cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry.
cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry.
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DOI：
10.1042/bcj20160648
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发表时间：
2016-10-01
期刊：
影响因子：
--
通讯作者：
Eyers PA
中科院分区：
文献类型：
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作者：
Byrne DP;Vonderach M;Ferries S;Brownridge PJ;Eyers CE;Eyers PA
CAMP依赖的蛋白激酶(PKA)是一种典型的生物信号模块，也是理解蛋白激酶调控的模型。在本研究中，我们将生物化学与差示扫描荧光(DSF)和离子迁移率-质谱仪(IM-MS)相结合，在体外评价了磷酸化和结构对异构体PKA蛋白复合体的配体结合、动力学和稳定性的影响。我们发现了动态的，构象不同的PKA催化亚基群体，具有不同的结构稳定性和对生理蛋白抑制剂PKI的敏感性。重组PKA R2C2全酶的天然MS显示了可变的亚基化学计量比和通过PKI结合的全酶消融。最后，我们发现，尽管一个‘死’的PKA催化结构域不能与溶液中的ATP结合，但它可以与几种重要的化学激酶抑制剂相互作用。这些数据显示了IM-MS和DSF在探测PKA动态和调节方面的组合能力，这些技术可以用于评估其他蛋白质-配体复合体，对细胞信号转导具有广泛的意义。
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R2C2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.