pr0600529_si_001.xls (24 kB)
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
dataset
posted on 2006-06-02, 00:00 authored by Arjen Scholten, Mee Kian Poh, Toon A. B. van Veen, Bas van Breukelen, Marc A. Vos, Albert J. R. HeckThe 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
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latter classbioinformatics analysiselute ADP6 AKAPsprotein kinases PKAChemical Proteomics Approachrat heartmass spectrometrycGMP binding proteinsagarose beadsPKGHEK 293 cellsAbundant AKAPThe cyclic nucleotide monophosphates cAMPeluting proteinsGDPsequential elution protocolDNA binding proteinschemical proteomics approachSKIPmessenger molecules
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