NERVE-SPECIFIC EXPRESSION OF NONMUSCLE MYOSIN HEAVY CHAINS

先前的工作已经证明，非肌肉肌球蛋白的独特亚型 重链-B(MHC-B)在鸡和人神经细胞中表达 (Takahashi et al.，J.Biol.化学。267：17864,1992年)。这些异构体， 它们是由前-mRNA的选择性剪接产生的，不同于 MHC-B亚型存在于大量的非肌肉细胞中，因为它们 在ATP结合区附近含有插入的氨基酸盒 和/或靠近肌动蛋白结合区。靠近ATP结合的插入物 该区域起始于第211个氨基酸之后，由10或16个氨基酸组成 酸性物质，并含有一种可磷酸化的丝氨酸，用于合成- 定向激活酶。肌动蛋白结合区附近的插入开始于 氨基酸621，由21个氨基酸组成。我们已经研究了 插入的MHC-B亚型的分布和表达。在 发育中的鸡脑，编码10个氨基酸插入片段的mRNA 受精后逐渐增加，高峰在10-14天的胚胎 然后就谢绝了。相比之下，编码21个氨基酸的mRNA 插入片段出现在出生前，并在成人体内大量表达 小脑。它们之间存在着明显的物种差异 插入的亚型在成体组织中的分布。10个氨基 在ATP结合区附近的酸插入在 鸡脑，但构成了人类表达的MHC-B mRNA的大部分 大脑和人视网膜中约90%的MHC-B基因。21世纪 氨基酸插入片段在鸡小脑和人小脑中大量表达 大脑，但在人的视网膜和培养的细胞中不存在。 使用各种神经来源的细胞系，我们研究了条件 这增加了10个氨基酸插入物的表达 ATP结合区。使用人类视网膜母细胞瘤(Y-79)和 神经母细胞瘤(SK-N-SH)细胞株，我们能够增加 使用多种激动剂或血清表达插入的异构体 剥夺。在每种情况下，插入的异构体的表达都与 具有细胞分化和抑制细胞分裂的作用。使用神经 生长因子和大鼠嗜铬细胞瘤细胞系(PC12)，我们能够 以显示插入的异构体的外观在时间上相关 并且可逆地长出轴突。使用牛脑肌球蛋白和 丁酸盐治疗的视网膜母细胞瘤中非肌肉型MHC-B的免疫沉淀 (Y-79)细胞提取物，我们能够磷酸化独特的位点， 存在于插入的氨基酸盒中，带有脯氨酸导向的 激活剂。

Previous work has demonstrated that unique isoforms of nonmuscle myosin heavy chain-B (MHC-B) are expressed in chicken and human neuronal cells (Takahashi et al., J. Biol. Chem. 267: 17864, 1992). These isoforms, which are generated by alternative splicing of pre-mRNA, differ from the MHC-B isoform present in a large number of nonmuscle cells in that they contain inserted cassettes of amino acids near the ATP binding region and/or near the actin binding region. The insert near the ATP binding region begins after amino acid 211 and consists of either 10 or 16 amino acids and contains a putative phosphorylatable serine for proline- directed kinases. The insert near the actin binding region begins after amino acid 621 and consists of 21 amino acids. We have studied the distribution and expression of the inserted MHC-B isoforms. In the developing chicken brain, mRNA encoding the 10 amino acid insert gradually increases after fertilization, peaks in the 10-14 day embryo and then declines. In contrast, the mRNA encoding the 21 amino acid insert appears just before birth and is abundantly expressed in the adult cerebellum. There is a marked species difference between the distribution of the inserted isoforms in adult tissues. The 10 amino acid insert near the ATP binding region is very poorly expressed in chicken brain, but makes up most of the MHC-B mRNA expressed in human cerebrum and approximately 90% of MHC-B mRNA in human retina. The 21 amino acid insert is abundantly expressed in chicken cerebellum and human cerebrum, but is absent from human retina and human cultured cells. Using a variety of neuronally derived cell lines, we studied conditions that increase the expression of the 10 amino acid insert present near the ATP binding region. Employing human retinoblastoma (Y-79) and neuroblastoma (SK-N-SH) cell lines, we are able to increase the expression of the inserted isoform using a number of agonists or serum deprivation. In each case, expression of the inserted isoform correlates with cell differentiation and inhibition of cell division. Using nerve growth factor and a rat pheochromocytoma cell line (PC12), we are able to show that the appearance of the inserted isoform correlates temporally and reversibly with neurite outgrowth. Using bovine brain myosin and immunoprecipitated nonmuscle MHC-B from a butyrate-treated retinoblastoma (Y-79) cell extract, we are able to phosphorylate the unique site, present in the inserted cassette of amino acids, with proline-directed kinases.