Okagaki T, Nakamura A, Suzuki T, Ohmi K, Kohama K
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.
J Cell Biol. 2000 Feb 21;148(4):653-63. doi: 10.1083/jcb.148.4.653.
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.
去磷酸化状态的平滑肌肌球蛋白在体外不会形成细丝。然而，由肌球蛋白和肌球蛋白结合蛋白组成的粗丝在平滑肌细胞中持续存在，即使肌球蛋白经历磷酸化-去磷酸化循环。将端激酶鉴定为肌球蛋白组装蛋白成功解释了这一差异。然而，已经观察到缺乏端激酶的平滑肌细胞。我们期望找到另一种具有类似作用的普遍存在的蛋白质，并试图从鸡胗中纯化它。这种38kDa的蛋白质与磷酸化和去磷酸化的肌球蛋白结合程度相似。其肌球蛋白结合活性的作用是将去磷酸化的肌球蛋白组装成细丝，尽管它对磷酸化的肌球蛋白没有影响。该38kDa的蛋白质与肌球蛋白结合，其COOH末端的20个残基和NH(2)末端的28个残基对于肌球蛋白结合都是必需的。该38kDa蛋白质的氨基酸序列与端激酶不同源，但与人p32同源，p32最初是在细胞核中作为前体mRNA剪接因子-2的一个亚基被发现的。蛋白质印迹法显示该蛋白质在各种平滑肌中表达。对培养的平滑肌细胞进行免疫荧光显微镜检查发现，该38kDa蛋白质与肌球蛋白以及其他细胞骨架成分共定位。讨论了缺乏核免疫染色与平滑肌分化的关系。
