一种新型丝氨酸苏氨酸激酶CPK的初步克隆及功能

Molecular Cloning of a Novel Serine Threonine Kinase (CPK) and Possibly Involved in Cell Proliferation Regulation

从人胎肝cDNA文库中分离到一种cDNA ,推测的蛋白氨基酸序列具有明显的丝氨酸苏氨酸激酶结构域,可能编码一种新的丝氨酸苏氨酸激酶(命名为CPK ,cellproliferationassociationkinase) .CPKmRNA全长约3 0kb .RT PCR检测表明CPKmRNA在增殖活跃组织或细胞如人胚胎组织、肿瘤细胞株中呈高丰度表达,在成人组织则呈低丰度或不表达.利用PCR技术扩增大鼠同源cDNA ,以此为探针,Northern杂交发现CPK表达于多种成年小鼠组织,以脑组织丰度最高.小鼠2 3肝部分切除可迅速诱导CPKmRNA的表达,在术后2 4～4 8h达高峰,与2 3肝部分切除后细胞增殖期吻合.以小鼠同源cDNA片段为模板,合成RNA探针,原位杂交显示在胚胎发育期CPK低丰度表达在大多数组织,以神经系统组织变化最大,在第8d胚胎神经管内皮细胞中即出现表达,11～13d在端脑、脑膜、间脑、脊神经节表皮细胞、海马、小脑神经胶质细胞等多种神经细胞中表达且丰度较高,16d后这些组织的表达迅速下降到较低水平,表明CPK可能与神经系统的生长发育有一定关联.利用信号通路检测技术,观察到CPK的表达对MAPK ,p38MAPK途径的激活有明显的影响,并可显著增强表皮生长因子对这两条途径的激活,提示该激酶可能参与细胞因子的信号转导.

The cDNA of a novel non-receptor type of serine threonine kinase(referred to as cell proliferation association kinase, CPK)was isolated from a human fetal liver cDNA library with representational difference analysis based on PCR.The predicted polypeptide contains all conserved subdomains characteristic of the protein serine threonine kinase family.Northern blot and RT-PCR analysis showed that the mRNA of 3.0 kb was detected abundantly in proliferating tissues or cells of adult mouse,particularly in brain.2/3 partial hepatectomy(PH) in mouse induced dramatically increased expression of CPK mRNA,and the mRNA level reached peak in 24—48 hours after PH.In situ hybridization with mRNA probe synthesized from mouse homological cDNA revealed that the mRNA positive signals of CPK gene is related to embryo development in mouse neural system; being low in early phase,localizing in neurotubules epidermis cells at 8th day,becoming abundant in variety of neurotic cells such as cerebrum,meninges,diencephalons,spiral ganglia,hippocampi and lelencephalon neuroglial cells at 11—13th days,and from 16th day of gestation the mRNA levels was rapidly down-regulated implying the CPK may be related to growth and development of embryonic neuron system.Using Path Detect Trans-reporting System with luciferase as reporter gene, it was found that CPK could sufficiently activate MAPK and p38MAPK signal transduction pathway in response to EGF stimulation.These results indicate that the novel serine threonine kinase,CPK,may be involved in the cytokine induced signal transduction.