14-3-3 antagonizes ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity
14-3-3 antagonizes ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity
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DOI：
10.1128/mcb.22.14.4984-4996.2002
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发表时间：
2002-07-01
影响因子：
5.3
通讯作者：
Marais, R
中科院分区：
文献类型：
--
作者：
Light, Y;Paterson, H;Marais, R
我们研究了S259磷酸化、S621磷酸化和14-3-3结合在调节Raf-1活性中的作用。我们发现，14-3-3结合，而不是Raf-1磷酸化，是正确调节激酶活性所必需的。S621的磷酸化不是活性所必需的，但14-3-3结合是必需的。当14-3-3与保守区2（CR2）的结合被破坏时，Raf-1基础激酶活性升高，并且它可以被（V12，G37）Ras、（V23）TC 21和R-V38-Ras进一步激活。在CR2处破坏14-3-3结合不能恢复Raf-1与（V12，G37）Ras的结合，但允许Raf-1更有效地募集到质膜并刺激其在S338上的磷酸化。最后，（V12）Ras，而不是（V12，G37）Ras，从全长Raf-1置换14-3-3，并且Raf-1与Ras结合。GTP在S259上仍被磷酸化。我们的数据表明，Raf-1与质膜的稳定结合需要Ras介导的14-3-3从CR2的置换。不能取代14-3-3的小G蛋白不能有效地将Raf-1募集到膜上，因此不能刺激激酶活性。
We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and R-V38-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37) Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.