Site determination and functional significance of phosphorylation of pX gene product of human T-cell leukemia virus type I.

点击翻译按钮获取中文摘要

1. HTLV-I-infected MT-2 cells were metabolically labeled with ^<32>-P orthophosphoric acid and used for p40^<tax> isolation by immunoprecipitation. The precipitate was partially digested with 0-10 units of trypsin axid separated by SDS-PAGE. Among several tryptic peptides, the label was found in 15 kd peptide, which suggested that the phosphorylation occurred in limited regions of p40^<tax> molecule. Further identification of proteolytic peptide containing the phosphorylation site should be carried out by using site specific monoclonal antibodies as described below.2. A plasmid carrying HTLV-I promoter connected to chrolamphenicol acetyltransferase (CAT) gene was constructed and transfected to HOS/PL cells expressing p40^<tax> whose transactivation activity was measured by CAT assay. By this assay system, the effect of a protein kinase inhibitor K252a was examined in a range 0-20 muM. The transactivation by p40^<tax> was inhibited by >0.2 muM K252a and suggested to require protein phosphorylation.3. BALB/C mice were immunized with a recombinant p40^<tax> to obtain four monoclonal antibodies whose epitopes were mapped within amino- and carboxy- proximal regions by using recombinant p40^<tax> with various deletions as antigens. These antibodies should be useful for the peptide mapping as described above. We are going to construct plasmids that allow the expression of mutated p40^<tax> in mammalian cells to examine their transactivation activities. Our final objects are to identify the particular serine residues that are phosphorylated and clarify the mechanism by which the phosphorylation affects the function of p40^<tax>.