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

Localization of the 38k protein in the cultured smooth muscle cell line AC01. a–c, Staining pattern with anti–38k protein (a) and that with anti-MHC (b) antibodies. To amplify colocalized region of the 38k protein and MHC, images of a and b were multiplied in c, i.e., c = a × b. d–f, The cell was stained with MitoTracker Red CMXRos to visualize position of mitochondria. Staining pattern with anti-38k protein (d) and that with MitoTracker Red CMXRos (e). To demonstrate colocalized region of the 38k protein with mitochondria, images of d and e were multiplied in f, i.e., f = d × e. Magnification is the same in all pictures. Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169363&req=5

Figure 8: Localization of the 38k protein in the cultured smooth muscle cell line AC01. a–c, Staining pattern with anti–38k protein (a) and that with anti-MHC (b) antibodies. To amplify colocalized region of the 38k protein and MHC, images of a and b were multiplied in c, i.e., c = a × b. d–f, The cell was stained with MitoTracker Red CMXRos to visualize position of mitochondria. Staining pattern with anti-38k protein (d) and that with MitoTracker Red CMXRos (e). To demonstrate colocalized region of the 38k protein with mitochondria, images of d and e were multiplied in f, i.e., f = d × e. Magnification is the same in all pictures. Bar, 30 μm.

Mentions: To determine whether the 38k protein is associated with myosin in smooth muscle cells, we examined its localization in the cultured smooth muscle cell line, AC01, by indirect immunofluorescence. Double staining (Fig. 8, a and b), followed by the image analysis (Fig. 8 c), indicates that the localization of the 38k protein coincides with myosin. Together with the same staining as shown in Fig. 8, a and b, the analysis indicates that the 38k protein is associated with myosin in the whole cells. In the case of the double staining of the 38k protein (Fig. 8 d) and mitochondria (Fig. 8 e, see below), the overlapping staining is limited to the central part of the cell (Fig. 8 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)

Localization of the 38k protein in the cultured smooth muscle cell line AC01. a–c, Staining pattern with anti–38k protein (a) and that with anti-MHC (b) antibodies. To amplify colocalized region of the 38k protein and MHC, images of a and b were multiplied in c, i.e., c = a × b. d–f, The cell was stained with MitoTracker Red CMXRos to visualize position of mitochondria. Staining pattern with anti-38k protein (d) and that with MitoTracker Red CMXRos (e). To demonstrate colocalized region of the 38k protein with mitochondria, images of d and e were multiplied in f, i.e., f = d × e. 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 8: Localization of the 38k protein in the cultured smooth muscle cell line AC01. a–c, Staining pattern with anti–38k protein (a) and that with anti-MHC (b) antibodies. To amplify colocalized region of the 38k protein and MHC, images of a and b were multiplied in c, i.e., c = a × b. d–f, The cell was stained with MitoTracker Red CMXRos to visualize position of mitochondria. Staining pattern with anti-38k protein (d) and that with MitoTracker Red CMXRos (e). To demonstrate colocalized region of the 38k protein with mitochondria, images of d and e were multiplied in f, i.e., f = d × e. Magnification is the same in all pictures. Bar, 30 μm.
Mentions: To determine whether the 38k protein is associated with myosin in smooth muscle cells, we examined its localization in the cultured smooth muscle cell line, AC01, by indirect immunofluorescence. Double staining (Fig. 8, a and b), followed by the image analysis (Fig. 8 c), indicates that the localization of the 38k protein coincides with myosin. Together with the same staining as shown in Fig. 8, a and b, the analysis indicates that the 38k protein is associated with myosin in the whole cells. In the case of the double staining of the 38k protein (Fig. 8 d) and mitochondria (Fig. 8 e, see below), the overlapping staining is limited to the central part of the cell (Fig. 8 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