Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains.
Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains.
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环状单磷酸腺苷依赖性蛋白激酶全酶的环状核苷酸选择性激活的切换揭示了串联环状核苷酸结合域的独特作用。
DOI：
10.1021/acschembio.7b00732
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发表时间：
2017
影响因子：
4
通讯作者：
Lim,ChintenJames
中科院分区：
文献类型：
--
作者：
He,Daniel;Lorenz,Robin;Kim,Choel;Herberg,FriedrichW;Lim,ChintenJames
环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）依赖性蛋白激酶（PKA和PKG）是环核苷酸信号传导的关键效应子。两者都具有包括串联环核苷酸结合（CNB）结构域CNB-A和CNB-B的结构特征，但它们的功能通过cAMP或cGMP的优先激活而分离。基于结构研究和建模，已经鉴定了两种激酶的关键CNB接触残基。在这项研究中，我们探讨了PKA激活从cAMP依赖性转换为cGMP依赖性的要求。利用纯化的重组蛋白，以及用野生型或突变型PKA-RI α基因重组的RIα缺陷小鼠胚胎成纤维细胞，在体外评估了PKA-RIα的CNB-A内T192 R/A212 T或CNB-B内G316 R/A336 T残基取代对环核苷酸结合和全酶激活的影响。当任一CNB结构域中的残基发生突变时，观察到结合和活化选择性的丧失，而两个CNB结构域中的突变导致选择性从cAMP完全转换为cGMP。选择性的转换也在体内重现，证实了它们在细胞中的功能作用。我们的研究结果突出了每个CNB结构域内的关键环核苷酸接触的重要性，并表明这些结构域可能已经从祖先的基因产物演变为产生两种不同的环核苷酸依赖性蛋白激酶。
The cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinases (PKA and PKG) are key effectors of cyclic nucleotide signaling. Both share structural features that include tandem cyclic nucleotide-binding (CNB) domains, CNB-A and CNB-B, yet their functions are separated through preferential activation by either cAMP or cGMP. Based on structural studies and modeling, key CNB contact residues have been identified for both kinases. In this study, we explored the requirements for conversion of PKA activation from cAMP-dependent to cGMP-dependent. The consequences of the residue substitutions T192R/A212T within CNB-A or G316R/A336T within CNB-B of PKA-RIα on cyclic nucleotide binding and holoenzyme activation were assessedin vitrousing purified recombinant proteins, andex vivousing RIα-deficient mouse embryonic fibroblasts genetically reconstituted with wild-type or mutant PKA-RIα.In vitro, a loss of binding and activation selectivity was observed when residues in either one of the CNB domains were mutated, while mutations in both CNB domains resulted in a complete switch of selectivity from cAMP to cGMP. The switch in selectivity was also recapitulatedex vivo, confirming their functional roles in cells. Our results highlight the importance of key cyclic nucleotide contacts within each CNB domain and suggest that these domains may have evolved from an ancestral gene product to yield two distinct cyclic nucleotide-dependent protein kinases.