Hu C D, Kariya K i, Kotani G, Shirouzu M, Yokoyama S, Kataoka T
Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
J Biol Chem. 1997 May 2;272(18):11702-5. doi: 10.1074/jbc.272.18.11702.
Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.
Raf-1是哺乳动物Ras的主要下游效应器。Ras的效应器结构域与Raf-1的Ras结合结构域的结合对于Ras依赖的Raf-1激活至关重要。然而，Rap1A具有与Ras相同的效应器结构域，却不能激活Raf-1，甚至在体内拮抗Ras的几种功能。最近，我们将Raf-1的富含半胱氨酸区域（CRR）鉴定为另一个Ras结合结构域。携带N26G和V45E突变的Ha-Ras蛋白无法与CRR结合，也无法激活Raf-1。由于这些突变将Ras残基替换为Rap1A的残基，我们研究了Rap1A是否缺乏与CRR结合的能力。与预期相反，与Ha-Ras相比，Rap1A与CRR的结合大大增强。在携带另一个Rap1A类型突变E31K的Ha-Ras中也发现了增强的CRR结合。Rap1A和Ha-Ras（E31K）突变体均无法激活Raf-1，并在Sf9细胞中干扰了Ha-Ras依赖的Raf-1激活。Rap1A与CRR结合的增强导致Rap1A和Ha-Ras分别通过与CRR和Ras结合结构域结合而与Raf-1 N端区域共缔合。这些结果表明，Rap1A通过抑制Ras与Raf-1 CRR的结合来干扰Ras依赖的Raf-1激活。
