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Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

Bang ML, Mudry RE, McElhinny AS, Trombitás K, Geach AJ, Yamasaki R, Sorimachi H, Granzier H, Gregorio CC, Labeit S - J. Cell Biol. (2001)

Bottom Line: Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A.Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity.Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg 69117, Germany.

ABSTRACT
We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

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Myopalladin interacts with the EF hand region of α–actinin-2. (A) The schematic structure of α–actinin-2 (ACTN2) with its four central rod domains, R1–R4, and its unique terminal sequence is shown. Numbers indicate the nucleotide or amino acid residue numbers deduced from the human full-length cDNA sequence. AAA indicates a poly(A+) tail. A series of ACTN2 deletion constructs (below) were tested for interaction with full-length myopalladin in the yeast two-hybrid system. This assigned the myopalladin-binding domain within α-actinin to its COOH-terminal region containing the two EF hands. (+) and (−) denote the presence or absence of the growth of yeast colonies on SD-/Trp-/Leu-/His-plates supplemented with 1.5 mM 3-AT. (B) Interaction of myopalladin with α-actinin in GST pull-down assays. Myopalladin's COOH-terminal 376 residues were translated in vitro (IVT-myopal-Δ6, left). When incubated together with expressed ACTN14-GST fusion peptides, their binding to glutathione-Sepharose 4B beads was observed (right). Middle lane, negative control pull-down performed without the addition of the α-actinin fragment (for details, see Materials and Methods).
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Figure 5: Myopalladin interacts with the EF hand region of α–actinin-2. (A) The schematic structure of α–actinin-2 (ACTN2) with its four central rod domains, R1–R4, and its unique terminal sequence is shown. Numbers indicate the nucleotide or amino acid residue numbers deduced from the human full-length cDNA sequence. AAA indicates a poly(A+) tail. A series of ACTN2 deletion constructs (below) were tested for interaction with full-length myopalladin in the yeast two-hybrid system. This assigned the myopalladin-binding domain within α-actinin to its COOH-terminal region containing the two EF hands. (+) and (−) denote the presence or absence of the growth of yeast colonies on SD-/Trp-/Leu-/His-plates supplemented with 1.5 mM 3-AT. (B) Interaction of myopalladin with α-actinin in GST pull-down assays. Myopalladin's COOH-terminal 376 residues were translated in vitro (IVT-myopal-Δ6, left). When incubated together with expressed ACTN14-GST fusion peptides, their binding to glutathione-Sepharose 4B beads was observed (right). Middle lane, negative control pull-down performed without the addition of the α-actinin fragment (for details, see Materials and Methods).

Mentions: To search for other myopalladin binding partners, we screened a human skeletal muscle cDNA library with a bait containing myopalladin's full-length coding sequence (BTM-myopalladin; Fig. 4 A). By screening of ∼1.2 × 106 colonies, 700 prey clones were identified, all of which were confirmed in the β-galactosidase assay. 65 β-galactosidase–expressing clones were randomly chosen and sequenced. Three of the prey clones were derived from the COOH-terminal part of nebulin, confirming again the nebulin SH3–myopalladin interaction. 13 of the prey clones contained parts of the ACTN2 gene, which encodes the α–actinin-2 isoform (Beggs et al. 1992). When hybridizing α–actinin-2 probes to the 700 yeast colonies from the myopalladin screen, 155 clones (22%) hybridized to ACTN2. Sequence analysis of 12 ACTN2-positive clones showed that they all share a region of overlap in the COOH-terminal region of sarcomeric α–actinin-2. To further narrow down the myopalladin-binding site within α–actinin-2, the interaction of myopalladin with various deletion constructs of α–actinin-2 (Sorimachi et al. 1997) was tested (Fig. 5 A). This analysis showed that α-actinin's binding site for myopalladin is located in the COOH-terminal part of α–actinin-2, from just after the spectrin repeats (before the two EF hands) up to the extreme COOH terminus. This interaction was confirmed by GST pull-down assays (Fig. 5 B).


Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

Bang ML, Mudry RE, McElhinny AS, Trombitás K, Geach AJ, Yamasaki R, Sorimachi H, Granzier H, Gregorio CC, Labeit S - J. Cell Biol. (2001)

Myopalladin interacts with the EF hand region of α–actinin-2. (A) The schematic structure of α–actinin-2 (ACTN2) with its four central rod domains, R1–R4, and its unique terminal sequence is shown. Numbers indicate the nucleotide or amino acid residue numbers deduced from the human full-length cDNA sequence. AAA indicates a poly(A+) tail. A series of ACTN2 deletion constructs (below) were tested for interaction with full-length myopalladin in the yeast two-hybrid system. This assigned the myopalladin-binding domain within α-actinin to its COOH-terminal region containing the two EF hands. (+) and (−) denote the presence or absence of the growth of yeast colonies on SD-/Trp-/Leu-/His-plates supplemented with 1.5 mM 3-AT. (B) Interaction of myopalladin with α-actinin in GST pull-down assays. Myopalladin's COOH-terminal 376 residues were translated in vitro (IVT-myopal-Δ6, left). When incubated together with expressed ACTN14-GST fusion peptides, their binding to glutathione-Sepharose 4B beads was observed (right). Middle lane, negative control pull-down performed without the addition of the α-actinin fragment (for details, see Materials and Methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169455&req=5

Figure 5: Myopalladin interacts with the EF hand region of α–actinin-2. (A) The schematic structure of α–actinin-2 (ACTN2) with its four central rod domains, R1–R4, and its unique terminal sequence is shown. Numbers indicate the nucleotide or amino acid residue numbers deduced from the human full-length cDNA sequence. AAA indicates a poly(A+) tail. A series of ACTN2 deletion constructs (below) were tested for interaction with full-length myopalladin in the yeast two-hybrid system. This assigned the myopalladin-binding domain within α-actinin to its COOH-terminal region containing the two EF hands. (+) and (−) denote the presence or absence of the growth of yeast colonies on SD-/Trp-/Leu-/His-plates supplemented with 1.5 mM 3-AT. (B) Interaction of myopalladin with α-actinin in GST pull-down assays. Myopalladin's COOH-terminal 376 residues were translated in vitro (IVT-myopal-Δ6, left). When incubated together with expressed ACTN14-GST fusion peptides, their binding to glutathione-Sepharose 4B beads was observed (right). Middle lane, negative control pull-down performed without the addition of the α-actinin fragment (for details, see Materials and Methods).
Mentions: To search for other myopalladin binding partners, we screened a human skeletal muscle cDNA library with a bait containing myopalladin's full-length coding sequence (BTM-myopalladin; Fig. 4 A). By screening of ∼1.2 × 106 colonies, 700 prey clones were identified, all of which were confirmed in the β-galactosidase assay. 65 β-galactosidase–expressing clones were randomly chosen and sequenced. Three of the prey clones were derived from the COOH-terminal part of nebulin, confirming again the nebulin SH3–myopalladin interaction. 13 of the prey clones contained parts of the ACTN2 gene, which encodes the α–actinin-2 isoform (Beggs et al. 1992). When hybridizing α–actinin-2 probes to the 700 yeast colonies from the myopalladin screen, 155 clones (22%) hybridized to ACTN2. Sequence analysis of 12 ACTN2-positive clones showed that they all share a region of overlap in the COOH-terminal region of sarcomeric α–actinin-2. To further narrow down the myopalladin-binding site within α–actinin-2, the interaction of myopalladin with various deletion constructs of α–actinin-2 (Sorimachi et al. 1997) was tested (Fig. 5 A). This analysis showed that α-actinin's binding site for myopalladin is located in the COOH-terminal part of α–actinin-2, from just after the spectrin repeats (before the two EF hands) up to the extreme COOH terminus. This interaction was confirmed by GST pull-down assays (Fig. 5 B).

Bottom Line: Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A.Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity.Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg 69117, Germany.

ABSTRACT
We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Show MeSH
Related in: MedlinePlus