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.

我们证明了细菌LP在鼠腹膜巨噬细胞中诱导一组特殊的磷酸化蛋白（PP），并纯化了最重的磷酸化底物蛋白，分子质量为65 kDa（pp65）。确定了两个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，包括与所有可能的密码子互补的DNA序列。 PP65基因通过该方法基于采用寡核苷酸和巨噬细胞cDNA的聚合酶链反应技术进行扩增。放大基因的序列通过Sanger的Dideoxy链终止方法进行。令人惊讶的是，该序列与人类Plastin的同源性有超过90％的同源性。最近确定的新型蛋白质，该蛋白质依赖于肿瘤的人类细胞中。为了确定PP65-磷酸化中涉及哪种丝氨酸激酶，我们从pp65中分离了 ^<32> p-serine continig肽，该肽是在 ^<32> p-p-laped且LPS刺激的巨噬细胞中产生的，并确定了序列。结果表明，具有序列序列的PP65可以通过cAMP依赖性激酶，蛋白激酶C或酪蛋白激酶II磷酸化。FURHERTHER研究正在弯曲。因此，对PP65函数的精确分析似乎应导致我们对分子水平巨噬细胞激活的理解。