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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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The amounts of the 38k protein coprecipitated by binding to myosin and its fragments, and striated muscle myosins. The assembly of unphosphorylated myosins was observed only in the presence of the 38k protein (see Fig. 3). The assembly of rod, LMM, skeletal, and cardiac myosins do not require the 38k protein. About 95% of total amount of rod, 85% of that of LMM, 90% of that of skeletal and cardiac myosins could be precipitatable under our assay conditions. The 38k protein was mixed with 1.1 μM of various types of myosin and its fragments and centrifuged to coprecipitate with them. The amounts of the 38k protein recovered in the precipitate were quantified by the densitometry. Rod (open circles), LMM (closed circles), unphosphorylated myosin (open triangles), skeletal (open squares), and cardiac myosins (closed squares). Data are from the average of two sets of independent experiments. Inset, Gel filtration pattern of the mixture of HMM and the 38k protein. The mixture of 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to Superose 6 equilibrated with buffer A and eluted with buffer A in a flow rate of 0.5 ml/min. A280 in arbitrary unit (ordinate) was plotted against elution time (abscissa). HMM and the 38k protein was separately eluted in the peak at 31 and 35 min, respectively. SDS-PAGE patterns of peak fractions of HMM (left) and the 38k protein (right) were also shown. Arrowheads indicate HMM (H) and the 38k protein (38).
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Figure 5: The amounts of the 38k protein coprecipitated by binding to myosin and its fragments, and striated muscle myosins. The assembly of unphosphorylated myosins was observed only in the presence of the 38k protein (see Fig. 3). The assembly of rod, LMM, skeletal, and cardiac myosins do not require the 38k protein. About 95% of total amount of rod, 85% of that of LMM, 90% of that of skeletal and cardiac myosins could be precipitatable under our assay conditions. The 38k protein was mixed with 1.1 μM of various types of myosin and its fragments and centrifuged to coprecipitate with them. The amounts of the 38k protein recovered in the precipitate were quantified by the densitometry. Rod (open circles), LMM (closed circles), unphosphorylated myosin (open triangles), skeletal (open squares), and cardiac myosins (closed squares). Data are from the average of two sets of independent experiments. Inset, Gel filtration pattern of the mixture of HMM and the 38k protein. The mixture of 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to Superose 6 equilibrated with buffer A and eluted with buffer A in a flow rate of 0.5 ml/min. A280 in arbitrary unit (ordinate) was plotted against elution time (abscissa). HMM and the 38k protein was separately eluted in the peak at 31 and 35 min, respectively. SDS-PAGE patterns of peak fractions of HMM (left) and the 38k protein (right) were also shown. Arrowheads indicate HMM (H) and the 38k protein (38).

Mentions: The centrifugation assay of the 38k protein bound to insoluble myosin fragments was also performed in a similar way. Various concentrations of the 38k protein were mixed with 1.1 μM rod or LMM in buffer A, centrifuged, and analyzed as above. The interaction of the 38k protein to soluble myosin-fragment of HMM was examined by the gel filtration (see Fig. 5, inset), i.e., 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to the column Superose 6 (Amersham-Pharmacia).


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)

The amounts of the 38k protein coprecipitated by binding to myosin and its fragments, and striated muscle myosins. The assembly of unphosphorylated myosins was observed only in the presence of the 38k protein (see Fig. 3). The assembly of rod, LMM, skeletal, and cardiac myosins do not require the 38k protein. About 95% of total amount of rod, 85% of that of LMM, 90% of that of skeletal and cardiac myosins could be precipitatable under our assay conditions. The 38k protein was mixed with 1.1 μM of various types of myosin and its fragments and centrifuged to coprecipitate with them. The amounts of the 38k protein recovered in the precipitate were quantified by the densitometry. Rod (open circles), LMM (closed circles), unphosphorylated myosin (open triangles), skeletal (open squares), and cardiac myosins (closed squares). Data are from the average of two sets of independent experiments. Inset, Gel filtration pattern of the mixture of HMM and the 38k protein. The mixture of 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to Superose 6 equilibrated with buffer A and eluted with buffer A in a flow rate of 0.5 ml/min. A280 in arbitrary unit (ordinate) was plotted against elution time (abscissa). HMM and the 38k protein was separately eluted in the peak at 31 and 35 min, respectively. SDS-PAGE patterns of peak fractions of HMM (left) and the 38k protein (right) were also shown. Arrowheads indicate HMM (H) and the 38k protein (38).
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Figure 5: The amounts of the 38k protein coprecipitated by binding to myosin and its fragments, and striated muscle myosins. The assembly of unphosphorylated myosins was observed only in the presence of the 38k protein (see Fig. 3). The assembly of rod, LMM, skeletal, and cardiac myosins do not require the 38k protein. About 95% of total amount of rod, 85% of that of LMM, 90% of that of skeletal and cardiac myosins could be precipitatable under our assay conditions. The 38k protein was mixed with 1.1 μM of various types of myosin and its fragments and centrifuged to coprecipitate with them. The amounts of the 38k protein recovered in the precipitate were quantified by the densitometry. Rod (open circles), LMM (closed circles), unphosphorylated myosin (open triangles), skeletal (open squares), and cardiac myosins (closed squares). Data are from the average of two sets of independent experiments. Inset, Gel filtration pattern of the mixture of HMM and the 38k protein. The mixture of 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to Superose 6 equilibrated with buffer A and eluted with buffer A in a flow rate of 0.5 ml/min. A280 in arbitrary unit (ordinate) was plotted against elution time (abscissa). HMM and the 38k protein was separately eluted in the peak at 31 and 35 min, respectively. SDS-PAGE patterns of peak fractions of HMM (left) and the 38k protein (right) were also shown. Arrowheads indicate HMM (H) and the 38k protein (38).
Mentions: The centrifugation assay of the 38k protein bound to insoluble myosin fragments was also performed in a similar way. Various concentrations of the 38k protein were mixed with 1.1 μM rod or LMM in buffer A, centrifuged, and analyzed as above. The interaction of the 38k protein to soluble myosin-fragment of HMM was examined by the gel filtration (see Fig. 5, inset), i.e., 8.5 μM HMM and 40 μM 38k protein in 300 μl buffer A was applied to the column Superose 6 (Amersham-Pharmacia).

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