Analysis of signal transduction mechanism through a novel tyrosine kinase receptor

在此之前，我们用c-fms探针低严格杂交方法克隆了从人红细胞白血病细胞系K562 cDNA文库中分离的ltk cDNA。为获得全长cDNA，筛选人胎盘cDNA文库，分离出5个阳性克隆。一个克隆（P2）包含最长的开放阅读框，它编码一个假定的864个氨基酸的酪氨酸激酶受体。对这5个克隆的分析表明，至少存在3个cDNA克隆。一个cDNA预测了221个氨基酸的蛋白质，并编码了一种可从细胞分泌的可溶性受体。另一个cDNA克隆（P3）在跨膜结构域的下游有一个替代插入，因此在跨膜结构域和酪氨酸激酶结构域之间产生帧内停止密码子，这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到COS细胞中，细胞裂解液进行western blot分析。用单克隆抗体在转染P2和P3的细胞裂解液中分别检测到大约100kD和50kD的蛋白。这两种产物都显示为双带，这可能是翻译后修饰的结果。这些结果表明，这些cdna并不代表未成熟的未剪接前mrna。表达全ltk cDNA克隆的COS细胞裂解物的免疫复合物使用[γ -^<32>P]ATP进行体外激酶测定。实验中检测到100kD、140kD、85kD和45kD蛋白的磷酸化，表明ltk蛋白具有蛋白激酶活性，能够磷酸化自身及其他相关蛋白。我们在抗ltk抗体沉淀的免疫复合物中检测到了PLC-gamma1、GAP、pi3 -激酶和c-raf蛋白。在35例人类恶性肿瘤的Northern blot分析中，ltk在白血病中优先表达（18例中的10例），没有细胞谱系特异性，但17例非白血病肿瘤中没有表达ltk基因。少

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