Diversity of cAMP-dependent protein kinase isoforms and their anchoring proteins in mouse ventricular tissue.
Using a chemical proteomics approach, we efficiently enriched for the generally low abundant cAMP signaling proteins, and their interactors, directly from mouse ventricular tissue. The presence of undesired contaminating (noncyclic) nucleotide-binding proteins was diminished using a tailored sequential elution protocol. Through further optimization of this affinity purification and elution protocol, we were able to detect all known protein kinase A regulatory isoforms (PKA-R). Furthermore, 11 different A-kinase anchoring proteins (AKAPs) were detected. A proposed fusion protein of paralemmin 2 and AKAP2 could be decisively established as a novel AKAP at the protein level in ventricular tissue. When comparing this dataset of cAMP-affinity purified proteins with earlier data obtained with immobilized cGMP from rat ventricular tissue, we observe a large overlap in the retained proteins but also some clear differences. Furthermore, implementation of an in-depth analysis of in vivo phosphorylation sites on PKA-R revealed the presence of several differentially phosphorylated PKA-R isoforms. This illustrates yet another layer of functional regulation in cyclic nucleotide signaling. In general, our improved chemical proteomics screen offers a broad, but detailed, view on nature's complex diversity in cyclic nucleotide signaling mechanisms. Possibly different AKAP-isoforms may direct differentially phosphorylated PKA-R isoforms to different cellular compartments, providing a multifaceted platform for just this kinase.
Scholten
Arjen
A
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.
van Veen
Toon A B
TA
Vos
Marc A
MA
Heck
Albert J R
AJ
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006352:Heart Ventricles / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D048868:Adaptor Proteins, Signal Transducing; D010455:Peptides; D020033:Protein Isoforms; D017868:Cyclic AMP-Dependent Protein Kinases