Mutational analysis of Raf-1 cysteine rich domain: Requirement for a cluster of basic aminoacids for interaction with phosphatidylserine
Mutational analysis of Raf-1 cysteine rich domain: Requirement for a cluster of basic aminoacids for interaction with phosphatidylserine
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DOI：
10.1023/a:1006981411691
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发表时间：
1999-08-01
影响因子：
4.3
通讯作者：
Bell, RM
中科院分区：
文献类型：
--
作者：
Improta-Brears, T;Ghosh, S;Bell, RM
Raf-1激酶的激活是由响应外部刺激的Raf-1易位到质膜引起的。Raf-1的膜定位是通过与活化的Ras和膜磷脂的相互作用而促进的。先前的证据表明，Raf-1与Ras的相互作用是由Raf-1 n端区域的两个不同结构域介导的，其中包括氨基酸残基51-131和残基139-184，后者编码含锌的富含半胱氨酸的结构域。据报道，Raf-1的富含半胱氨酸的结构域还与其他蛋白质（如14-3-3）结合，并选择性地结合酸性磷脂，特别是磷脂酰丝氨酸（PS）。在本研究中，我们研究了Raf-1富半胱氨酸结构域内的进行性缺失和点突变对其结合PS能力的影响。在体外实验中，我们观察到所有Raf-1缺失突变体与PS的相互作用减少，无论是作为全长蛋白表达，还是作为含有分离的富半胱氨酸结构域的片段表达。特别是，碱性氨基酸R-143、K-144和K-148似乎对与PS的相互作用至关重要，因为将所有三个残基替换为丙氨酸导致蛋白质无法与富含PS的脂质体相互作用。体内富含半胱氨酸结构域的Raf-1表达导致截断多肽，缺乏Ras和PS结合位点，在血清刺激下不能再转运到质膜上。这些结果表明，Raf-1富半胱氨酸结构域前半部分的碱性残基143、144和148参与了与脂质双分子层的关联，并可能与蛋白质稳定性有关，因此它们可能与Raf-1的定位和随后的激活有关。
Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R-143, K-144, and K-148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.