DIK, a novel protein kinase that interacts with protein kinase Cδ -: Cloning, characterization, and gene analysis
DIK, a novel protein kinase that interacts with protein kinase Cδ -: Cloning, characterization, and gene analysis
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DOI：
10.1074/jbc.m004771200
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发表时间：
2000-11-17
影响因子：
4.8
通讯作者：
Gschwendt, M
中科院分区：
文献类型：
--
作者：
Bähr, C;Rohwer, A;Gschwendt, M
以大鼠蛋白激酶C6（PKC delta（cat））的催化结构域cDNA为诱饵，利用酵母双杂交系统从人角质形成细胞系Ha-CaT的cDNA文库中克隆了一种新的丝氨酸/苏氨酸激酶DIK。预测的784个氨基酸的多肽，计算的分子量为86 kDa，包含一个催化激酶结构域和一个推定的调节结构域与锚定样重复序列和一个核定位信号。DIK在mRNA和蛋白水平的表达可以在几个细胞系中被证明。DIK基因位于染色体21q22.3上，具有8个外显子和7个内含子，DIK在体外转录/翻译系统中合成，并在细菌、HEK、COS-7和杆状病毒感染的昆虫细胞中表达为重组蛋白。在体外系统和细胞中，而不是在细菌中，产生了各种后修饰形式的DIK。DM被证明表现出蛋白激酶活性朝向自磷酸化和底物磷酸化。PKC δ（cat）和PKC δ与DIK的相互作用通过来自用PRC δ（cat）或PKC γ和DIK表达构建体瞬时转染的HEK细胞的蛋白质的共免疫沉淀来证实。
A novel serine/threonine kinase, termed DIK, was cloned-using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line Ha-CaT with the catalytic domain of rat protein kinase C6 (PKC delta (cat)) CDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a-catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns, DIK was synthesized in an in vitro transcription/translation system and expressed:as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DM was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKC delta (cat) and PKC delta with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PRC delta (cat) or PKC gamma and DIK expression constructs.