Analysis of signal transduction mechanism through a novel tyrosine kinase receptor

此前，我们通过与c-FMS探针的低严格杂交，从人红白血病细胞系K562的cDNA库中克隆了一个ltk基因。为获得人胎盘全长cDNA，对人胎盘基因文库进行了筛选，获得了5个阳性克隆。其中一个克隆(P2)含有最长的开放阅读框，编码一个由864个氨基酸组成的酪氨酸激酶受体。对这5个克隆的分析表明，至少有3个克隆存在。其中一个cdna预测了一个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。另一个克隆(P3)在跨膜区下游有一个可选择的插入片段，因此在跨膜区和酪氨酸激酶结构域之间产生了框内终止密码子，这表明该克隆编码的是一个缺乏激活域的受体蛋白。我们将含有不同ltk cdna克隆的表达载体导入cos细胞，细胞裂解产物为亚…。更多地受到西方印迹分析的影响。抗P2和P3细胞裂解物中分别检测到约100kD和50kD的蛋白质。这两种产物都显示为双条带，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的前mRNAs。表达完整的ltk cdna克隆的COS细胞裂解产物免疫复合物在体外用[Gamma-^&lt；32&gt；P]ATPase检测。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，并对自身和其他相关蛋白进行磷酸化。我们已经在抗Ltk抗体沉淀的免疫复合体中检测到PLC-Gamma1、GAP、PI3-激酶和c-RAF蛋白。在35例人类恶性肿瘤的Northern印迹分析中，ltk基因在白血病(10/18例)中优先表达，但在17例非白血病肿瘤中均不表达。较少

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