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

Henk W.P Van Den Toorn,Toon A.B Van Veen,Albert J.R Heck,Shabaz Mohammed,Thin Thin Aye,Arjen Scholten,Marcel A.G Van Der Heyden

doi:10.1074/mcp.m800226-mcp200

Henk W.P Van Den Toorn, Toon A.B Van Veen + Show 5 more

Open Access

https://doi.org/10.1074/mcp.m800226-mcp200

Copy DOI

Abstract

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.

Highlights

Proteins Can be Bead-specific—In our affinity pull-downs we detected some high abundance “background” proteins such as actin, hemoglobin, alpha 2-globulin, L-lactose dehydrogenase B-chain and glyceraldehyde-3phosphate dehydrogenase (GAPDH)
We use a common strategy in proteomics, namely stable isotope labeling, whereby we introduce the label via reductive amination (18 –20)
A very selective enrichment of PKA RI isoforms can be achieved by using cAMP-agarose beads in which the hydroxyl group at the 2Ј position on the ribose was replaced with a methoxyl group

Summary

Introduction

Proteins Can be Bead-specific—In our affinity pull-downs we detected some high abundance “background” proteins such as actin, hemoglobin, alpha 2-globulin, L-lactose dehydrogenase B-chain and glyceraldehyde-3phosphate dehydrogenase (GAPDH). When analyzing the differential binding behavior of these proteins we observed that actin, hemoglobin and alpha 2-globulin were abundant no matter which of the four beads (C8, C2, C8_OCH3, and control beads) we used. Several “background” proteins revealed to some extent specific enrichments. L-lactose dehydrogenase B-chain bound to C8 beads with a relative affinity ratio of 100, compared with C2. GAPDH was enriched by the C8 beads with a relative affinity ratio of 4:1 to both C2 and C8_OCH3. Various ribosomal proteins were found to be preferentially enriched using the C2 beads (supplemental Table S2)

Results

Discussion

Conclusion