Okada T, Hu C D, Jin T G, Kariya K, Yamawaki-Kataoka Y, Kataoka T
Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
Mol Cell Biol. 1999 Sep;19(9):6057-64. doi: 10.1128/MCB.19.9.6057.
To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.
为了在质膜上被完全激活，Raf-1必须与Ras建立两种不同的相互作用模式，一种通过其Ras结合结构域，另一种通过其富含半胱氨酸的结构域（CRD）。Ras同源物Rap1A无法激活Raf-1，甚至拮抗Raf-1的Ras依赖性激活。我们之前提出，Rap1A的这种特性可能归因于其与Raf-1 CRD的相互作用比Ras大大增强。另一方面，另一个Raf家族成员B-Raf可被Ras和Rap1A激活。当测量与Ras和Rap1A的相互作用时，B-Raf CRD与Rap1A的相互作用并未增强，这表明CRD处相互作用的强度可能解释了Rap1A对Raf-1和B-Raf的不同作用。Raf-1和B-Raf之间的结构域交换实验进一步支持了CRD处相互作用的重要性，该实验清楚地表明CRD的性质决定了对Rap1A反应的特异性：CRD被B-Raf CRD取代的Raf-1可被Rap1A激活，而CRD被Raf-1 CRD取代的B-Raf失去了对Rap1A的反应。最后，分离出一个与Rap1A相互作用选择性增强的B-Raf CRD突变体，发现其具有双突变K252E/M278T。携带此突变的B-Raf未被Rap1A激活，但保留了对Ras的反应。这些结果表明，在其CRD处与Ras和Rap1A相互作用的强度可能是Ras家族小GTP酶调节Raf激酶活性的关键决定因素。
