| Co-Investigator(Kenkyū-buntansha) |
CHIBA Shigeru Faculty of Medicine, University of Tokyo, Assistant Prof., 医学部(病), 助手
TOYOSHIMA Hideo Faculty of Medicine, University of Tokyo, Assistant Prof., 医学部(病), 助手 (20197966)
MANO Hiroyuki Faculty of Medicine, University of Tokyo, Assistant Prof., 医学部(病), 助手 (90240704)
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| Research Abstract |
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
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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
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