Department of Biomedical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Biochem Soc Trans. 2013 Aug;41(4):981-6. doi: 10.1042/BST20130120.
The catalytic (C) subunit of PKA was the first protein kinase structure to be solved, and it continues to serve as the prototype for the protein kinase superfamily. In contrast, by comparing many active and inactive kinases, we developed a novel 'spine' concept where every active kinase is composed of two hydrophobic spines anchored to a hydrophobic F-helix. The R-spine (regulatory spine) is dynamically assembled, typically by activation loop phosphorylation, whereas the C-spine (catalytic spine) is completed by the adenine ring of ATP. In the present paper, we show how the spine concept can be applied to B-Raf, specifically to engineer a kinase-dead pseudokinase. To achieve this, we mutated one of the C-spine residues in the N-lobe (N-terminal lobe), Ala481, to phenylalanine. This mutant cannot bind ATP and is thus kinase-dead, presumably because the phenylalanine ring fills the adenine-binding pocket. The C-spine is thus fused. However, the A481F mutant is still capable of binding wild-type B-Raf and wild-type C-Raf, and dimerization with a wild-type Raf leads to downstream activation of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK. The mutant requires dimerization, but is independent of Ras and does not require enzymatic activity. By distinguishing between catalytic and scaffold functions of B-Raf, we define kinases as being bifunctional and show that, at least in some cases, the scaffold function is sufficient for downstream signalling. Since this alanine residue is one of the most highly conserved residues in the kinome, we suggest that this may be a general strategy for engineering kinase-dead pseudokinases and exploring biological functions that are independent of catalysis.
蛋白激酶 A 的催化 (C) 亚基是第一个被解析的蛋白激酶结构，它仍然是蛋白激酶超家族的原型。相比之下，通过比较许多有活性和无活性的激酶，我们提出了一个新的“脊柱”概念，即每个有活性的激酶都是由两个锚定在疏水性 F 螺旋上的疏水性脊柱组成的。R 脊柱（调节脊柱）是动态组装的，通常通过激活环磷酸化，而 C 脊柱（催化脊柱）则由 ATP 的腺嘌呤环完成。在本文中，我们展示了如何将脊柱概念应用于 B-Raf，特别是设计激酶失活的假激酶。为了实现这一点，我们突变了 N 端结构域 (N-末端结构域) 中的一个 C 脊柱残基 (Ala481) 为苯丙氨酸。这种突变体不能结合 ATP，因此是激酶失活的，大概是因为苯丙氨酸环填充了腺嘌呤结合口袋。C 脊柱因此融合了。然而，A481F 突变体仍然能够结合野生型 B-Raf 和野生型 C-Raf，并且与野生型 Raf 的二聚化导致 MEK [MAPK (丝裂原激活的蛋白激酶)/ERK (细胞外信号调节激酶) 激酶] 和 ERK 的下游激活。该突变体需要二聚化，但不依赖 Ras，也不需要酶活性。通过区分 B-Raf 的催化和支架功能，我们将激酶定义为双功能，并表明，至少在某些情况下，支架功能足以进行下游信号传递。由于这个丙氨酸残基是激酶组中最保守的残基之一，我们建议这可能是一种设计激酶失活假激酶和探索独立于催化的生物学功能的通用策略。
