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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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Myosin assembly by human p32. a, Electron micrographs of 0.2 μM unphosphorylated myosin alone (1) or mixed with 0.6 μM human p32 (2). b, Length distribution of myosin filaments formed by human p32. The histogram was obtained by measurement of 150 filaments. An arrow indicates average length. c, Quantification of myosin assembly by the sedimentation assay. Myosin at 1.1 μM was mixed with human p32 and subjected to the centrifugation assay of assembly. Amounts of assembled myosin were plotted against concentration of human p32. Open and closed circles denote unphosphorylated and phosphorylated myosins, respectively.
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Figure 7: Myosin assembly by human p32. a, Electron micrographs of 0.2 μM unphosphorylated myosin alone (1) or mixed with 0.6 μM human p32 (2). b, Length distribution of myosin filaments formed by human p32. The histogram was obtained by measurement of 150 filaments. An arrow indicates average length. c, Quantification of myosin assembly by the sedimentation assay. Myosin at 1.1 μM was mixed with human p32 and subjected to the centrifugation assay of assembly. Amounts of assembled myosin were plotted against concentration of human p32. Open and closed circles denote unphosphorylated and phosphorylated myosins, respectively.

Mentions: To demonstrate the colocalization of the 38k protein/myosin and that of the 38k protein/mitochondria in the smooth muscle cells (see Fig. 7), they were subjected to the double staining with anti-38k protein antibody, and with anti-MHC antibody or with MitoTracker Red CMXRos. Each pair of images were incorporated into Adobe Photoshop (version 4.0) and then processed to create product images (see Fig. 7c and Fig. f).


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)

Myosin assembly by human p32. a, Electron micrographs of 0.2 μM unphosphorylated myosin alone (1) or mixed with 0.6 μM human p32 (2). b, Length distribution of myosin filaments formed by human p32. The histogram was obtained by measurement of 150 filaments. An arrow indicates average length. c, Quantification of myosin assembly by the sedimentation assay. Myosin at 1.1 μM was mixed with human p32 and subjected to the centrifugation assay of assembly. Amounts of assembled myosin were plotted against concentration of human p32. Open and closed circles denote unphosphorylated and phosphorylated myosins, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169363&req=5

Figure 7: Myosin assembly by human p32. a, Electron micrographs of 0.2 μM unphosphorylated myosin alone (1) or mixed with 0.6 μM human p32 (2). b, Length distribution of myosin filaments formed by human p32. The histogram was obtained by measurement of 150 filaments. An arrow indicates average length. c, Quantification of myosin assembly by the sedimentation assay. Myosin at 1.1 μM was mixed with human p32 and subjected to the centrifugation assay of assembly. Amounts of assembled myosin were plotted against concentration of human p32. Open and closed circles denote unphosphorylated and phosphorylated myosins, respectively.
Mentions: To demonstrate the colocalization of the 38k protein/myosin and that of the 38k protein/mitochondria in the smooth muscle cells (see Fig. 7), they were subjected to the double staining with anti-38k protein antibody, and with anti-MHC antibody or with MitoTracker Red CMXRos. Each pair of images were incorporated into Adobe Photoshop (version 4.0) and then processed to create product images (see Fig. 7c and Fig. f).

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