Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.
Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.
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DOI：
10.1074/jbc.m114.561787
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发表时间：
2014-08-22
期刊：
影响因子：
--
通讯作者：
Overduin M
中科院分区：
文献类型：
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作者：
Lenoir M;Sugawara M;Kaur J;Ball LJ;Overduin M
背景：Lbc癌蛋白刺激RhoA中GT3活性失调。结果如下：虽然Lbc DH结构域可以独立地通过RhoA激活GTP交换，但其PH结构域也呈现用于DH和激活的RhoA相互作用的表面。结论：Lbc支架的两个结构域上的多个位点控制RhoA。意义：揭示了基于机制设计Lbc作用调节剂的新位点。小的GTdR RhoA在与淋巴母细胞危象（Lbc）（A激酶锚定蛋白13（AKAP 13）的致癌形式）相互作用后促进失调的信号传导。癌细胞Lbc蛋白是Rho特异性的鸟嘌呤核苷酸交换因子（GEF），其结构与心肌肥厚和肿瘤的发生有关，但其作用机制尚未见报道。Lbc的普列克底物蛋白同源性（PH）结构域位于蛋白质的C末端，并且在此显示特异性识别活化的RhoA而不是脂质。分离的dbl同源（DH）结构域可以作为具有增强活性的独立激活剂起作用。然而，DH结构域通常不充当与RhoA-GDP的单独Lbc界面。相反，它由PH结构域负控制。特别地，DH螺旋束通过螺旋接头与结构依赖性PH结构域偶联，这降低了其活性。这两个域一起在溶液中形成刚性支架，如通过小角X射线散射和基于1H、13 C、15 N的NMR光谱所证明的。这两个结构域呈“椅子”形状，其背部具有独立的GEF活性，PH结构域为RhoA-GTP对接而不是膜识别提供了广阔的座位。这为DH和PH结构域如何在溶液中一起工作以支持调节的RhoA活性提供了结构和动力学见解。突变分析支持DH-RhoA相互作用的双功能PH结构域介导，并解释了为什么控制GEF信号传导需要串联结构域。
Background: The Lbc oncoprotein stimulates deregulated GTPase activity in RhoA. Results: Although the Lbc DH domain can independently activate GTP exchange by RhoA, its PH domain also presents surfaces for DH and activated RhoA interaction. Conclusion: Multiple sites on both structural domains of the Lbc scaffold control RhoA. Significance: New sites for mechanism-based design of modulators of Lbc action are revealed. The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and 1H,13C,15N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.