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

1. HTLV-I感染的MT-2细胞用^<32>-P正磷酸进行代谢标记，并用于<tax>通过免疫沉淀分离p40^。沉淀物用0-10单位胰蛋白酶部分消化，通过SDS-PAGE分离。在几种胰蛋白酶肽中，在15 kd肽中发现了标记，这表明磷酸化发生在p40^分子的有限区域<tax>。含有磷酸化位点的蛋白水解肽的进一步鉴定应通过使用位点特异性单克隆抗体进行，如下所述。将HTLV-Ⅰ启动子与氯霉素乙酰转移酶（CAT）基因连接，构建质粒，转染表达p40 α的HOS/PL细胞，<tax>用CAT法检测p40 α的反式激活活性。通过该测定系统，在0-20 μ M范围内检测蛋白激酶抑制剂K252 a的作用。&gt; <tax>0.2 μ M K252 a可抑制p40 α的反式激活，并提示需要蛋白磷酸化。用重组p40 α免疫BALB/C小鼠<tax>，获得4株单克隆抗体，它们的抗原表位分别定位于氨基和羧基近端区域<tax>。这些抗体应可用于如上所述的肽图谱。我们将构建允许突变的p40^<tax>在哺乳动物细胞中表达的质粒，以检测它们的反式激活活性。我们的最终目标是鉴定磷酸化的特定丝氨酸残基，并阐明磷酸化影响p40功能的机制<tax>。

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>.