Angelastro J M, Ho C L, Frappier T, Liem R K, Greene L A
Department of Pathology and Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
J Neurochem. 1998 Feb;70(2):540-9. doi: 10.1046/j.1471-4159.1998.70020540.x.
Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim- cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim- cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim- cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.
外周蛋白是一种III型中间丝，存在于外周神经元和某些中枢神经系统神经元中。我们在此报告，外周蛋白含有一个磷酸酪氨酸残基，因此是已知唯一以这种方式被修饰的中间丝蛋白。针对磷酸酪氨酸的抗血清可识别PC12细胞（无论有无神经生长因子处理）、大鼠坐骨神经中的外周蛋白，以及在Sf-9细胞和SW-13 cl. 2 vim-细胞中表达的外周蛋白。通过免疫沉淀、二维等电聚焦/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及磷酸氨基酸分析，证实了PC12细胞中外周蛋白作为酪氨酸磷酸化蛋白的身份。与丝氨酸/苏氨酸磷酸化不同，外周蛋白的酪氨酸磷酸化不受去极化或神经生长因子处理的调节。为了确定酪氨酸磷酸化位点，大鼠外周蛋白在几个酪氨酸残基处发生突变，并在SW-13 cl. 2 vim-细胞中表达。只有羧基末端酪氨酸（Y474）发生突变的外周蛋白突变体选择性地失去了酪氨酸磷酸化。间接免疫荧光染色表明，野生型外周蛋白和外周蛋白Y474F在SW-13 cl. 2 vim-细胞中均形成丝状网络。这表明外周蛋白C末端残基的酪氨酸磷酸化对于组装不是必需的，并且留下了这种修饰可能具有其他功能的可能性。
