Diversity of cAMP-Dependent Protein Kinase Isoforms and Their Anchoring Proteins in Mouse Ventricular Tissue

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• 作者：Arjen Scholten ; Toon A. B. van Veen ; Marc A. Vos ; Albert J. R. Heck
• 刊名：Journal of Proteome Research
• 出版年：2007
• 出版时间：May 2007
• 年：2007
• 卷：6
• 期：5
• 页码：1705 - 1717
• 全文大小：1526K
• 年卷期：v.6,no.5(May 2007)
• ISSN：1535-3907

文摘

Using a chemical proteomics approach, we efficiently enriched for the generally low abundant cAMPsignaling proteins, and their interactors, directly from mouse ventricular tissue. The presence ofundesired contaminating (noncyclic) nucleotide-binding proteins was diminished using a tailoredsequential 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 differentA-kinase anchoring proteins (AKAPs) were detected. A proposed fusion protein of paralemmin 2 andAKAP2 could be decisively established as a novel AKAP at the protein level in ventricular tissue. Whencomparing this dataset of cAMP-affinity purified proteins with earlier data obtained with immobilizedcGMP from rat ventricular tissue, we observe a large overlap in the retained proteins but also someclear differences. Furthermore, implementation of an in-depth analysis of in vivo phosphorylation siteson PKA-R revealed the presence of several differentially phosphorylated PKA-R isoforms. This illustratesyet another layer of functional regulation in cyclic nucleotide signaling. In general, our improvedchemical proteomics screen offers a broad, but detailed, view on nature's complex diversity in cyclicnucleotide signaling mechanisms. Possibly different AKAP-isoforms may direct differentially phosphorylated PKA-R isoforms to different cellular compartments, providing a multifaceted platform for justthis kinase.