Limits...
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.

Show MeSH

Related in: MedlinePlus

Sedimentation assay of myosin assembly. Myosin (1.1 μM) and the 38k protein were mixed in buffer A, and the mixture was allowed to assemble. The assembled myosin was precipitated by centrifugation and quantified. a, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) after centrifugation of the mixture of unphosphorylated myosin and the 38k protein. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). b, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the sedimentation assay with phosphorylated myosin. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). c, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the binding assay of the 38k protein to actin filaments. Actin filaments alone at 10 μM (1 and 2); 10 μM actin filaments and 5.4 μM of the 38k protein (3 and 4); and 10 μM actin filaments, 1.4 μM tropomyosin, and 5.4 μM of the 38k protein (5 and 6). d, The amount of assembled myosin was plotted against concentration of the 38k protein mixed with 1.1 μM myosin. Open and closed circles indicate unphosphorylated and phosphorylated myosin, respectively. e, The amount of the 38k protein bound to myosin was plotted against the 38k protein mixed with 1.1 μM myosin. Open and closed circles denote the unphosphorylated and phosphorylated myosin, respectively. MHC, 38k, A, α-TM, and β-TM denote the bands of MHC, the 38k protein, actin, and α- and β-tropomyosins, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169363&req=5

Figure 3: Sedimentation assay of myosin assembly. Myosin (1.1 μM) and the 38k protein were mixed in buffer A, and the mixture was allowed to assemble. The assembled myosin was precipitated by centrifugation and quantified. a, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) after centrifugation of the mixture of unphosphorylated myosin and the 38k protein. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). b, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the sedimentation assay with phosphorylated myosin. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). c, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the binding assay of the 38k protein to actin filaments. Actin filaments alone at 10 μM (1 and 2); 10 μM actin filaments and 5.4 μM of the 38k protein (3 and 4); and 10 μM actin filaments, 1.4 μM tropomyosin, and 5.4 μM of the 38k protein (5 and 6). d, The amount of assembled myosin was plotted against concentration of the 38k protein mixed with 1.1 μM myosin. Open and closed circles indicate unphosphorylated and phosphorylated myosin, respectively. e, The amount of the 38k protein bound to myosin was plotted against the 38k protein mixed with 1.1 μM myosin. Open and closed circles denote the unphosphorylated and phosphorylated myosin, respectively. MHC, 38k, A, α-TM, and β-TM denote the bands of MHC, the 38k protein, actin, and α- and β-tropomyosins, respectively.

Mentions: The relationship of the amount of the 38k protein bound to unphosphorylated myosin is shown (see Fig. 3 e). Unphosphorylated myosin remains in the supernatant if insufficient 38k protein is added. Therefore, it is possible that part of the 38k protein remains in the supernatant, despite its binding to myosin. However, we believe that this is unlikely and that the 38k protein bound to unphosphorylated myosin is mostly recovered in the precipitate (compare open and closed circles in Fig. 3 e). The binding of 5.4 μM of 38k protein to 10 μM actin filaments in the presence and absence of tropomyosin was examined by a similar centrifugation assay (Ishikawa et al. 1989).


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)

Sedimentation assay of myosin assembly. Myosin (1.1 μM) and the 38k protein were mixed in buffer A, and the mixture was allowed to assemble. The assembled myosin was precipitated by centrifugation and quantified. a, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) after centrifugation of the mixture of unphosphorylated myosin and the 38k protein. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). b, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the sedimentation assay with phosphorylated myosin. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). c, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the binding assay of the 38k protein to actin filaments. Actin filaments alone at 10 μM (1 and 2); 10 μM actin filaments and 5.4 μM of the 38k protein (3 and 4); and 10 μM actin filaments, 1.4 μM tropomyosin, and 5.4 μM of the 38k protein (5 and 6). d, The amount of assembled myosin was plotted against concentration of the 38k protein mixed with 1.1 μM myosin. Open and closed circles indicate unphosphorylated and phosphorylated myosin, respectively. e, The amount of the 38k protein bound to myosin was plotted against the 38k protein mixed with 1.1 μM myosin. Open and closed circles denote the unphosphorylated and phosphorylated myosin, respectively. MHC, 38k, A, α-TM, and β-TM denote the bands of MHC, the 38k protein, actin, and α- and β-tropomyosins, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Sedimentation assay of myosin assembly. Myosin (1.1 μM) and the 38k protein were mixed in buffer A, and the mixture was allowed to assemble. The assembled myosin was precipitated by centrifugation and quantified. a, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) after centrifugation of the mixture of unphosphorylated myosin and the 38k protein. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). b, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the sedimentation assay with phosphorylated myosin. Concentrations of the 38k protein were 0 μM (1 and 2), 1.4 μM (3 and 4), and 5.4 μM (5 and 6). c, SDS-PAGE pattern of supernatants (1, 3, and 5) and precipitates (2, 4, and 6) from the binding assay of the 38k protein to actin filaments. Actin filaments alone at 10 μM (1 and 2); 10 μM actin filaments and 5.4 μM of the 38k protein (3 and 4); and 10 μM actin filaments, 1.4 μM tropomyosin, and 5.4 μM of the 38k protein (5 and 6). d, The amount of assembled myosin was plotted against concentration of the 38k protein mixed with 1.1 μM myosin. Open and closed circles indicate unphosphorylated and phosphorylated myosin, respectively. e, The amount of the 38k protein bound to myosin was plotted against the 38k protein mixed with 1.1 μM myosin. Open and closed circles denote the unphosphorylated and phosphorylated myosin, respectively. MHC, 38k, A, α-TM, and β-TM denote the bands of MHC, the 38k protein, actin, and α- and β-tropomyosins, respectively.
Mentions: The relationship of the amount of the 38k protein bound to unphosphorylated myosin is shown (see Fig. 3 e). Unphosphorylated myosin remains in the supernatant if insufficient 38k protein is added. Therefore, it is possible that part of the 38k protein remains in the supernatant, despite its binding to myosin. However, we believe that this is unlikely and that the 38k protein bound to unphosphorylated myosin is mostly recovered in the precipitate (compare open and closed circles in Fig. 3 e). The binding of 5.4 μM of 38k protein to 10 μM actin filaments in the presence and absence of tropomyosin was examined by a similar centrifugation assay (Ishikawa et al. 1989).

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