Gao Z H, Metherall J, Virshup D M
Department of Oncological Sciences, University of Utah, Salt Lake City, Utah 84132, USA.
Biochem Biophys Res Commun. 2000 Feb 16;268(2):562-6. doi: 10.1006/bbrc.2000.2168.
Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates.
酪蛋白激酶I（CKI）是一种广泛表达的蛋白激酶家族，参与多种细胞过程，包括膜运输、DNA修复和昼夜节律。尽管有大量的CKI基因，但已鉴定出的生物学相关底物却很少。为了更好地确定CKI底物的范围，我们扩展了最近描述的体外表达克隆（IVEC）策略。通过筛选多肽库以寻找激酶依赖性的电泳迁移率变化。从3000个随机cDNA克隆的初始样本中分离出10个假定的CKI底物。候选底物包括参与RNA代谢的蛋白质（一种假定的RNA解旋酶、核仁蛋白hNOP56和hnRNP A1，以及核糖体蛋白L4、L8和L13），以及角蛋白17、一种与necdin相关的蛋白质，和钙结合蛋白桥粒芯糖蛋白2和膜联蛋白II。还用活性ERK2筛选了相同的库，并鉴定出4种底物：醛缩酶、NSD样蛋白、尿嘧啶-DNA糖基化酶和HHR23A。IVEC是鉴定新型蛋白激酶底物的有效方法。
