Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid - Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells
Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid - Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells
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DOI：
10.1074/jbc.271.14.8472
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发表时间：
1996-04-05
影响因子：
4.8
通讯作者：
Bell, RM
中科院分区：
文献类型：
--
作者：
Ghosh, S;Strum, JC;Bell, RM
先前的研究表明Raf-1激酶的富含半胱氨酸的氨基末端结构域选择性地与磷脂酰丝氨酸相互作用（Ghosh，S.，Xie，W.问：Quest，A. F. G.，Mabrouk，G. M.，Strum，J.C.，和Bell，R. M.（1994）J.Biol.Chem.269，10000-10007）。进一步的分析表明，全长Raf-1结合磷脂酰丝氨酸和磷脂酸（PA）。具体而言，Raf-1激酶的羧基末端结构域（RafC;人Raf-1的残基295-648）与磷脂酸强烈相互作用。RafC与PA的结合显示出正协同性，Hill数在3.3和6.2之间;表观Kd范围为4.9 +/- 0.6至7.8 +/- 0.9 mol % PA。RafC与PA的相互作用显示出与Raf-1的富含半胱氨酸的结构域与PA之间的相互作用不同的pH依赖性。此外，RafC-PA相互作用在高离子强度下不受影响。在所有测试的脂质中，仅PA和心磷脂表现出高亲和力结合;其他酸性脂质无效或弱有效。通过缺失诱变，RafC内的PA结合位点被缩小到残基389和423之间的35个氨基酸的区段。RafC没有结合磷脂醇，也，抑制PA形成在Madin-Darby犬肾细胞治疗1%乙醇显着减少了Raf-1的易位从细胞质到膜刺激后，12-O-十四烷酰佛波醇-13-乙酸酯。这些结果表明，脂质第二信使，PA，在体内的调节易位和随后的激活Raf-1的潜在作用。
Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295-648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent K-d ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.