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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 in myopodin mediate 14-3-3 binding. (A) SDS-PAGE analysis showing reduced molecular weight of purified FLAG-myopodin after dephosphorylation with λ-PPase. (B) Dephosphorylation of myopodin abrogates binding to GST–14-3-3β. The interaction is also prevented by the 14-3-3–blocking peptide R18. (C) Binding of purified FLAG-myopodin to endogenous 14-3-3β from C2C12 myoblasts is abrogated by dephosphorylation of myopodin (top). In contrast, binding of endogenous myopodin to purified FLAG–14-3-3β is not affected by the dephosphorylation of 14-3-3β (bottom). (D) Dephosphorylation with λ-PPase abrogates the interaction of FLAG-myopodin (bottom) with endogenous 14-3-3β (top) and importin α (middle) from X. laevis extracts. (E) Putative phosphoacceptor sites within the 14-3-3–binding motifs of myopodin. S225 in motif#1, as well as T272 and S273 in motif#2, were substituted with alanine to remove putative phosphorylation sites. Replacement with aspartic acid or glutamic acid was done to mimic phosphorylation. (F) Purified FLAG-tagged, wild-type myopodin interacts with GST–14-3-3β. Substitution of S225 or T272 with alanine (S225A, T272A, and S225AT272A) abrogates 14-3-3β binding. Replacement of S273 with alanine (S273A) does not interfere with the binding of myopodin to 14-3-3β. (G) Substitutions of S225 or T272 with aspartic or glutamic acid, respectively, does not alter the binding of myopodin to 14-3-3 (top). However, after dephosphorylation, only S225DT272E retains strong binding to GST–14-3-3β. Single mutations (S225D and T272E) bind significantly less, and wild-type binding is abrogated.
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fig4: Two phosphorylated residues in myopodin mediate 14-3-3 binding. (A) SDS-PAGE analysis showing reduced molecular weight of purified FLAG-myopodin after dephosphorylation with λ-PPase. (B) Dephosphorylation of myopodin abrogates binding to GST–14-3-3β. The interaction is also prevented by the 14-3-3–blocking peptide R18. (C) Binding of purified FLAG-myopodin to endogenous 14-3-3β from C2C12 myoblasts is abrogated by dephosphorylation of myopodin (top). In contrast, binding of endogenous myopodin to purified FLAG–14-3-3β is not affected by the dephosphorylation of 14-3-3β (bottom). (D) Dephosphorylation with λ-PPase abrogates the interaction of FLAG-myopodin (bottom) with endogenous 14-3-3β (top) and importin α (middle) from X. laevis extracts. (E) Putative phosphoacceptor sites within the 14-3-3–binding motifs of myopodin. S225 in motif#1, as well as T272 and S273 in motif#2, were substituted with alanine to remove putative phosphorylation sites. Replacement with aspartic acid or glutamic acid was done to mimic phosphorylation. (F) Purified FLAG-tagged, wild-type myopodin interacts with GST–14-3-3β. Substitution of S225 or T272 with alanine (S225A, T272A, and S225AT272A) abrogates 14-3-3β binding. Replacement of S273 with alanine (S273A) does not interfere with the binding of myopodin to 14-3-3β. (G) Substitutions of S225 or T272 with aspartic or glutamic acid, respectively, does not alter the binding of myopodin to 14-3-3 (top). However, after dephosphorylation, only S225DT272E retains strong binding to GST–14-3-3β. Single mutations (S225D and T272E) bind significantly less, and wild-type binding is abrogated.

Mentions: 14-3-3 proteins are phosphoserine/threonine-binding proteins (Muslin et al., 1996), and many 14-3-3 interactions depend on the phosphorylation of the target protein (Yaffe, 2002). Therefore, we examined whether the myopodin–14-3-3 interaction is regulated by phosphorylation. To this end, purified FLAG-myopodin was dephosphorylated with λ protein phosphatase (λ-PPase), which reduced the molecular size of myopodin (Fig. 4 A). Moreover, λ-PPase treatment converted the fuzzier signal of the phosphorylated protein into a more compact and sharper band (Fig. 4 A, top). These findings show that myopodin is phosphorylated in vivo.


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 in myopodin mediate 14-3-3 binding. (A) SDS-PAGE analysis showing reduced molecular weight of purified FLAG-myopodin after dephosphorylation with λ-PPase. (B) Dephosphorylation of myopodin abrogates binding to GST–14-3-3β. The interaction is also prevented by the 14-3-3–blocking peptide R18. (C) Binding of purified FLAG-myopodin to endogenous 14-3-3β from C2C12 myoblasts is abrogated by dephosphorylation of myopodin (top). In contrast, binding of endogenous myopodin to purified FLAG–14-3-3β is not affected by the dephosphorylation of 14-3-3β (bottom). (D) Dephosphorylation with λ-PPase abrogates the interaction of FLAG-myopodin (bottom) with endogenous 14-3-3β (top) and importin α (middle) from X. laevis extracts. (E) Putative phosphoacceptor sites within the 14-3-3–binding motifs of myopodin. S225 in motif#1, as well as T272 and S273 in motif#2, were substituted with alanine to remove putative phosphorylation sites. Replacement with aspartic acid or glutamic acid was done to mimic phosphorylation. (F) Purified FLAG-tagged, wild-type myopodin interacts with GST–14-3-3β. Substitution of S225 or T272 with alanine (S225A, T272A, and S225AT272A) abrogates 14-3-3β binding. Replacement of S273 with alanine (S273A) does not interfere with the binding of myopodin to 14-3-3β. (G) Substitutions of S225 or T272 with aspartic or glutamic acid, respectively, does not alter the binding of myopodin to 14-3-3 (top). However, after dephosphorylation, only S225DT272E retains strong binding to GST–14-3-3β. Single mutations (S225D and T272E) bind significantly less, and wild-type binding is abrogated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171942&req=5

fig4: Two phosphorylated residues in myopodin mediate 14-3-3 binding. (A) SDS-PAGE analysis showing reduced molecular weight of purified FLAG-myopodin after dephosphorylation with λ-PPase. (B) Dephosphorylation of myopodin abrogates binding to GST–14-3-3β. The interaction is also prevented by the 14-3-3–blocking peptide R18. (C) Binding of purified FLAG-myopodin to endogenous 14-3-3β from C2C12 myoblasts is abrogated by dephosphorylation of myopodin (top). In contrast, binding of endogenous myopodin to purified FLAG–14-3-3β is not affected by the dephosphorylation of 14-3-3β (bottom). (D) Dephosphorylation with λ-PPase abrogates the interaction of FLAG-myopodin (bottom) with endogenous 14-3-3β (top) and importin α (middle) from X. laevis extracts. (E) Putative phosphoacceptor sites within the 14-3-3–binding motifs of myopodin. S225 in motif#1, as well as T272 and S273 in motif#2, were substituted with alanine to remove putative phosphorylation sites. Replacement with aspartic acid or glutamic acid was done to mimic phosphorylation. (F) Purified FLAG-tagged, wild-type myopodin interacts with GST–14-3-3β. Substitution of S225 or T272 with alanine (S225A, T272A, and S225AT272A) abrogates 14-3-3β binding. Replacement of S273 with alanine (S273A) does not interfere with the binding of myopodin to 14-3-3β. (G) Substitutions of S225 or T272 with aspartic or glutamic acid, respectively, does not alter the binding of myopodin to 14-3-3 (top). However, after dephosphorylation, only S225DT272E retains strong binding to GST–14-3-3β. Single mutations (S225D and T272E) bind significantly less, and wild-type binding is abrogated.
Mentions: 14-3-3 proteins are phosphoserine/threonine-binding proteins (Muslin et al., 1996), and many 14-3-3 interactions depend on the phosphorylation of the target protein (Yaffe, 2002). Therefore, we examined whether the myopodin–14-3-3 interaction is regulated by phosphorylation. To this end, purified FLAG-myopodin was dephosphorylated with λ protein phosphatase (λ-PPase), which reduced the molecular size of myopodin (Fig. 4 A). Moreover, λ-PPase treatment converted the fuzzier signal of the phosphorylated protein into a more compact and sharper band (Fig. 4 A, top). These findings show that myopodin is phosphorylated in vivo.

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