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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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Yeast two-hybrid screens identify myopalladin and desmin as Z-line nebulin-binding proteins. Schematic structure of the COOH-terminal region of nebulin and its location within the sarcomere is shown based on previous immunoelectron microscopy studies (Millevoi et al. 1998). The nebulin COOH-terminal region constructs pAS2-M160-M185+Ser+SH3C and pAS2-M160-M183 identified myopalladin and desmin prey clones in yeast two-hybrid screens. Nebulin bait deletion constructs (NebΔ1–NebΔ3) allowed us to further map the myopalladin-binding region on nebulin to within the nebulin SH3 domain. The desmin-binding site on nebulin was found to be located within the nebulin M160-M183 modules. (+) 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.
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Figure 1: Yeast two-hybrid screens identify myopalladin and desmin as Z-line nebulin-binding proteins. Schematic structure of the COOH-terminal region of nebulin and its location within the sarcomere is shown based on previous immunoelectron microscopy studies (Millevoi et al. 1998). The nebulin COOH-terminal region constructs pAS2-M160-M185+Ser+SH3C and pAS2-M160-M183 identified myopalladin and desmin prey clones in yeast two-hybrid screens. Nebulin bait deletion constructs (NebΔ1–NebΔ3) allowed us to further map the myopalladin-binding region on nebulin to within the nebulin SH3 domain. The desmin-binding site on nebulin was found to be located within the nebulin M160-M183 modules. (+) 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.

Mentions: To search for proteins interacting with the Z-disc region of nebulin, a fragment corresponding to its COOH-terminal 2,687 kb was amplified from rabbit psoas skeletal muscle and inserted into the yeast two-hybrid bait vector pAS2-1. The bait, referred to as pAS2-M160-M185+Ser+SH3, included the nebulin repeats, M160–M176 and M182–M185, the Ser-rich region, and the SH3 domain (Fig. 1). The nebulin repeats M177–M181 are absent from rabbit psoas muscle nebulin (Millevoi et al. 1998). When screening 700,000 clones from a human skeletal library with pAS2-M160-M185+Ser+SH3, >1,000 potentially interacting clones were identified. From these, 100 clones were randomly picked and analyzed for β-galactosidase expression. 12 clones with the highest levels of β-galactosidase expression were sequenced. One clone corresponded to a partial cDNA, which encoded one Ig repeat and was derived from a palladin-homologous gene (Parast and Otey 2000) described here as “myopalladin.” The full-length cDNA sequence of myopalladin was obtained by the screening of both human skeletal and heart muscle cDNA libraries and performing extensions to reach the 5′ and 3′ end. This identified a 5.7-kb full-length cDNA for myopalladin (see Materials and Methods), which contains a single open reading frame and encodes a protein of 1,320 amino acids with a predicted molecular mass of 145 kD (see Fig. 4 A). We found no evidence for differentially spliced myopalladin isoforms in heart and skeletal muscle (not shown).


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)

Yeast two-hybrid screens identify myopalladin and desmin as Z-line nebulin-binding proteins. Schematic structure of the COOH-terminal region of nebulin and its location within the sarcomere is shown based on previous immunoelectron microscopy studies (Millevoi et al. 1998). The nebulin COOH-terminal region constructs pAS2-M160-M185+Ser+SH3C and pAS2-M160-M183 identified myopalladin and desmin prey clones in yeast two-hybrid screens. Nebulin bait deletion constructs (NebΔ1–NebΔ3) allowed us to further map the myopalladin-binding region on nebulin to within the nebulin SH3 domain. The desmin-binding site on nebulin was found to be located within the nebulin M160-M183 modules. (+) 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169455&req=5

Figure 1: Yeast two-hybrid screens identify myopalladin and desmin as Z-line nebulin-binding proteins. Schematic structure of the COOH-terminal region of nebulin and its location within the sarcomere is shown based on previous immunoelectron microscopy studies (Millevoi et al. 1998). The nebulin COOH-terminal region constructs pAS2-M160-M185+Ser+SH3C and pAS2-M160-M183 identified myopalladin and desmin prey clones in yeast two-hybrid screens. Nebulin bait deletion constructs (NebΔ1–NebΔ3) allowed us to further map the myopalladin-binding region on nebulin to within the nebulin SH3 domain. The desmin-binding site on nebulin was found to be located within the nebulin M160-M183 modules. (+) 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.
Mentions: To search for proteins interacting with the Z-disc region of nebulin, a fragment corresponding to its COOH-terminal 2,687 kb was amplified from rabbit psoas skeletal muscle and inserted into the yeast two-hybrid bait vector pAS2-1. The bait, referred to as pAS2-M160-M185+Ser+SH3, included the nebulin repeats, M160–M176 and M182–M185, the Ser-rich region, and the SH3 domain (Fig. 1). The nebulin repeats M177–M181 are absent from rabbit psoas muscle nebulin (Millevoi et al. 1998). When screening 700,000 clones from a human skeletal library with pAS2-M160-M185+Ser+SH3, >1,000 potentially interacting clones were identified. From these, 100 clones were randomly picked and analyzed for β-galactosidase expression. 12 clones with the highest levels of β-galactosidase expression were sequenced. One clone corresponded to a partial cDNA, which encoded one Ig repeat and was derived from a palladin-homologous gene (Parast and Otey 2000) described here as “myopalladin.” The full-length cDNA sequence of myopalladin was obtained by the screening of both human skeletal and heart muscle cDNA libraries and performing extensions to reach the 5′ and 3′ end. This identified a 5.7-kb full-length cDNA for myopalladin (see Materials and Methods), which contains a single open reading frame and encodes a protein of 1,320 amino acids with a predicted molecular mass of 145 kD (see Fig. 4 A). We found no evidence for differentially spliced myopalladin isoforms in heart and skeletal muscle (not shown).

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