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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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Myopodin interacts with 14-3-3 via two consensus 14-3-3–binding motifs. (A) Myopodin contains two NLSs, a PPXY motif, two 14-3-3–binding motifs, and an actin-binding site. Arrows indicate potential interactions with other proteins or with protein domains (WW). The Myo-2 fragment (aa 187–420) was used as bait in a yeast two-hybrid screen. (B) 14-3-3β colocalizes with the Z-disc marker α-actinin in mouse skeletal muscle. (C) In undifferentiated C2C12 myoblasts, myopodin is predominantly found in the nucleus (top left), whereas 14-3-3β is preferentially found in the cytoplasm (top right). Blocking nuclear export with LMB does not affect the nuclear localization of myopodin (bottom left), but leads to the nuclear accumulation of 14-3-3β (bottom right). (D) Heat shock causes nuclear accumulation of 14-3-3β in differentiated myotubes, as visualized by double labeling with DAPI. (E) Myopodin from mouse skeletal muscle (left) and C2C12 myoblast extracts (right) specifically binds to GST–14-3-3β, but not to GST alone. (F) Coimmunoprecipitation experiments show that endogenous myopodin interacts with 14-3-3β in adult mouse heart. (left) IP with anti–14-3-3β; (right) IP with antimyopodin. No binding was found with a control IgG. (G) Myopodin binds to all 14-3-3 isoforms except 14-3-3τ. 14-3-3β lacking the NH2-terminal dimerization domain (βΔN) can bind to myopodin, albeit to a lesser extent. In contrast, 14-3-3ɛ carrying a point mutation (ɛK49E) that is known to abrogate target binding does not interact with myopodin. (H) The two consensus 14-3-3–binding motifs, 14-3-3#1 (sequence RSLASVP) and 14-3-3#2 (sequence RSVTSP), were deleted separately (Δ14-3-3#1 and Δ14-3-3#2) and together (Δ14-3-3#1 + 2). Both FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 display dramatically reduced binding to 14-3-3β as compared with full-length myopodin (full-length FLAG). Deletion of both 14-3-3–binding motifs (FLAG–Δ14-3-3#1 + 2) abrogates binding to 14-3-3β.
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fig1: Myopodin interacts with 14-3-3 via two consensus 14-3-3–binding motifs. (A) Myopodin contains two NLSs, a PPXY motif, two 14-3-3–binding motifs, and an actin-binding site. Arrows indicate potential interactions with other proteins or with protein domains (WW). The Myo-2 fragment (aa 187–420) was used as bait in a yeast two-hybrid screen. (B) 14-3-3β colocalizes with the Z-disc marker α-actinin in mouse skeletal muscle. (C) In undifferentiated C2C12 myoblasts, myopodin is predominantly found in the nucleus (top left), whereas 14-3-3β is preferentially found in the cytoplasm (top right). Blocking nuclear export with LMB does not affect the nuclear localization of myopodin (bottom left), but leads to the nuclear accumulation of 14-3-3β (bottom right). (D) Heat shock causes nuclear accumulation of 14-3-3β in differentiated myotubes, as visualized by double labeling with DAPI. (E) Myopodin from mouse skeletal muscle (left) and C2C12 myoblast extracts (right) specifically binds to GST–14-3-3β, but not to GST alone. (F) Coimmunoprecipitation experiments show that endogenous myopodin interacts with 14-3-3β in adult mouse heart. (left) IP with anti–14-3-3β; (right) IP with antimyopodin. No binding was found with a control IgG. (G) Myopodin binds to all 14-3-3 isoforms except 14-3-3τ. 14-3-3β lacking the NH2-terminal dimerization domain (βΔN) can bind to myopodin, albeit to a lesser extent. In contrast, 14-3-3ɛ carrying a point mutation (ɛK49E) that is known to abrogate target binding does not interact with myopodin. (H) The two consensus 14-3-3–binding motifs, 14-3-3#1 (sequence RSLASVP) and 14-3-3#2 (sequence RSVTSP), were deleted separately (Δ14-3-3#1 and Δ14-3-3#2) and together (Δ14-3-3#1 + 2). Both FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 display dramatically reduced binding to 14-3-3β as compared with full-length myopodin (full-length FLAG). Deletion of both 14-3-3–binding motifs (FLAG–Δ14-3-3#1 + 2) abrogates binding to 14-3-3β.

Mentions: Myopodin contains two classic nuclear localization signals, a PPXY motif, an actin-binding site (Weins et al., 2001), and two consensus 14-3-3–binding domains (Fig. 1 A). To identify myopodin-interacting proteins, we screened a mouse embryonic cDNA library using the yeast two-hybrid system. Because full-length myopodin was autoactive, we used the myopodin fragment Myo-2 (aa 187–420) as bait (Fig. 1 A). Among several others, we found a cDNA clone containing the COOH-terminal part of 14-3-3β (aa 143–246), which creates the binding cleft of 14-3-3 (Fu et al., 2000).


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)

Myopodin interacts with 14-3-3 via two consensus 14-3-3–binding motifs. (A) Myopodin contains two NLSs, a PPXY motif, two 14-3-3–binding motifs, and an actin-binding site. Arrows indicate potential interactions with other proteins or with protein domains (WW). The Myo-2 fragment (aa 187–420) was used as bait in a yeast two-hybrid screen. (B) 14-3-3β colocalizes with the Z-disc marker α-actinin in mouse skeletal muscle. (C) In undifferentiated C2C12 myoblasts, myopodin is predominantly found in the nucleus (top left), whereas 14-3-3β is preferentially found in the cytoplasm (top right). Blocking nuclear export with LMB does not affect the nuclear localization of myopodin (bottom left), but leads to the nuclear accumulation of 14-3-3β (bottom right). (D) Heat shock causes nuclear accumulation of 14-3-3β in differentiated myotubes, as visualized by double labeling with DAPI. (E) Myopodin from mouse skeletal muscle (left) and C2C12 myoblast extracts (right) specifically binds to GST–14-3-3β, but not to GST alone. (F) Coimmunoprecipitation experiments show that endogenous myopodin interacts with 14-3-3β in adult mouse heart. (left) IP with anti–14-3-3β; (right) IP with antimyopodin. No binding was found with a control IgG. (G) Myopodin binds to all 14-3-3 isoforms except 14-3-3τ. 14-3-3β lacking the NH2-terminal dimerization domain (βΔN) can bind to myopodin, albeit to a lesser extent. In contrast, 14-3-3ɛ carrying a point mutation (ɛK49E) that is known to abrogate target binding does not interact with myopodin. (H) The two consensus 14-3-3–binding motifs, 14-3-3#1 (sequence RSLASVP) and 14-3-3#2 (sequence RSVTSP), were deleted separately (Δ14-3-3#1 and Δ14-3-3#2) and together (Δ14-3-3#1 + 2). Both FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 display dramatically reduced binding to 14-3-3β as compared with full-length myopodin (full-length FLAG). Deletion of both 14-3-3–binding motifs (FLAG–Δ14-3-3#1 + 2) abrogates binding to 14-3-3β.
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fig1: Myopodin interacts with 14-3-3 via two consensus 14-3-3–binding motifs. (A) Myopodin contains two NLSs, a PPXY motif, two 14-3-3–binding motifs, and an actin-binding site. Arrows indicate potential interactions with other proteins or with protein domains (WW). The Myo-2 fragment (aa 187–420) was used as bait in a yeast two-hybrid screen. (B) 14-3-3β colocalizes with the Z-disc marker α-actinin in mouse skeletal muscle. (C) In undifferentiated C2C12 myoblasts, myopodin is predominantly found in the nucleus (top left), whereas 14-3-3β is preferentially found in the cytoplasm (top right). Blocking nuclear export with LMB does not affect the nuclear localization of myopodin (bottom left), but leads to the nuclear accumulation of 14-3-3β (bottom right). (D) Heat shock causes nuclear accumulation of 14-3-3β in differentiated myotubes, as visualized by double labeling with DAPI. (E) Myopodin from mouse skeletal muscle (left) and C2C12 myoblast extracts (right) specifically binds to GST–14-3-3β, but not to GST alone. (F) Coimmunoprecipitation experiments show that endogenous myopodin interacts with 14-3-3β in adult mouse heart. (left) IP with anti–14-3-3β; (right) IP with antimyopodin. No binding was found with a control IgG. (G) Myopodin binds to all 14-3-3 isoforms except 14-3-3τ. 14-3-3β lacking the NH2-terminal dimerization domain (βΔN) can bind to myopodin, albeit to a lesser extent. In contrast, 14-3-3ɛ carrying a point mutation (ɛK49E) that is known to abrogate target binding does not interact with myopodin. (H) The two consensus 14-3-3–binding motifs, 14-3-3#1 (sequence RSLASVP) and 14-3-3#2 (sequence RSVTSP), were deleted separately (Δ14-3-3#1 and Δ14-3-3#2) and together (Δ14-3-3#1 + 2). Both FLAG–Δ14-3-3#1 and FLAG–Δ14-3-3#2 display dramatically reduced binding to 14-3-3β as compared with full-length myopodin (full-length FLAG). Deletion of both 14-3-3–binding motifs (FLAG–Δ14-3-3#1 + 2) abrogates binding to 14-3-3β.
Mentions: Myopodin contains two classic nuclear localization signals, a PPXY motif, an actin-binding site (Weins et al., 2001), and two consensus 14-3-3–binding domains (Fig. 1 A). To identify myopodin-interacting proteins, we screened a mouse embryonic cDNA library using the yeast two-hybrid system. Because full-length myopodin was autoactive, we used the myopodin fragment Myo-2 (aa 187–420) as bait (Fig. 1 A). Among several others, we found a cDNA clone containing the COOH-terminal part of 14-3-3β (aa 143–246), which creates the binding cleft of 14-3-3 (Fu et al., 2000).

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