Analysis of the cGMP/cAMP Interactome Using a Chemical Proteomics Approach in Mammalian Heart Tissue Validates Sphingosine Kinase Type 1-interacting Protein as a Genuine and Highly Abundant AKAP

The cyclic nucleotide monophosphates cAMP and cGMP play an essential role in many signaling
pathways. To analyze which proteins do interact with these second messenger molecules, we developed
a chemical proteomics approach using cAMP and cGMP immobilized onto agarose beads, via flexible
linkers in the 2- and 8-position of the nucleotide. Optimization of the affinity pull-down procedures in
lysates of HEK293 cells revealed that a large variety of proteins could be pulled down specifically.
Identification of these proteins by mass spectrometry showed that many of these proteins were indeed
genuine cAMP or cGMP binding proteins. However, additionally many of the pulled-down proteins
were more abundant AMP/ADP/ATP, GMP/GDP/GTP, or general DNA/RNA binding proteins. Therefore,
a sequential elution protocol was developed, eluting proteins from the beads using solutions containing
ADP, GDP, cGMP, and/or cAMP, respectively. Using this protocol, we were able to sequentially and
selectively elute ADP, GDP, and DNA binding proteins. The fraction left on the beads was further
enriched, for cAMP/cGMP binding proteins. Transferring this protocol to the analysis of the cGMP/cAMP “interactome” in rat heart ventricular tissue enabled the specific pull-down of known cAMP/cGMP binding proteins such as cAMP and cGMP dependent protein kinases PKA and PKG, several
phosphodiesterases and 6 AKAPs, that interact with PKA. Among the latter class of proteins was the
highly abundant sphingosine kinase type1-interating protein (SKIP), recently proposed to be a potential
AKAP. Further bioinformatics analysis endorses that SKIP is indeed a genuine PKA interacting protein,
which is highly abundant in heart ventricular tissue.
Keywords: cAMP • cGMP • PKA • PKG • AKAP • chemical proteomics

环核苷酸单磷酸类分子cAMP（Cyclic adenosine monophosphate，环腺苷酸）与cGMP（Cyclic guanosine monophosphate，环鸟苷酸）在诸多信号通路中发挥着至关重要的作用。为解析可与这类第二信使分子相互作用的蛋白质种类，我们开发了一种化学蛋白质组学方法：将cAMP与cGMP通过柔性连接子固定于琼脂糖微珠上，固定位点选在核苷酸的2位与8位。
我们对HEK293细胞裂解液中的亲和下拉实验流程进行优化，结果显示可特异性富集得到多种蛋白质。通过质谱法对这些蛋白质进行鉴定，证实其中多数确实为真正的cAMP或cGMP结合蛋白。
但与此同时，诸多被富集的蛋白质同时为丰度较高的AMP/ADP/ATP结合蛋白、GMP/GDP/GTP结合蛋白，或是通用型DNA/RNA结合蛋白。为此，我们开发了一套分步洗脱方案：分别使用含有ADP、GDP、cGMP以及cAMP的溶液从微珠上洗脱结合的蛋白质。
利用该方案，我们能够依次选择性洗脱ADP、GDP与DNA结合蛋白，残留在微珠上的组分可进一步富集cAMP/cGMP结合蛋白。
将该方案应用于大鼠心室心肌组织中cGMP/cAMP"相互作用组"的分析，可特异性富集得到已知的cAMP/cGMP结合蛋白，例如cAMP与cGMP依赖的蛋白激酶PKA（Protein Kinase A，蛋白激酶A）、PKG（Protein Kinase G，蛋白激酶G），多种磷酸二酯酶，以及6种可与PKA相互作用的AKAP（A-kinase anchoring proteins，A激酶锚定蛋白）。
在这类蛋白质中，丰度极高的鞘氨醇激酶1相互作用蛋白SKIP（sphingosine kinase type1-interacting protein）近期被提出为潜在AKAP。进一步的生物信息学分析证实，SKIP确实为真正的PKA相互作用蛋白，且在心室心肌组织中丰度极高。
关键词：cAMP • cGMP • PKA • PKG • AKAP • 化学蛋白质组学