McPherson R A, Harding A, Roy S, Lane A, Hancock J F
Queensland Cancer Fund Laboratory for Experimental Oncology, Department of Pathology, University of Queensland Medical School, Herston, Australia.
Oncogene. 1999 Jul 1;18(26):3862-9. doi: 10.1038/sj.onc.1202730.
Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.
Raf-1的激活发生在质膜上。我们最近发现，14-3-3必须与Raf-1形成复合物，才能有效地募集到质膜并被Ras激活，但在质膜结合后，14-3-3会从Raf-1上完全解离。我们在此表明，Raf-1锌指对于14-3-3的结合并非绝对必需，但对于稳定Raf-1与14-3-3之间的相互作用是必需的。用磷脂酰丝氨酸（一种质膜内层磷脂）孵育Raf-1，会导致14-3-3的去除以及Raf-1激酶活性的增加，而使用特定磷酸肽从Raf-1上去除14-3-3则会显著降低Raf-1的基础激酶活性。如果首先将Raf-1从溶液中去除，磷酸肽从活化的Raf-1上置换14-3-3对激酶活性没有影响，但会完全消除可溶性活化Raf-1的激酶活性。这些结果提示了一种在质膜上从Raf-1去除14-3-3的机制，并表明根据实验条件，从Raf-1上去除14-3-3会产生明显不同的效果。
