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.
Scholten
Arjen
A
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Poh
Mee Kian
MK
van Veen
Toon A B
TA
van Breukelen
Bas
B
Vos
Marc A
MA
Heck
Albert J R
AJ
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006352:Heart Ventricles / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D000099157:Sphingosine Kinase
D048868:Adaptor Proteins, Signal Transducing; D000242:Cyclic AMP; D006152:Cyclic GMP; D004268:DNA-Binding Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D016601:RNA-Binding Proteins; D000099157:Sphingosine Kinase; C466999:SPHKAP protein, human