cells (n=120–225). ***P<0.001 by Student's t-test. (b) Cell lysates from HeLa cells transfected with F-3(WT) or H1068A_H1079A mutant were subjected to immunoprecipitation by using anti-GFP antibody, which was followed by immunoblotting with importin β antibody. Bar graph in right shows the relative binding tendency of importin β with cargo protein compared to F-3(WT). (c) HeLa cells were transfected with wild-type F-3 or indicated histidine-to-lysine mutant constructs, and subjected to immunocytochemistry by using anti-GFP antibodies. Bar graph displaying on right upper panel shows the percentage of subcellular distribution of cells (n=102–156) and on the right lower panel shows the relative nucleo-cytoplasmic ratio of green fluorescence intensity. Middle panel shows the intensity changes revealed by line scan analysis performed following the arrow. Error bars; standard deviations of three independent experiments. *P<0.05, **P<0.01 by Student's t-test, n=25.

Mentions: Classical bipartite NLSs comprise two stretches of basic amino acids (lysines or arginines) separated by a 10- to 12-amino-acid spacer, as previously described for many nuclear proteins, including nucleoplasmin26,27 (Supplementary Table 1). In bipartite NLSs, the positive charges provided by lysines or arginines are essential because they enable the electrostatic interaction with negatively charged amino acid residues on importin α, resulting in binding between the importin and the cargo protein.33, 34, 35 We hypothesized that the putative NLS found in c-Met (H1068-H1079) might be a variant of this classical bipartite NLS, with two histidines replacing the lysines or arginines found in classical bipartite NLSs. To test this, we created point mutants of the F-3 protein wherein one or both histidines of the putative NLS motif were substituted with alanine (H1068A, H1079A and H1068A_H1079A), and examined the importance of the histidine residues for the nuclear localization of c-Met. Indeed, the mutant constructs all yielded significantly higher fractions of cells showing cytoplasm-only localization (from ~10% to 70–80%), indicating that nuclear translocation was blocked in the mutant-expressing cells (Figure 3a). Next, we performed biochemical analysis to confirm the essential role of these histidine residues for nuclear translocation. Since mammalian cells may express six different forms of importin α but only one form of importin β,36,37 and association with importin β is an essential process for the cargo proteins to translocate into nucleus, we examined whether the association of cargo with importin β changes depending on the histidine residues. Clearly, the wild-type F-3 fragment co-immunoprecipitated with importin β, and substitution of both histidines with alanine significantly decreased this protein–protein interaction (Figure 3b). These results are consistent with our hypothesis that these two histidines are essential for the function of the NLS of c-Met. Moreover, considering that positive charges of lysine or arginine residues in classical bipartite NLS enable electrostatic interaction with importin α, the histidine residues might also confer positivity needed for the interaction between cargo and importin α, which subsequently enhance the formation of cargo-importin α-importin β ternary complex.


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)

Two histidine residues are crucial for the nuclear translocation of c-Met fragment. (a) HeLa cells were transfected with wild-type F-3, indicated histidine-to-alanine mutant constructs or pEGFP as a vector control. Immunocytochemistry by using anti-GFP antibody was performed as in Figure 2. Representative images were taken from fluorescence microscopy at × 40 objective. Bar graph on right panel shows the percentage of subcellular distribution of cells (n=120–225). ***P<0.001 by Student's t-test. (b) Cell lysates from HeLa cells transfected with F-3(WT) or H1068A_H1079A mutant were subjected to immunoprecipitation by using anti-GFP antibody, which was followed by immunoblotting with importin β antibody. Bar graph in right shows the relative binding tendency of importin β with cargo protein compared to F-3(WT). (c) HeLa cells were transfected with wild-type F-3 or indicated histidine-to-lysine mutant constructs, and subjected to immunocytochemistry by using anti-GFP antibodies. Bar graph displaying on right upper panel shows the percentage of subcellular distribution of cells (n=102–156) and on the right lower panel shows the relative nucleo-cytoplasmic ratio of green fluorescence intensity. Middle panel shows the intensity changes revealed by line scan analysis performed following the arrow. Error bars; standard deviations of three independent experiments. *P<0.05, **P<0.01 by Student's t-test, n=25.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig3: Two histidine residues are crucial for the nuclear translocation of c-Met fragment. (a) HeLa cells were transfected with wild-type F-3, indicated histidine-to-alanine mutant constructs or pEGFP as a vector control. Immunocytochemistry by using anti-GFP antibody was performed as in Figure 2. Representative images were taken from fluorescence microscopy at × 40 objective. Bar graph on right panel shows the percentage of subcellular distribution of cells (n=120–225). ***P<0.001 by Student's t-test. (b) Cell lysates from HeLa cells transfected with F-3(WT) or H1068A_H1079A mutant were subjected to immunoprecipitation by using anti-GFP antibody, which was followed by immunoblotting with importin β antibody. Bar graph in right shows the relative binding tendency of importin β with cargo protein compared to F-3(WT). (c) HeLa cells were transfected with wild-type F-3 or indicated histidine-to-lysine mutant constructs, and subjected to immunocytochemistry by using anti-GFP antibodies. Bar graph displaying on right upper panel shows the percentage of subcellular distribution of cells (n=102–156) and on the right lower panel shows the relative nucleo-cytoplasmic ratio of green fluorescence intensity. Middle panel shows the intensity changes revealed by line scan analysis performed following the arrow. Error bars; standard deviations of three independent experiments. *P<0.05, **P<0.01 by Student's t-test, n=25.
Mentions: Classical bipartite NLSs comprise two stretches of basic amino acids (lysines or arginines) separated by a 10- to 12-amino-acid spacer, as previously described for many nuclear proteins, including nucleoplasmin26,27 (Supplementary Table 1). In bipartite NLSs, the positive charges provided by lysines or arginines are essential because they enable the electrostatic interaction with negatively charged amino acid residues on importin α, resulting in binding between the importin and the cargo protein.33, 34, 35 We hypothesized that the putative NLS found in c-Met (H1068-H1079) might be a variant of this classical bipartite NLS, with two histidines replacing the lysines or arginines found in classical bipartite NLSs. To test this, we created point mutants of the F-3 protein wherein one or both histidines of the putative NLS motif were substituted with alanine (H1068A, H1079A and H1068A_H1079A), and examined the importance of the histidine residues for the nuclear localization of c-Met. Indeed, the mutant constructs all yielded significantly higher fractions of cells showing cytoplasm-only localization (from ~10% to 70–80%), indicating that nuclear translocation was blocked in the mutant-expressing cells (Figure 3a). Next, we performed biochemical analysis to confirm the essential role of these histidine residues for nuclear translocation. Since mammalian cells may express six different forms of importin α but only one form of importin β,36,37 and association with importin β is an essential process for the cargo proteins to translocate into nucleus, we examined whether the association of cargo with importin β changes depending on the histidine residues. Clearly, the wild-type F-3 fragment co-immunoprecipitated with importin β, and substitution of both histidines with alanine significantly decreased this protein–protein interaction (Figure 3b). These results are consistent with our hypothesis that these two histidines are essential for the function of the NLS of c-Met. Moreover, considering that positive charges of lysine or arginine residues in classical bipartite NLS enable electrostatic interaction with importin α, the histidine residues might also confer positivity needed for the interaction between cargo and importin α, which subsequently enhance the formation of cargo-importin α-importin β ternary complex.

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