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Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor-2.

Okagaki T, Nakamura A, Suzuki T, Ohmi K, Kohama K - J. Cell Biol. (2000)

Bottom Line: Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro.The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy.The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.

ABSTRACT
Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation- dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH(2)-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.

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Amino acid sequence of the 38k protein deduced from cDNA sequencing. a, The sequences of the 38k protein as denoted by chicken 38k (GenBank/EMBL/DDBJ accession number AB029946) are aligned with human p32 (GenBank/EMBL/DDBJ identification number, g338043) and mouse YL2 (GenBank/EMBL/DDBJ identification number, g743485). Two sequences of the 38k protein determined by direct amino acid sequencing are underlined. Hatched sequences indicate identical amino acids. b, Expression of human p32 in E. coli. SDS-PAGE pattern of total cell homogenate before (1) and after (2) IPTG induction. Purified human p32 (3) and the 38k protein (4). c, Immunoblotting with anti-38k protein antibody. Total homogenate of chicken gizzard (1, 2, and 3) and the purified 38k protein (4, 5, and 6). Protein staining (1 and 4) and immunostaining with anti-38k protein antibody (2 and 5), and with preimmune serum (3 and 6). 38k and h-p32 denote the bands of the 38k protein and human p32, respectively. d, Expression of the 38k protein in various muscles. The 38k protein was detected in the homogenate of various smooth muscle tissues of rat. Lanes: esophagus (1), stomach (2), duodenum (3), small intestine (4), cecum (5), rectum (6), uterus (7), aorta (8), air way (9), skeletal muscle (10), and cardiac muscle (11). 10 μg of each homogenate were applied.
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Figure 6: Amino acid sequence of the 38k protein deduced from cDNA sequencing. a, The sequences of the 38k protein as denoted by chicken 38k (GenBank/EMBL/DDBJ accession number AB029946) are aligned with human p32 (GenBank/EMBL/DDBJ identification number, g338043) and mouse YL2 (GenBank/EMBL/DDBJ identification number, g743485). Two sequences of the 38k protein determined by direct amino acid sequencing are underlined. Hatched sequences indicate identical amino acids. b, Expression of human p32 in E. coli. SDS-PAGE pattern of total cell homogenate before (1) and after (2) IPTG induction. Purified human p32 (3) and the 38k protein (4). c, Immunoblotting with anti-38k protein antibody. Total homogenate of chicken gizzard (1, 2, and 3) and the purified 38k protein (4, 5, and 6). Protein staining (1 and 4) and immunostaining with anti-38k protein antibody (2 and 5), and with preimmune serum (3 and 6). 38k and h-p32 denote the bands of the 38k protein and human p32, respectively. d, Expression of the 38k protein in various muscles. The 38k protein was detected in the homogenate of various smooth muscle tissues of rat. Lanes: esophagus (1), stomach (2), duodenum (3), small intestine (4), cecum (5), rectum (6), uterus (7), aorta (8), air way (9), skeletal muscle (10), and cardiac muscle (11). 10 μg of each homogenate were applied.

Mentions: The binding of the 38k protein to skeletal muscle myosin was much weaker than that of smooth muscle myosin (see Fig. 5). The same was observed with cardiac myosin (see Fig. 5). These observations indicate that its binding is specific for smooth muscle myosin and agree with the observation that it is absent from skeletal muscle cells (see Fig. 6 d).


Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor-2.

Okagaki T, Nakamura A, Suzuki T, Ohmi K, Kohama K - J. Cell Biol. (2000)

Amino acid sequence of the 38k protein deduced from cDNA sequencing. a, The sequences of the 38k protein as denoted by chicken 38k (GenBank/EMBL/DDBJ accession number AB029946) are aligned with human p32 (GenBank/EMBL/DDBJ identification number, g338043) and mouse YL2 (GenBank/EMBL/DDBJ identification number, g743485). Two sequences of the 38k protein determined by direct amino acid sequencing are underlined. Hatched sequences indicate identical amino acids. b, Expression of human p32 in E. coli. SDS-PAGE pattern of total cell homogenate before (1) and after (2) IPTG induction. Purified human p32 (3) and the 38k protein (4). c, Immunoblotting with anti-38k protein antibody. Total homogenate of chicken gizzard (1, 2, and 3) and the purified 38k protein (4, 5, and 6). Protein staining (1 and 4) and immunostaining with anti-38k protein antibody (2 and 5), and with preimmune serum (3 and 6). 38k and h-p32 denote the bands of the 38k protein and human p32, respectively. d, Expression of the 38k protein in various muscles. The 38k protein was detected in the homogenate of various smooth muscle tissues of rat. Lanes: esophagus (1), stomach (2), duodenum (3), small intestine (4), cecum (5), rectum (6), uterus (7), aorta (8), air way (9), skeletal muscle (10), and cardiac muscle (11). 10 μg of each homogenate were applied.
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Related In: Results  -  Collection

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Figure 6: Amino acid sequence of the 38k protein deduced from cDNA sequencing. a, The sequences of the 38k protein as denoted by chicken 38k (GenBank/EMBL/DDBJ accession number AB029946) are aligned with human p32 (GenBank/EMBL/DDBJ identification number, g338043) and mouse YL2 (GenBank/EMBL/DDBJ identification number, g743485). Two sequences of the 38k protein determined by direct amino acid sequencing are underlined. Hatched sequences indicate identical amino acids. b, Expression of human p32 in E. coli. SDS-PAGE pattern of total cell homogenate before (1) and after (2) IPTG induction. Purified human p32 (3) and the 38k protein (4). c, Immunoblotting with anti-38k protein antibody. Total homogenate of chicken gizzard (1, 2, and 3) and the purified 38k protein (4, 5, and 6). Protein staining (1 and 4) and immunostaining with anti-38k protein antibody (2 and 5), and with preimmune serum (3 and 6). 38k and h-p32 denote the bands of the 38k protein and human p32, respectively. d, Expression of the 38k protein in various muscles. The 38k protein was detected in the homogenate of various smooth muscle tissues of rat. Lanes: esophagus (1), stomach (2), duodenum (3), small intestine (4), cecum (5), rectum (6), uterus (7), aorta (8), air way (9), skeletal muscle (10), and cardiac muscle (11). 10 μg of each homogenate were applied.
Mentions: The binding of the 38k protein to skeletal muscle myosin was much weaker than that of smooth muscle myosin (see Fig. 5). The same was observed with cardiac myosin (see Fig. 5). These observations indicate that its binding is specific for smooth muscle myosin and agree with the observation that it is absent from skeletal muscle cells (see Fig. 6 d).

Bottom Line: Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro.The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy.The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.

ABSTRACT
Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation- dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH(2)-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.

Show MeSH
Related in: MedlinePlus