Molecular Basis for Ser/Thr Specificity in PKA Signaling.

cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to ß-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.

Divalent Metal Ions Mg²âº and Ca²âº Have Distinct Effects on Protein Kinase A Activity and Regulation.

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca(2+), do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca(2+), in contrast to Mg(2+), does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca(2+) works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg(2+)ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg(2+), Ca(2+) apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca(2+) is not as efficient as Mg(2+) in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg(2+) easily releases the phospho-substrate while Ca(2+) traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca(2+). Furthermore, Ca(2+) is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg(2+). We therefore hypothesize that the physiological metal ions Mg(2+) and Ca(2+) both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca(2+), our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.

Systematic interpretation of cyclic nucleotide binding studies using KinetXBase.

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.