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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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14-3-3 binding to myopodin is required for the interaction between myopodin and importin α. (A) Endogenous 14-3-3β from C2C12 myoblasts coimmmunoprecipitates with myopodin (FLAG-full-length). FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 show impaired binding to 14-3-3β. No binding to 14-3-3β is found for FLAG–Δ14-3-3#1 + 2. In contrast, deletion of one or both NLSs (FLAG-ΔNLS#1, FLAG-ΔNLS#2, and FLAG-ΔNLS#1 + 2) does not interfere with the binding of myopodin to 14-3-3β. Combined deletion of all four motifs (FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2) abrogates the binding of myopodin to 14-3-3β. (B) Binding of 14-3-3β (top) and importin α (middle) to FLAG-tagged myopodin (bottom). Single NLS deletions (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) do not impair the expression of myopodin or the interaction of myopodin with 14-3-3β or importin α. In contrast, the absence of both NLSs (FLAG-ΔNLS#1 + 2) causes loss of importin α binding, whereas 14-3-3β binding is preserved. Deletion of 14-3-3–binding motifs in myopodin (FLAG–Δ14-3-3#1, FLAG–Δ14-3-3#2, and FLAG–Δ14-3-3#1 + 2) abrogates the interaction of myopodin with 14-3-3β and with importin α. (C) The 14-3-3 inhibitory peptide R18 abrogates the binding of myopodin to 14-3-3β (top) and to importin α (bottom). (D) Immobilized GST–importin α, but not GST alone, binds purified FLAG-myopodin in the presence of purified FLAG–14-3-3β. In contrast, no interaction is found between GST–importin α and FLAG–14-3-3β or FLAG-myopodin alone.
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fig3: 14-3-3 binding to myopodin is required for the interaction between myopodin and importin α. (A) Endogenous 14-3-3β from C2C12 myoblasts coimmmunoprecipitates with myopodin (FLAG-full-length). FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 show impaired binding to 14-3-3β. No binding to 14-3-3β is found for FLAG–Δ14-3-3#1 + 2. In contrast, deletion of one or both NLSs (FLAG-ΔNLS#1, FLAG-ΔNLS#2, and FLAG-ΔNLS#1 + 2) does not interfere with the binding of myopodin to 14-3-3β. Combined deletion of all four motifs (FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2) abrogates the binding of myopodin to 14-3-3β. (B) Binding of 14-3-3β (top) and importin α (middle) to FLAG-tagged myopodin (bottom). Single NLS deletions (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) do not impair the expression of myopodin or the interaction of myopodin with 14-3-3β or importin α. In contrast, the absence of both NLSs (FLAG-ΔNLS#1 + 2) causes loss of importin α binding, whereas 14-3-3β binding is preserved. Deletion of 14-3-3–binding motifs in myopodin (FLAG–Δ14-3-3#1, FLAG–Δ14-3-3#2, and FLAG–Δ14-3-3#1 + 2) abrogates the interaction of myopodin with 14-3-3β and with importin α. (C) The 14-3-3 inhibitory peptide R18 abrogates the binding of myopodin to 14-3-3β (top) and to importin α (bottom). (D) Immobilized GST–importin α, but not GST alone, binds purified FLAG-myopodin in the presence of purified FLAG–14-3-3β. In contrast, no interaction is found between GST–importin α and FLAG–14-3-3β or FLAG-myopodin alone.

Mentions: To determine whether the various myopodin mutant forms (Fig. 2 C) can bind to endogenous 14-3-3β, FLAG-tagged proteins were expressed in C2C12 myoblasts and were immunoprecipitated with anti-FLAG antibody (Fig. 3 A, bottom). Similar to full-length myopodin (FLAG-full-length), FLAG–Δ14-3-3#1 and FLAG-Δ14-3-3#2 coprecipitated endogenous 14-3-3β, albeit to a lesser extent (Fig. 3 A, top). In contrast, FLAG-Δ14-3-3#1 + 2 did not bind to myopodin. These results corroborate the interaction studies in transfected HEK-293 cells (Fig. 1 H) and suggest that binding to 14-3-3β is required for the nuclear import of myopodin. The deletion of the two NLSs separately (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) or together (FLAG-ΔNLS#1 + 2) did not abrogate the myopodin–14-3-3β interaction. Therefore, the two NLSs in myopodin are not necessary for its binding to 14-3-3β. As expected, FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2 did not interact with endogenous 14-3-3β (Fig. 3 A).


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)

14-3-3 binding to myopodin is required for the interaction between myopodin and importin α. (A) Endogenous 14-3-3β from C2C12 myoblasts coimmmunoprecipitates with myopodin (FLAG-full-length). FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 show impaired binding to 14-3-3β. No binding to 14-3-3β is found for FLAG–Δ14-3-3#1 + 2. In contrast, deletion of one or both NLSs (FLAG-ΔNLS#1, FLAG-ΔNLS#2, and FLAG-ΔNLS#1 + 2) does not interfere with the binding of myopodin to 14-3-3β. Combined deletion of all four motifs (FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2) abrogates the binding of myopodin to 14-3-3β. (B) Binding of 14-3-3β (top) and importin α (middle) to FLAG-tagged myopodin (bottom). Single NLS deletions (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) do not impair the expression of myopodin or the interaction of myopodin with 14-3-3β or importin α. In contrast, the absence of both NLSs (FLAG-ΔNLS#1 + 2) causes loss of importin α binding, whereas 14-3-3β binding is preserved. Deletion of 14-3-3–binding motifs in myopodin (FLAG–Δ14-3-3#1, FLAG–Δ14-3-3#2, and FLAG–Δ14-3-3#1 + 2) abrogates the interaction of myopodin with 14-3-3β and with importin α. (C) The 14-3-3 inhibitory peptide R18 abrogates the binding of myopodin to 14-3-3β (top) and to importin α (bottom). (D) Immobilized GST–importin α, but not GST alone, binds purified FLAG-myopodin in the presence of purified FLAG–14-3-3β. In contrast, no interaction is found between GST–importin α and FLAG–14-3-3β or FLAG-myopodin alone.
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fig3: 14-3-3 binding to myopodin is required for the interaction between myopodin and importin α. (A) Endogenous 14-3-3β from C2C12 myoblasts coimmmunoprecipitates with myopodin (FLAG-full-length). FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 show impaired binding to 14-3-3β. No binding to 14-3-3β is found for FLAG–Δ14-3-3#1 + 2. In contrast, deletion of one or both NLSs (FLAG-ΔNLS#1, FLAG-ΔNLS#2, and FLAG-ΔNLS#1 + 2) does not interfere with the binding of myopodin to 14-3-3β. Combined deletion of all four motifs (FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2) abrogates the binding of myopodin to 14-3-3β. (B) Binding of 14-3-3β (top) and importin α (middle) to FLAG-tagged myopodin (bottom). Single NLS deletions (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) do not impair the expression of myopodin or the interaction of myopodin with 14-3-3β or importin α. In contrast, the absence of both NLSs (FLAG-ΔNLS#1 + 2) causes loss of importin α binding, whereas 14-3-3β binding is preserved. Deletion of 14-3-3–binding motifs in myopodin (FLAG–Δ14-3-3#1, FLAG–Δ14-3-3#2, and FLAG–Δ14-3-3#1 + 2) abrogates the interaction of myopodin with 14-3-3β and with importin α. (C) The 14-3-3 inhibitory peptide R18 abrogates the binding of myopodin to 14-3-3β (top) and to importin α (bottom). (D) Immobilized GST–importin α, but not GST alone, binds purified FLAG-myopodin in the presence of purified FLAG–14-3-3β. In contrast, no interaction is found between GST–importin α and FLAG–14-3-3β or FLAG-myopodin alone.
Mentions: To determine whether the various myopodin mutant forms (Fig. 2 C) can bind to endogenous 14-3-3β, FLAG-tagged proteins were expressed in C2C12 myoblasts and were immunoprecipitated with anti-FLAG antibody (Fig. 3 A, bottom). Similar to full-length myopodin (FLAG-full-length), FLAG–Δ14-3-3#1 and FLAG-Δ14-3-3#2 coprecipitated endogenous 14-3-3β, albeit to a lesser extent (Fig. 3 A, top). In contrast, FLAG-Δ14-3-3#1 + 2 did not bind to myopodin. These results corroborate the interaction studies in transfected HEK-293 cells (Fig. 1 H) and suggest that binding to 14-3-3β is required for the nuclear import of myopodin. The deletion of the two NLSs separately (FLAG-ΔNLS#1 and FLAG-ΔNLS#2) or together (FLAG-ΔNLS#1 + 2) did not abrogate the myopodin–14-3-3β interaction. Therefore, the two NLSs in myopodin are not necessary for its binding to 14-3-3β. As expected, FLAG–Δ14-3-3#1 + 2 + ΔNLS#1 + 2 did not interact with endogenous 14-3-3β (Fig. 3 A).

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