FUNCTIONAL MAPPING OF THE N-TERMINAL REGULATORY DOMAIN IN THE HUMAN RAF-1 PROTEIN-KINASE
FUNCTIONAL MAPPING OF THE N-TERMINAL REGULATORY DOMAIN IN THE HUMAN RAF-1 PROTEIN-KINASE
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DOI：
10.1074/jbc.270.23.14100
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发表时间：
1995-06-09
影响因子：
4.8
通讯作者：
JOVE, R
中科院分区：
文献类型：
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作者：
CHOW, YH;PUMIGLIA, K;JOVE, R
Raf-1 是一种丝氨酸/苏氨酸激酶，位于细胞表面至细胞核的有丝分裂信号转导途径的关键中继点。 Raf-1 转化潜力的激活与 N 端截短和/或与其他蛋白质的融合有关，表明 Raf-1 N 端半部具有负调节结构域。生成了七个内部缺失突变体，它们一起扫描人 Raf-1 蛋白的整个 N 端半部，以绘制该调节结构域中的功能区域。使用杆状病毒/昆虫细胞过表达系统和使用 Raf-1 的已知生理底物（丝裂原激活蛋白激酶激酶）进行的体外激酶测定，评估了缺失突变对 Raf-1 激酶活性的影响。删除保守区 2 和 3 之间的独特序列中的氨基酸 276-323 会导致 Raf-1 基础激酶活性适度升高，而删除保守区 1 中的氨基酸 133-180 会导致激酶活性降低。令人惊讶的是，没有一个 Raf-1 N 端缺失突变体，包括在啮齿动物成纤维细胞中转化的截短版本，表现出基础激酶活性水平大大增加。此外，虽然 Ras 激活 Raf-1 激酶需要保守区 1 中的序列，但 Src 激活只需要包含 Raf-1 激酶结构域的 C 端一半。这些发现表明，Raf-1 中的 N 端缺失不一定会导致基础激酶活性组成性升高，并且 N 端调节结构域对于 Src 激活 Raf-1 来说是完全可有可无的。
Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways hom the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain, Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src, These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.