Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.
Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.
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DOI：
10.1083/jcb.127.1.161
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发表时间：
1994-10
期刊：
影响因子：
--
通讯作者：
Omary MB
中科院分区：
文献类型：
--
作者：
Ku NO;Omary MB
有大量的体外证据表明，包括角蛋白在内的中间纤维的磷酸化在纤维重组中起着重要作用。为了更好地了解中间丝磷酸化的功能，我们试图确定人角蛋白多肽18（K18）的主要磷酸化位点，并研究其在丝组装或重组中的作用。我们在潜在的磷酸化位点产生了一系列K18 ser->ala突变，然后在昆虫细胞中表达并比较所产生的构建体的胰蛋白酶32 PO 4标记模式。使用这种方法，加上Edman降解的32 PO 4标记的胰蛋白酶肽，并与标记正常人结肠组织后分析的胰蛋白酶肽比较，我们确定了ser-52作为主要的K18生理磷酸化位点。K18中的Ser-52不被糖基化，并且与CAM激酶、S6激酶和蛋白激酶C磷酸化的共有序列匹配，并且所有这些激酶都可以在体外主要在该位点磷酸化K18。哺乳动物细胞中K18 ser-52-->ala突变体的表达显示出最小的磷酸化，但与野生型K18相比，在细丝组装中没有可区分的差异.相比之下，Ser-52突变在细丝重组中发挥了明确但非排他性的作用，基于对用冈田酸处理的细胞或在细胞周期的G2/M期被阻滞的细胞中细丝改变的分析。我们的研究结果表明，ser-52是人K18在间期细胞中的主要生理磷酸化位点，并且其磷酸化可能在体内细丝重组中发挥作用。
There is ample in vitro evidence that phosphorylation of intermediate filaments, including keratins, plays an important role in filament reorganization. In order to gain a better understanding of the function of intermediate filament phosphorylation, we sought to identify the major phosphorylation site of human keratin polypeptide 18 (K18) and study its role in filament assembly or reorganization. We generated a series of K18 ser-->ala mutations at potential phosphorylation sites, followed by expression in insect cells and comparison of the tryptic 32PO4-labeled patterns of the generated constructs. Using this approach, coupled with Edman degradation of the 32PO4-labeled tryptic peptides, and comparison with tryptic peptides analyzed after labeling normal human colonic tissues, we identified ser-52 as the major K18 physiologic phosphorylation site. Ser-52 in K18 is not glycosylated and matches consensus sequences for phosphorylation by CAM kinase, S6 kinase and protein kinase C, and all these kinases can phosphorylate K18 in vitro predominantly at that site. Expression of K18 ser-52-->ala mutant in mammalian cells showed minimal phosphorylation but no distinguishable difference in filament assembly when compared with wild- type K18. In contrast, the ser-52 mutation played a clear but nonexclusive role in filament reorganization, based on analysis of filament alterations in cells treated with okadaic acid or arrested at the G2/M stage of the cell cycle. Our results show that ser-52 is the major physiologic phosphorylation site of human K18 in interphase cells, and that its phosphorylation may play an in vivo role in filament reorganization.