Analysis of the plastin-pathway involved in macrophage activation.

分析参与巨噬细胞激活的塑性蛋白途径。

基本信息

批准号：
04670247
负责人：
SHINOMIYA Hiroto
金额：
$ 1.28万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We demonstrated that bacterial LPS induced a special set of phosphorylated protein (pp) in murine peritoneal macrophages, and purified the most heavily phosphorylated substrate protein with a molecular mass of 65-kDa(pp65) to homogeneity. Amino acid sequences of the two pp65-derived peptides were determined. According to the amino acid sequences, we synthesized oligonucleotide primer pools including DNA sequences complementary to all possible codons. The pp65 gene was amplified by the method based on the polymerase chain reaction technique employing the oligonucleotides and the macrophage cDNA.Sequence of the amplified gene was performed by Sanger's dideoxy chain termination method. It was surprising to us that the sequence has more than 90% of homology with that of human plastin ; a recently identified novel protein that transformation-dependently appears in neoplastic human cells.We already clarified that serine residues on pp65 are exclusively phosphorylated, which means that serine kinases are activated in macrophages after LPS-stimulation. In order to determine what kind of serine kinases are involed in the pp65-phosphorylation, We isolated the ^<32>P-serine containig peptide from pp65 which had generated in the ^<32>P-labeled and LPS-stimulated macrophages and determined the sequence. The result indicated that pp65 having sequence could be phosphorylated by cAMP-dependent kinase, protein kinase C or casein kinase II.further investigations are currenly being done. It therefore seemed that precise analysis on the pp65 functions should lead to progress in our understanding of the macrophage activation at the molecular level.

我们证明细菌 LPS 在小鼠腹腔巨噬细胞中诱导一组特殊的磷酸化蛋白 (pp)，并将分子量为 65 kDa (pp65) 的磷酸化程度最高的底物蛋白纯化至均质。确定了两种 pp65 衍生肽的氨基酸序列。根据氨基酸序列，我们合成了寡核苷酸引物池，其中包括与所有可能的密码子互补的DNA序列。采用基于聚合酶链式反应技术的方法，利用寡核苷酸和巨噬细胞cDNA扩增pp65基因。扩增基因的测序通过Sanger双脱氧链终止法进行。令我们惊讶的是，该序列与人plastin的序列具有90%以上的同源性；最近发现的一种新型蛋白质，其转化依赖性地出现在人类肿瘤细胞中。我们已经阐明，pp65 上的丝氨酸残基完全被磷酸化，这意味着丝氨酸激酶在 LPS 刺激后在巨噬细胞中被激活。为了确定哪种丝氨酸激酶参与pp65-磷酸化，我们从pp65中分离出含有^ 32 P-丝氨酸的肽，该肽在^ 32 P标记和LPS刺激的巨噬细胞中产生并确定顺序。结果表明，具有序列的pp65可以被cAMP依赖性激酶、蛋白激酶C或酪蛋白激酶II磷酸化。目前正在做进一步的研究。因此，对 pp65 功能的精确分析似乎应该有助于我们在分子水平上理解巨噬细胞活化。