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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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Colocalization of the 38k protein with components of contractile apparatus in the cultured smooth muscle cell line, AC01. a–h, Double staining of AC01 cell with anti-38k protein antibody, as detected by FITC-labeled second antibody and with various antibodies as detected by rhodamine-labeled second antibody. Staining pattern with anti-38k protein antibody (a, c, e, and g) and with antibodies against MHC (b), α-actin (d), β-actin (f), or desmin (h). Magnification is the same in all pictures. Bar, 30 μm.
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Figure 9: Colocalization of the 38k protein with components of contractile apparatus in the cultured smooth muscle cell line, AC01. a–h, Double staining of AC01 cell with anti-38k protein antibody, as detected by FITC-labeled second antibody and with various antibodies as detected by rhodamine-labeled second antibody. Staining pattern with anti-38k protein antibody (a, c, e, and g) and with antibodies against MHC (b), α-actin (d), β-actin (f), or desmin (h). Magnification is the same in all pictures. Bar, 30 μm.

Mentions: To examine whether the 38k protein is localized in the contractile apparatus composed of myosin and/or actin, we performed further double staining with antibodies against components of the contractile apparatus other than myosin. At least two types of actin isoforms are detected in AC01 cells by Western blotting, α- and β-actin (data not shown). Localization of the 38k protein in the cells is close to that of α-actin (Fig. 9c and Fig. d), but does not necessarily match that of β-actin (Fig. 9e and Fig. f). The localization of the 38k protein overlaps that of desmin, a major component of dense bodies (Fig. 9g and Fig. h).


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)

Colocalization of the 38k protein with components of contractile apparatus in the cultured smooth muscle cell line, AC01. a–h, Double staining of AC01 cell with anti-38k protein antibody, as detected by FITC-labeled second antibody and with various antibodies as detected by rhodamine-labeled second antibody. Staining pattern with anti-38k protein antibody (a, c, e, and g) and with antibodies against MHC (b), α-actin (d), β-actin (f), or desmin (h). Magnification is the same in all pictures. Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Colocalization of the 38k protein with components of contractile apparatus in the cultured smooth muscle cell line, AC01. a–h, Double staining of AC01 cell with anti-38k protein antibody, as detected by FITC-labeled second antibody and with various antibodies as detected by rhodamine-labeled second antibody. Staining pattern with anti-38k protein antibody (a, c, e, and g) and with antibodies against MHC (b), α-actin (d), β-actin (f), or desmin (h). Magnification is the same in all pictures. Bar, 30 μm.
Mentions: To examine whether the 38k protein is localized in the contractile apparatus composed of myosin and/or actin, we performed further double staining with antibodies against components of the contractile apparatus other than myosin. At least two types of actin isoforms are detected in AC01 cells by Western blotting, α- and β-actin (data not shown). Localization of the 38k protein in the cells is close to that of α-actin (Fig. 9c and Fig. d), but does not necessarily match that of β-actin (Fig. 9e and Fig. f). The localization of the 38k protein overlaps that of desmin, a major component of dense bodies (Fig. 9g and Fig. h).

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