Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase - Mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos
Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase - Mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos
复制标题
DOI：
10.1074/jbc.m302933200
复制
发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Dush, M
中科院分区：
文献类型：
--
作者：
Ghosh, S;Moore, S;Dush, M
以前，我们和其他人在人Raf-1激酶中鉴定了一个35个氨基酸的片段，该片段优先结合磷脂酸。磷脂酸的存在被认为是必要的Raf-1的易位到质膜。我们现在已经采用了丙氨酸扫描和缺失突变的组合，以确定关键的氨基酸残基Raf-1与磷脂酸的相互作用所必需的。四肽基序（人Raf-1的残基398-401）内的进行性突变减少并最终消除Raf-1与磷脂酸的结合。然后，我们将编码野生型Raf-1激酶或四肽基序中具有三重丙氨酸突变的突变体版本的RNA注射到斑马鱼胚胎中，并跟踪胚胎发育的形态命运。胚胎与突变体，但不是野生型Raf-1表现出的缺陷，在后轴的形成，例如弯曲的躯干和尾部结构。通过在Raf-1下游起作用的ntl（（n）在baro（t）在bar ail下）基因的异常原位杂交获得了缺乏通过突变的Raf-1的信号传导的分子证据。我们的研究结果表明，一个功能性的磷脂酸结合位点是必需的Raf-1在胚胎发育中的功能。
Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl ((n) under baro (t) under bar ail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.