Light Yvonne, Paterson Hugh, Marais Richard
Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
Mol Cell Biol. 2002 Jul;22(14):4984-96. doi: 10.1128/MCB.22.14.4984-4996.2002.
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 (V38)R-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.
我们研究了S259磷酸化、S621磷酸化及14-3-3结合在调节Raf-1活性中所起的作用。我们发现，激酶活性的正确调节需要14-3-3结合，而非Raf-1磷酸化。活性并不需要S621磷酸化，但14-3-3结合至关重要。当14-3-3与保守区域2（CR2）的结合被破坏时，Raf-1基础激酶活性升高，并且它能被（V12,G37）Ras、（V23）TC21和（V38）R-Ras进一步激活。CR2处14-3-3结合的破坏并未恢复Raf-1与（V12,G37）Ras的结合，但使Raf-1更有效地募集到质膜并刺激其S338位点的磷酸化。最后，（V12）Ras而非（V12,G37）Ras将14-3-3从全长Raf-1及与Ras结合的Raf-1上置换下来。GTP在S259处仍被磷酸化。我们的数据表明，Raf-1与质膜的稳定结合需要Ras介导14-3-3从CR2上的置换。无法置换14-3-3的小G蛋白不能有效地将Raf-1募集到膜上，因此无法刺激激酶活性。
