Liao J, Lowthert L A, Ku N O, Fernandez R, Omary M B
VA Palo Alto Health Care System, California 94304, USA.
J Cell Biol. 1995 Dec;131(5):1291-301. doi: 10.1083/jcb.131.5.1291.
Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization.
角蛋白多肽8和18（K8/18）以及其他中间丝蛋白的磷酸化会导致它们在体外和体内发生重组。为了研究人K18磷酸化的功能方面，我们制备并纯化了一种多克隆抗体（称为3055），它能特异性识别人类K18的一个主要磷酸化位点（Ser52），但不能识别去磷酸化的K18或Ser52突变为Ala的K18突变体。进行脉冲追踪实验，随后对体内32PO4标记的K8/18进行免疫沉淀和肽图谱分析，结果表明K18的总体磷酸化周转速度比K8快，并且K18的Ser52是一个高度动态的磷酸化位点。对32PO4标记的K18进行等电聚焦，然后用3055进行免疫印迹分析，结果显示主要的磷酸化K18物种含有Ser52磷酸化，但也存在一些K18分子，它们在Ser52以外的K18位点上优先被磷酸化。对处于细胞周期不同阶段的细胞获得的总细胞裂解物进行免疫印迹分析，结果显示在细胞周期的S期和G2/M期，Ser52磷酸化增加了三到四倍。使用3055或其他识别总角蛋白池的抗体，对有丝分裂不同阶段的细胞进行免疫荧光染色，结果导致3055抗体优先结合重组的角蛋白部分。对表达人K18的人体组织或转基因小鼠组织进行染色，结果显示磷酸化的Ser52 - K18物种分别优先位于肝脏的基底外侧和胰腺的顶端区域，但在检查的其他简单上皮器官中未发现优先定位。我们的结果支持一种模型，即磷酸化的中间丝根据组织类型和磷酸化位点定位于特定的细胞区域。此外，人K18的Ser52是一个高度动态的磷酸化位点，在细胞周期的S期和G2/M期与丝的重组相关联而受到调节。
