Distal Recognition Sites in Substrates Are Required for Efficient Phosphorylation by the cAMP-Dependent Protein Kinase
Distal Recognition Sites in Substrates Are Required for Efficient Phosphorylation by the cAMP-Dependent Protein Kinase
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DOI：
10.1534/genetics.109.102178
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发表时间：
2009-06-01
期刊：
影响因子：
3.3
通讯作者：
Herman, Paul K.
中科院分区：
文献类型：
--
作者：
Deminoff, Stephen J.;Ramachandran, Vidhya;Herman, Paul K.
蛋白激酶是真核细胞信号转导的重要介体，识别这些酶的底物对于全面了解大多数信号网络是至关重要的。在这份报告中，新的底物结合变体的cAMP依赖的蛋白激酶(PKA)被用来确定在体内有效的磷酸化所需的底物结构域。大多数野生型蛋白激酶，包括PKA，只与其底物短暂地相互作用。鉴定的底物结构域位于磷酸化位点的远端，并被发现与PKA的C-末端区域相互作用，该区域本身从活性部位移除。只有一小部分PKA改变导致与底物的稳定结合，所鉴定的残基聚集在该酶的疏水核心内。有趣的是，这些残基从酶的活性部位延伸到这里确定的C末端底物结合结构域。这种空间组织在整个真核蛋白激酶家族中是保守的，而这些残基在第二个无关的蛋白激酶中的变化也导致了与底物的稳定结合。总之，这项研究确定了PKA底物上对该酶识别重要的远端位点，并表明这些结构域与PKA的相互作用可能影响底物结合和/或释放的特定方面。
Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.