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

Abstract

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.

A PKA-ezrin-connexin 43 signaling complex controls gap junction communication and thereby trophoblast cell fusion

“…Pull-down assays cAMP pulldown assays (8-AHA-cAMP-agarose beads) were performed as described previously (Oberprieler et al, 2010;Scholten et al, 2006). Cell lysate (500 mg proteins) was incubated with purified RI-FLAG or RII-FLAG proteins (100 mg).…”

A-kinase anchoring proteins: scaffolding proteins in the heart

“…ezrin (36), sphingosine kinase type 1-interacting protein (61,96), gravin (81), synemin (95), myospryn (92), troponin T (109), and the phosphoinositide 3-kinase p110␥ (90) have been shown to be expressed in cardiac tissues (Table 1). In this context, it is now clear that in the heart, AKAP-based transduction complexes play a crucial role in coordinating signaling pathways that control physiological functions such as Ca 2ϩ cycling, cardiac contractility, and action potential duration, as well as pathophysiological processes including arrhythmias, cardiomyocyte hypertrophy, heart failure, and the adaptive response to hypoxia.…”

Suction blister fluid as potential body fluid for biomarker proteins

“…For this semi-quantitative analysis, the amount of uniquely detected peptides in suction blister fluid and serum were compared for individual proteins. Therefore, the relative extent to which the proteins are present in suction blister fluid and serum was very roughly estimated by the number of uniquely detected peptides found per protein in both body fluids according to similar principles as used by Scholten et al, Sanders et al, and Ishihama et al [34][35][36] for rough determination of protein abundances. The ratios between the number of uniquely detected peptides per protein found in suction blister fluid and serum were plotted from high to low ratios using a logarithmic scale (Fig.…”