Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF
Mutation that blocks ATP binding creates a pseudokinase stabilizing the scaffolding function of kinase suppressor of Ras, CRAF and BRAF
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DOI：
10.1073/pnas.1102554108
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发表时间：
2011-04-12
影响因子：
11.1
通讯作者：
Shaw, Andrey S.
中科院分区：
文献类型：
--
作者：
Hu, Jiancheng;Yu, Haiyang;Shaw, Andrey S.
由于RAS和BRAF突变代表了人类肿瘤中最常见的突变，因此抑制剂的鉴定一直是一个主要目标。令人惊讶的是，新的致癌BRAF特异性抑制剂抑制用突变的BRAF转化的细胞，但矛盾地刺激用RAS转化的细胞的生长。在这里，我们表明，激活的机制是通过药物诱导的CRAF和激酶抑制因子Ras（KSR）1之间的二聚体形成。为了了解KSR1的功能，我们产生了一个KSR1突变体，它不能结合ATP，但稳定了KSR1的封闭活性构象。分子模拟表明，突变体稳定的两个疏水性的刺封闭的活性构象的关键。因此，我们可以使用突变体来区分KSR1的支架功能与激酶功能。KSR 1突变体组成型结合RAF和丝裂原活化蛋白激酶激酶（MEK），但不能重建活性，表明KSR 1的催化活性是其功能所必需的。BRAF和CRAF中的类似突变使我们能够测试模型的通用性。突变诱导的变化与两种激酶的活性、闭合构象一致，并证实BRAF在MAP激酶途径中与CRAF功能不同。这项工作不仅表明KSR1可能作为激酶发挥作用，我们还预计我们产生的突变可能广泛适用于稳定其他激酶的闭合构象，其中许多激酶也可能形成二聚体。
Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.