cDNA cloning of novel tyrosine kinases expressed in neuronal cells

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We established the C3KSP and Mel-ret cell lines which are derived from the neural tube of a 8.5 day mouse embryo and a melanocytic tumor developed in a transgenic mouse with the ret oncogene, respectively. When tyrosine-Phosphorylated proteins in these cells were analyzed by Western blotting with anti-phosphotyrosine antibody, a 160 kDa tyrosine-phosphorylated protein in C3KSP cells and a 85 kDa protein in Mel-ret cells were detected. Cell fractionation experiments showed that both proteins localize in the membrane component. We succeeded in purifying the 85 kDa proteins from Mel-ret cells using an anti-phosphotyrosine antibody-bound column. The purity of the 85 kDa protein was more than 90%. In vitro kinase assay suggested that the 85 kDa protein contains the strong kinase activity in the presence of Mn^<2+> While it was weak in the presence of Mg^<2+>. The 85 kDa protein also showed the kinase activity in "ingel" kinase assay. The phosphorylated amino acids of the 85 kDa protein in "invitro" kinase assay were tyrosine in the presence of Mn^<2+> while they were serine and tyrosine in the presence of Mg^<2+>. We are planning to purify a large amount of the 85 kDa protein and determine its amino acid sequences to isolate its cDNA.