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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454521/
• 关键词: ltk protein; Phosphorylation; Receptor-type protein tyrosine kinase; Alternative splicing; 翻訳開始点; 造血細胞; 細胞外領域; レセプタ-型チロシンキナ-ゼ; alternative splicing

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

在此之前，我们克隆了ltk的cDNA分离自人红白血病细胞系K562的cDNA文库的低严格杂交与c-fms探针。为获得全长cDNA，从人胎盘cDNA文库中筛选出5个阳性克隆。一个克隆（P2）含有最长的开放阅读框，其编码864个氨基酸的推定酪氨酸激酶受体。对这五个克隆的分析表明存在至少三个cDNA克隆。一个cDNA预测221个氨基酸的蛋白质，并编码可从细胞分泌的可溶性类型的受体。另一个cDNA克隆（P3）在跨膜结构域的下游具有替代插入物，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，表明该克隆编码缺乏激酶结构域的受体蛋白。我们将含有不同ltk cDNA克隆的表达质粒转染COS细胞， ...更多信息 进行蛋白质印迹分析。P2和P3转染的细胞裂解液中，抗胞外区的单克隆抗体分别检测到约100 kD和50 kD的蛋白。两种产物均显示为双条带，可能是翻译后修饰所致。这些结果表明，这些cDNA不代表未成熟的未剪接的前mRNA。来自表达完整ltk cDNA克隆的COS细胞裂解物的免疫复合物使用[γ-β P]ATP进行体外激酶测定<32>。在实验中检测到100 kD、140 kD、85 kD和45 kD蛋白的磷酸化，表明ltk蛋白具有蛋白激酶活性，并磷酸化自身和其他相关蛋白。我们已经检测到PLC-γ 1，GAP，PI 3-激酶，和c-raf蛋白的免疫复合物沉淀的抗ltk抗体。北方印迹分析表明ltk基因在18例白血病中有10例优先表达，无细胞系特异性，而在17例非白血病肿瘤中均无ltk基因表达。少

项目成果

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Toyoshima H 等人：“人类 ltk 受体酪氨酸酶的不同剪接 cDNA 可以预测有或没有酪氨酸酶 sowain 和 sdubli 受体蛋白的受体蛋白。”

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