Cyclic nucleotides as affinity tools: Phosphorothioate cAMP analogues address specific PKA subproteomes
Cyclic nucleotides as affinity tools: Phosphorothioate cAMP analogues address specific PKA subproteomes
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DOI：
10.1016/j.nbt.2010.12.001
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发表时间：
2011-07-01
影响因子：
5.4
通讯作者：
Herberg, Friedrich W.
中科院分区：
文献类型：
--
作者：
Hanke, Susanne E.;Bertinetti, Daniela;Herberg, Friedrich W.
cAMP（Adenosine-3 '，5'-cyclic monophosphate）是真核细胞中控制不同靶点的第二信使。在（亚）蛋白质组学方法中，使用两类硫代磷酸cAMP亲和工具来分离和鉴定主要cAMP靶点cAMP依赖性蛋白激酶（PKA）的信号传导复合物。激动剂类似物（此处：Sp-cAMPS）与PKA的调节亚基（PKA-R）以及它们的相互作用配偶体结合，并引起包含PKA-R和PKA的催化亚基（PKA-C）的全酶复合物的解离。拮抗剂类似物（此处：Rp-cAMPS）结合全酶而不解离复合物，并被开发用于鉴定结合整个复合物或PKA-C的相互作用配偶体。从组织提取物中分离出80多种不同的蛋白质，包括几种PKA同工型和已知的以及潜在的新的相互作用伙伴。然而，一般核苷酸结合蛋白的非特异性结合限制了这种化学蛋白质组学方法的结果。采用表面等离子体共振（SPR）来优化下拉蛋白质组学的整个工作流程，并定量不同核苷酸（ATP、ADP、GTP和NADH）对PKA-R与亲和材料结合的影响。我们可以证明向裂解物中添加NADH改善了下拉实验中的特异性。使用SPR研究和拉下实验的组合，明确地表明，可以从cAMPS类似物基质中特异性地将蛋白质复合物与cAMP或cGMP结合。PKA-R相互作用组和与相互作用蛋白质复合的全酶的并排分析将有助于进一步解剖多方面的PKA信号网络。
cAMP (Adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C). Antagonist analogues (here: Rp-cAMPS) bind to the holoenzyme without dissociating the complex and were developed to identify interaction partners that bind to the entire complex or to PKA-C. More than 80 different proteins were isolated from tissue extracts including several PKA isoforms and known as well as potentially new interaction partners. Nevertheless, unspecific binding of general nucleotide binding proteins limited the outcome of this chemical proteomics approach. Surface plasmon resonance (SPR) was employed to optimise the entire workflow of pull down proteomics and to quantify the effects of different nucleotides (ATP, ADP, GTP and NADH) on PKA-R binding to affinity material. We could demonstrate that the addition of NADH to lysates improved specificity in pull down experiments. Using a combination of SPR studies and pull down experiments it was shown unambiguously that it is possible to specifically elute protein complexes with cAMP or cGMP from cAMPS analogue matrices. The side-by-side analysis of the PKA-R interactome and the holoenzyme complexed with interacting proteins will contribute to a further dissection of the multifaceted PKA signalling network.