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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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14-3-3β is required for nuclear import of myopodin. (A) Overexpression of full-length GFP–14-3-3β does not change the nuclear localization of endogenous myopodin (top left), whereas the dominant negative form GFP–14-3-3βΔN causes the nuclear exclusion of myopodin (bottom left). LMB does not reverse the cytoplasmic localization of myopodin that is caused by GFP–14-3-3βΔN (bottom right). (B) Confocal microscopy reveals a predominantly nuclear localization of GFP-tagged, full-length myopodin (top). Deletion of both NLSs (ΔNLS#1 + 2) or both 14-3-3–binding sites (Δ14-3-3#1 + 2) dramatically decreases the nuclear localization of GFP–myopodin. The combined deletion of all four binding motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2; bottom) virtually abrogates the nuclear localization of myopodin (bottom). (C) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 73.4% of GFP full-length myopodin is found in the nucleus. Deletion of both NLSs separately (ΔNLS#1 and ΔNLS#2) or together (ΔNLS#1 + 2) decreases the amount of nuclear GFP–myopodin to 14.8%, 13.4%, and 14.2%, respectively. Removal of 14-3-3–binding sites (Δ14-3-3#1, Δ14-3-3#2, and Δ14-3-3#1 + 2) reduces nuclear myopodin to 20.0%, 21.8%, and 19.1%, respectively. Combined deletion of all four motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2) causes a further reduction of nuclear myopodin to 6.4%. 60.5% of GFP (control) is found in the nucleus. Statistical significance was confirmed by analysis of variance between groups (ANOVA; P < 0.001). Error bars indicate standard deviation.
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fig2: 14-3-3β is required for nuclear import of myopodin. (A) Overexpression of full-length GFP–14-3-3β does not change the nuclear localization of endogenous myopodin (top left), whereas the dominant negative form GFP–14-3-3βΔN causes the nuclear exclusion of myopodin (bottom left). LMB does not reverse the cytoplasmic localization of myopodin that is caused by GFP–14-3-3βΔN (bottom right). (B) Confocal microscopy reveals a predominantly nuclear localization of GFP-tagged, full-length myopodin (top). Deletion of both NLSs (ΔNLS#1 + 2) or both 14-3-3–binding sites (Δ14-3-3#1 + 2) dramatically decreases the nuclear localization of GFP–myopodin. The combined deletion of all four binding motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2; bottom) virtually abrogates the nuclear localization of myopodin (bottom). (C) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 73.4% of GFP full-length myopodin is found in the nucleus. Deletion of both NLSs separately (ΔNLS#1 and ΔNLS#2) or together (ΔNLS#1 + 2) decreases the amount of nuclear GFP–myopodin to 14.8%, 13.4%, and 14.2%, respectively. Removal of 14-3-3–binding sites (Δ14-3-3#1, Δ14-3-3#2, and Δ14-3-3#1 + 2) reduces nuclear myopodin to 20.0%, 21.8%, and 19.1%, respectively. Combined deletion of all four motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2) causes a further reduction of nuclear myopodin to 6.4%. 60.5% of GFP (control) is found in the nucleus. Statistical significance was confirmed by analysis of variance between groups (ANOVA; P < 0.001). Error bars indicate standard deviation.

Mentions: 14-3-3 proteins can regulate the subcellular localization of binding partners (Muslin and Xing, 2000), and myopodin is a dual-compartment protein (Weins et al., 2001). This raises the possibility that 14-3-3β participates in regulating the subcellular localization of myopodin. A dominant negative mutant of 14-3-3θ that lacks the NH2-terminal dimerization domain (14-3-3θΔN) redistributes the catalytic subunit of human telomerase from the nucleus to the cytoplasm (Seimiya et al., 2000). Therefore, we tested the effect of 14-3-3βΔN (Fig. 1 G) on the subcellular localization of myopodin in myoblasts. In cells expressing GFP–14-3-3βΔN, myopodin showed a cytoplasmic localization (Fig. 2 A, bottom left), whereas in cells expressing full-length GFP–14-3-3β (Fig. 2 A, top left) or GFP alone (not depicted), myopodin was localized in the nucleus.


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)

14-3-3β is required for nuclear import of myopodin. (A) Overexpression of full-length GFP–14-3-3β does not change the nuclear localization of endogenous myopodin (top left), whereas the dominant negative form GFP–14-3-3βΔN causes the nuclear exclusion of myopodin (bottom left). LMB does not reverse the cytoplasmic localization of myopodin that is caused by GFP–14-3-3βΔN (bottom right). (B) Confocal microscopy reveals a predominantly nuclear localization of GFP-tagged, full-length myopodin (top). Deletion of both NLSs (ΔNLS#1 + 2) or both 14-3-3–binding sites (Δ14-3-3#1 + 2) dramatically decreases the nuclear localization of GFP–myopodin. The combined deletion of all four binding motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2; bottom) virtually abrogates the nuclear localization of myopodin (bottom). (C) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 73.4% of GFP full-length myopodin is found in the nucleus. Deletion of both NLSs separately (ΔNLS#1 and ΔNLS#2) or together (ΔNLS#1 + 2) decreases the amount of nuclear GFP–myopodin to 14.8%, 13.4%, and 14.2%, respectively. Removal of 14-3-3–binding sites (Δ14-3-3#1, Δ14-3-3#2, and Δ14-3-3#1 + 2) reduces nuclear myopodin to 20.0%, 21.8%, and 19.1%, respectively. Combined deletion of all four motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2) causes a further reduction of nuclear myopodin to 6.4%. 60.5% of GFP (control) is found in the nucleus. Statistical significance was confirmed by analysis of variance between groups (ANOVA; P < 0.001). Error bars indicate standard deviation.
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Related In: Results  -  Collection

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fig2: 14-3-3β is required for nuclear import of myopodin. (A) Overexpression of full-length GFP–14-3-3β does not change the nuclear localization of endogenous myopodin (top left), whereas the dominant negative form GFP–14-3-3βΔN causes the nuclear exclusion of myopodin (bottom left). LMB does not reverse the cytoplasmic localization of myopodin that is caused by GFP–14-3-3βΔN (bottom right). (B) Confocal microscopy reveals a predominantly nuclear localization of GFP-tagged, full-length myopodin (top). Deletion of both NLSs (ΔNLS#1 + 2) or both 14-3-3–binding sites (Δ14-3-3#1 + 2) dramatically decreases the nuclear localization of GFP–myopodin. The combined deletion of all four binding motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2; bottom) virtually abrogates the nuclear localization of myopodin (bottom). (C) Quantitative analysis is presented as a percentage of nuclear GFP–myopodin. 73.4% of GFP full-length myopodin is found in the nucleus. Deletion of both NLSs separately (ΔNLS#1 and ΔNLS#2) or together (ΔNLS#1 + 2) decreases the amount of nuclear GFP–myopodin to 14.8%, 13.4%, and 14.2%, respectively. Removal of 14-3-3–binding sites (Δ14-3-3#1, Δ14-3-3#2, and Δ14-3-3#1 + 2) reduces nuclear myopodin to 20.0%, 21.8%, and 19.1%, respectively. Combined deletion of all four motifs (Δ14-3-3#1 + 2 + ΔNLS#1 + 2) causes a further reduction of nuclear myopodin to 6.4%. 60.5% of GFP (control) is found in the nucleus. Statistical significance was confirmed by analysis of variance between groups (ANOVA; P < 0.001). Error bars indicate standard deviation.
Mentions: 14-3-3 proteins can regulate the subcellular localization of binding partners (Muslin and Xing, 2000), and myopodin is a dual-compartment protein (Weins et al., 2001). This raises the possibility that 14-3-3β participates in regulating the subcellular localization of myopodin. A dominant negative mutant of 14-3-3θ that lacks the NH2-terminal dimerization domain (14-3-3θΔN) redistributes the catalytic subunit of human telomerase from the nucleus to the cytoplasm (Seimiya et al., 2000). Therefore, we tested the effect of 14-3-3βΔN (Fig. 1 G) on the subcellular localization of myopodin in myoblasts. In cells expressing GFP–14-3-3βΔN, myopodin showed a cytoplasmic localization (Fig. 2 A, bottom left), whereas in cells expressing full-length GFP–14-3-3β (Fig. 2 A, top left) or GFP alone (not depicted), myopodin was localized in the nucleus.

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