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Molecular & Cellular Proteomics 8:1016-1028, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins^*,S

Thin Thin Aye, Shabaz Mohammed, Henk W. P. van den Toorn, Toon A. B. van Veen^,¶, Marcel A. G. van der Heyden, Arjen Scholten and Albert J. R. Heck^,||

From the Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and the Department of Medical Physiology, University Medical Centre Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.

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