Analysis of signal transduction mechanism through a novel tyrosine kinase receptor

新型酪氨酸激酶受体信号转导机制分析

基本信息

批准号：
03454521
负责人：
HIRAI Hisamaru
金额：
$ 3.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Previously, we cloned an ltk cDNA isolated from a human erythroleukemia cell line K562 cDNA library by low stringency hybridization with a c-fms probe. To obtain a full length cDNA, a cDNA library of human placenta was screened, and five positive clones were isolated. One clone (P2) contains the longest open reading frame which encodes a putative tyrosine kinase receptor of 864 amino acids. The analysis of the five clones revealed the existence of at least three spieces of cDNA clones. One cDNA predicts a protein of 221 amino acids and encodes a soluble type of receptor which can be secreted from the cell. Another cDNA clone (P3) has an alternative insert just downstream of the transmembrane domain and thus produce in-frame stop codon between the transmembrane domain and the tyrosine kinase domain, suggesting that this clone encodes a receptor protein lacking the kinase domain. We transfected expression plasmids containing various ltk cDNA clones to COS cells, and cell lysates were sub … More jected to western blot analysis. Proteins of approximately 100kD and 50kD were detected by the monoclonal antibody against the extracellular domain in the cell lysates transfected with P2 and P3, respectively. Both products are shown in double bands which may result from posttranslational modification. These results suggest that these cDNAs do not represent immature unspliced pre-mRNAs. Immune complexes from lysates of COS cells expressing the full ltk cDNA clone were subjected to in vitro kinase assay using [gamma-^<32>P]ATP. Phosphorylation of 100kD, 140kD, 85kD and 45kD proteins was detected in the experiment, suggesting that ltk protein has protein kinase activity and phosphorylates itself and other associated proteins. We have detected PLC-gamma1, GAP, PI3-kinase, and c-raf protein in the immune complex precipitated by anti-ltk antibody. In Northern blot analysis of 35 human malignancies, ltk is preferentially expressed in leukemias (10 out of 18 cases) with no cell lineage specificity, but none of 17 nonleukemic neoplasms expressed ltk gene. Less

上一堂，我们通过低严格的杂交与C-FMS探针从人红血球细胞系K562 cDNA库中分离出来的LTK cDNA。克隆（P2）包含编码864个氨基酸的推定酪氨酸激酶受体的阅读框，这使AT的存在cDNA克隆的cDNA的存在预测221个氨基酸的蛋白质。 p3）具有另一个cDNA克隆（P3）跨膜结构域，因此在跨膜结构域和酪氨酸激酶结构域之间产生框内终止密码子，这表明该克隆编码包含各种LTK克隆的variocted expressids，这些exprone含有S细胞，细胞裂解液和细胞裂解物为子…对细胞外的抗体进行了更大的抗体。在实验中检测到完整的LTK克隆使用[Gamma-^32> p] ATP进行激酶测定。抗LTK抗体沉淀出35种人类恶性肿瘤的抗LTK抗体，LTK在白血病（18例中的10个）中优先表达，没有细胞谱系SS，LTK优先表达，抗LTK抗体在35例人类恶性肿瘤中，抗LTK抗体沉淀。