Abstract
BackgroundIn the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins.ResultsTwo sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. These compounds were tested via direct surface plasmon resonance (SPR) analyses for their binding properties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinity purification approach to analyse their binding and elution properties for the enrichment and improvement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR, all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding to other proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing the capability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within 8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can be employed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS, displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intact PKA holoenzyme both from recombinant and endogenous sources.ConclusionIn summary, all tested cAMP analogs were useful for their respective application as an affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS was considered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstrated incomplete elution from the matrix, as well as retaining notable amounts of bound protein contaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.
Highlights
In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP
3. provide a purification procedure with mild but efficient elution conditions while retaining high yields of protein; 4. obtain nucleotide-free proteins which can be used for further interaction studies and biochemical assays; 5. provide an easy-to-use procedure applicable in chemical proteomics
Improved cAMP analogs as novel tools for PKA R-subunit purification For our developments we used optimised binders based on Sp-cAMPS (Fig. 1A left panel, 1B), a cAMP analog where the axial exocyclic oxygen atom of the cyclic phosphate is replaced by a sulphur atom, resulting in an approximately 10-fold reduced affinity towards the R-subunit when compared to cAMP alone [15,16,17]
Summary
In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. The cAMP-dependent protein kinase (PKA) is a key regulator protein in eukaryotic signal transduction and is involved in several cellular processes during growth and development. Via phosphorylation of its substrate proteins PKA controls metabolic processes, cAMP mediated gene expression, cell differentiation and/or apoptosis [1]. The enzymatic activity of the PKA catalytic (C) subunit is controlled by a set of four different regulatory (R) subunit isoforms, i.e. type I and type II, both with two isoforms (α and β) each. The PKA holoenzyme is activated upon cooperative binding of four molecules of the second messenger cAMP to the R-subunits, releasing the active C-subunits [2]. PKA provides an important model system for kinases, allowing investigation of the molecular mechanisms of kinase function as well as the development of tools for diagnostic purposes which in turn enables their use as biomarkers [3]
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