Departments of Pharmacology and Medicine (J.A.B., M.G., M.S.-A., M.-C.B., S.-E.O.), and Division of Metabolism, Endocrinology and Nutrition (K.E.B.), University of Washington, Seattle, Washington
Departments of Pharmacology and Medicine (J.A.B., M.G., M.S.-A., M.-C.B., S.-E.O.), and Division of Metabolism, Endocrinology and Nutrition (K.E.B.), University of Washington, Seattle, Washington.
Mol Pharmacol. 2021 May;99(5):342-357. doi: 10.1124/molpharm.120.000197. Epub 2021 Feb 11.
In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone-producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.
近年来，基于高灵敏度质谱的磷酸化蛋白质组学分析开始被应用于鉴定 cAMP 增加后改变的蛋白激酶底物。这些研究确定了大量受 cAMP 增加调节的磷酸化位点。因此，我们现在面临的挑战是确定这些改变的磷酸化位点中有多少与细胞功能的调节有关。这篇综述描述了使用磷酸化蛋白质组学分析来监测环核苷酸磷酸二酯酶 (PDE) 抑制剂对 cAMP 依赖性磷酸化事件的影响。更具体地说，它描述了作者实验室使用选择性 PDE 抑制剂方法进行的这一方法的两个示例。在简短讨论了这些分析对类固醇激素产生细胞和 T 细胞中 cAMP 功能的几个可能结论后，它扩展到讨论了对这些数据的一些更新颖和更推测性的解释。这些解释包括这样一种观点，即多个磷酸化位点而不是单个限速步骤可能调节这些磷酸化，并且通过类比，许多其他 cAMP 依赖性途径也是如此。此外，还讨论了这样一种观点，即有意义的调节需要高磷酸化化学计量比才重要，并提出在许多情况下这一观点并不成立。这些新的解释对药物设计具有重要意义，特别是对靶向途径激动剂。
磷酸化蛋白质组学分析在药物治疗后确定了数千个改变的磷酸化位点，提供了许多可能的调节靶点，但也突出了哪些磷酸化位点在功能上重要的问题。这些数据表明，多步过程是通过磷酸化而不是一个而是许多位点来调节的。以前的大多数研究假设磷酸化改变了一个或很少几个限速步骤。这个概念应该改变。以前的解释还假设亚化学计量比的磷酸化没有调节重要性。这个假设也应该改变。
