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Promotion of importin alpha-mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3.

Faul C, Hüttelmaier S, Oh J, Hachet V, Singer RH, Mundel P - J. Cell Biol. (2005)

Bottom Line: We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3.These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding.They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

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Two phosphorylated residues regulate nuclear localization of myopodin. (A) Confocal imaging of GFP-tagged myopodin in C2C12 myoblasts. Wild-type myopodin shows a predominantly nuclear localization (top), and S225AT272A displays a dramatically decreased nuclear localization (middle). In contrast, S225DT272E shows a primarily nuclear localization (bottom). Rhodamine-labeled phalloidin identifies myopodin-induced actin bundles, and DAPI visualizes nuclei. (B) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 77.4% of wild-type myopodin is detected in the nucleus. S225A and T272A show 20.1% and 20.5% nuclear myopodin, respectively. S225AT272A shows a further decrease to 10.5%. In contrast, S273A does not alter nuclear localization (78.8%). S225D, S273D, and T272E show 75.6%, 75.7%, and 80.3% nuclear localization, respectively. S225DT272E displays 83.5% nuclear localization. Statistical significance was confirmed by ANOVA (P < 0.001). Error bars indicate standard deviation.
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fig5: Two phosphorylated residues regulate nuclear localization of myopodin. (A) Confocal imaging of GFP-tagged myopodin in C2C12 myoblasts. Wild-type myopodin shows a predominantly nuclear localization (top), and S225AT272A displays a dramatically decreased nuclear localization (middle). In contrast, S225DT272E shows a primarily nuclear localization (bottom). Rhodamine-labeled phalloidin identifies myopodin-induced actin bundles, and DAPI visualizes nuclei. (B) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 77.4% of wild-type myopodin is detected in the nucleus. S225A and T272A show 20.1% and 20.5% nuclear myopodin, respectively. S225AT272A shows a further decrease to 10.5%. In contrast, S273A does not alter nuclear localization (78.8%). S225D, S273D, and T272E show 75.6%, 75.7%, and 80.3% nuclear localization, respectively. S225DT272E displays 83.5% nuclear localization. Statistical significance was confirmed by ANOVA (P < 0.001). Error bars indicate standard deviation.

Mentions: To determine whether the aa S225 and T272 of myopodin are not only required for 14-3-3 binding but are also required for the nuclear import of myopodin, the subcellular distribution of GFP-tagged wild-type and mutant myopodin was analyzed by confocal microscopy (Fig. 5 A) and was quantified as described above (Fig. 2 C). As shown previously (Fig. 2 B; Weins et al., 2001), wild-type myopodin was mainly localized in the nucleus (Fig. 5 A). The quantitative analysis showed 77.4% nuclear localization (Fig. 5 B). The removal of single phosphorylation sites (S225A and T272A) resulted in a significantly decreased nuclear localization (20.1% for S225A and 20.5% for T272A; Fig. 5 B) versus 77.4% for wild-type myopodin (P < 0.01; t test). The combined replacement of S225 and T272 (Fig. 5 A, S225AT272A) further decreased nuclear myopodin to 10.5% (Fig. 5 B), a significant difference from the single mutations S225A and T272A (P < 0.01; t test). In contrast, S273A showed normal nuclear localization (78.8% nuclear; Fig. 5 B). The active mutants S225D, T272E, or S273D showed 75.6%, 80.3%, and 75.7% nuclear localization, respectively. The highest percentage of nuclear myopodin was found for S225DT272E (Fig. 5 A). However, the difference to wild type (83.5% for S225DT272E versus 77.4% for wild-type myopodin) was not significant. Altogether, these experiments show that the phosphorylation of S225 and T272 is required for the efficient nuclear import of myopodin.


Promotion of importin alpha-mediated nuclear import by the phosphorylation-dependent binding of cargo protein to 14-3-3.

Faul C, Hüttelmaier S, Oh J, Hachet V, Singer RH, Mundel P - J. Cell Biol. (2005)

Two phosphorylated residues regulate nuclear localization of myopodin. (A) Confocal imaging of GFP-tagged myopodin in C2C12 myoblasts. Wild-type myopodin shows a predominantly nuclear localization (top), and S225AT272A displays a dramatically decreased nuclear localization (middle). In contrast, S225DT272E shows a primarily nuclear localization (bottom). Rhodamine-labeled phalloidin identifies myopodin-induced actin bundles, and DAPI visualizes nuclei. (B) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 77.4% of wild-type myopodin is detected in the nucleus. S225A and T272A show 20.1% and 20.5% nuclear myopodin, respectively. S225AT272A shows a further decrease to 10.5%. In contrast, S273A does not alter nuclear localization (78.8%). S225D, S273D, and T272E show 75.6%, 75.7%, and 80.3% nuclear localization, respectively. S225DT272E displays 83.5% nuclear localization. Statistical significance was confirmed by ANOVA (P < 0.001). Error bars indicate standard deviation.
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fig5: Two phosphorylated residues regulate nuclear localization of myopodin. (A) Confocal imaging of GFP-tagged myopodin in C2C12 myoblasts. Wild-type myopodin shows a predominantly nuclear localization (top), and S225AT272A displays a dramatically decreased nuclear localization (middle). In contrast, S225DT272E shows a primarily nuclear localization (bottom). Rhodamine-labeled phalloidin identifies myopodin-induced actin bundles, and DAPI visualizes nuclei. (B) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 77.4% of wild-type myopodin is detected in the nucleus. S225A and T272A show 20.1% and 20.5% nuclear myopodin, respectively. S225AT272A shows a further decrease to 10.5%. In contrast, S273A does not alter nuclear localization (78.8%). S225D, S273D, and T272E show 75.6%, 75.7%, and 80.3% nuclear localization, respectively. S225DT272E displays 83.5% nuclear localization. Statistical significance was confirmed by ANOVA (P < 0.001). Error bars indicate standard deviation.
Mentions: To determine whether the aa S225 and T272 of myopodin are not only required for 14-3-3 binding but are also required for the nuclear import of myopodin, the subcellular distribution of GFP-tagged wild-type and mutant myopodin was analyzed by confocal microscopy (Fig. 5 A) and was quantified as described above (Fig. 2 C). As shown previously (Fig. 2 B; Weins et al., 2001), wild-type myopodin was mainly localized in the nucleus (Fig. 5 A). The quantitative analysis showed 77.4% nuclear localization (Fig. 5 B). The removal of single phosphorylation sites (S225A and T272A) resulted in a significantly decreased nuclear localization (20.1% for S225A and 20.5% for T272A; Fig. 5 B) versus 77.4% for wild-type myopodin (P < 0.01; t test). The combined replacement of S225 and T272 (Fig. 5 A, S225AT272A) further decreased nuclear myopodin to 10.5% (Fig. 5 B), a significant difference from the single mutations S225A and T272A (P < 0.01; t test). In contrast, S273A showed normal nuclear localization (78.8% nuclear; Fig. 5 B). The active mutants S225D, T272E, or S273D showed 75.6%, 80.3%, and 75.7% nuclear localization, respectively. The highest percentage of nuclear myopodin was found for S225DT272E (Fig. 5 A). However, the difference to wild type (83.5% for S225DT272E versus 77.4% for wild-type myopodin) was not significant. Altogether, these experiments show that the phosphorylation of S225 and T272 is required for the efficient nuclear import of myopodin.

Bottom Line: We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3.These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding.They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
14-3-3 proteins are phosphoserine/threonine-binding proteins that play important roles in many regulatory processes, including intracellular protein targeting. 14-3-3 proteins can anchor target proteins in the cytoplasm and in the nucleus or can mediate their nuclear export. So far, no role for 14-3-3 in mediating nuclear import has been described. There is also mounting evidence that nuclear import is regulated by the phosphorylation of cargo proteins, but the underlying mechanism remains elusive. Myopodin is a dual-compartment, actin-bundling protein that functions as a tumor suppressor in human bladder cancer. In muscle cells, myopodin redistributes between the nucleus and the cytoplasm in a differentiation-dependent and stress-induced fashion. We show that importin alpha binding and the subsequent nuclear import of myopodin are regulated by the serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. These results establish a novel paradigm for the promotion of nuclear import by 14-3-3 binding. They provide a molecular explanation for the phosphorylation-dependent nuclear import of nuclear localization signal-containing cargo proteins.

Show MeSH
Related in: MedlinePlus