INTERACTION OF NONMUSCLE MYOSIN II WITH PLASMA MEMBRANES

在果蝇中，肿瘤的基因产物（Dlgl） 抑制基因l（2）gl与非肌肉肌球蛋白II（NMII）结合， 该结合由与Dlgl结合的激酶调节（Kalmes 例如，《细胞科学杂志》109，1359，1996）。哺乳动物细胞含有 一种密切相关的蛋白质，也结合一种激酶调节 与NMII合作。由于激酶身份未知，我们 构建了含有氨基酸617 - 731的GST融合蛋白 Dlgl的小鼠同源物Mgl，以帮助纯化Dlgl的小鼠同源物Mgl。 激酶。该融合蛋白含有4个高度保守的丝氨酸， S650、654、658和662，它们被认为是 激酶当GST-Mg1与PC12裂解物一起孵育时， 用谷胱甘肽-琼脂糖沉淀并进行体外 激酶测定，沉淀物含有紧密结合的激酶 磷酸化GST-Mgl的活性;丝氨酸的突变 丙氨酸残基阻断磷酸化。利用 GST-Mgl与激酶紧密结合，激酶活性 在下拉测定中使用融合蛋白作为 亲和配体和底物。激酶活性得到部分纯化 从25 - 60%的硫酸铵级分的大鼠脑高速 上清液。Sephacryl 300柱层析后进行离子交换。 Q-Sepharose亲和交换色谱法 在Blue Sepharose上层析和在Superose上凝胶过滤 6，导致比活性增加700倍。的主要蛋白 提取物中80 kD的条带与GST-Mgl结合， 与ATP孵育时磷酸化;两者的磷酸化 融合蛋白和80kD条带被蛋白抑制 激酶C（PKC）抑制剂GF109203X。蛋白质印迹 与GST-Mgl结合显示了几种PKC亚型的结合 与融合蛋白。未来的实验将研究 PKC同工型与全长Mgl的结合和磷酸化 的全长蛋白质及其调控在体外和体内。

In Drosophila, the gene product (Dlgl) of the tumor suppressor gene l(2)gl binds to nonmuscle myosin II (NMII) and the association is regulated by a kinase which binds to Dlgl (Kalmes et al., J. Cell Sci. 109, 1359, 1996). Mammalian cells contain a closely related protein which also binds a kinase regulating association with NMII. Since the kinase identity is unknown, we constructed a GST-fusion protein containing amino acids 617-731 of Mgl, the mouse homolog of Dlgl, to assist in purification of the kinase. The fusion protein contains 4 highly conserved serines, S650, 654, 658 and 662, which have been implicated as targets for the kinase. When GST-Mgl was incubated with PC12 lysates, precipitated with glutathione-agarose and subjected to in vitro kinase assay, the precipitates contained a tightly-bound kinase activity which phosphorylated GST-Mgl; mutation of the serine residues to alanine blocked phosphorylation. Taking advantage of the tight association of GST-Mgl and kinase, kinase activity was assayed in a pull-down assay using the fusion protein as affinity-ligand and substrate. Kinase activity was partially purified from a 25-60% ammonium sulfate fraction of rat brain high-speed supernatants. Sephacryl 300 chromatography was followed by ion exchange chromatography on Q-Sepharose, affinity chromatography on Blue Sepharose and gel filtration on Superose 6, leading to a 700-fold increase in specific activity. A major protein band of 80 kD in the extract binds to GST-Mgl and is phosphorylated on incubation with ATP; phosphorylation of both the fusion protein and the 80 kD band is inhibited by the protein kinase C (PKC) inhibitor GF109203X. Western blotting of proteins bound to GST-Mgl shows association of several PKC isoforms with the fusion protein. Future experiments will examine the binding of PKC isoforms to full-length Mgl and the phosphorylation of the full-length protein and its regulation in vitro and in vivo.