Divalent Metal Ions Mg2+ and Ca2+ Have Distinct Effects on Protein Kinase A Activity and Regulation

• Published in: ACS chemical biology Vol. 10; no. 10; pp. 2303 - 2315
• Main Authors: Knape, Matthias J, Ahuja, Lalima G, Bertinetti, Daniela, Burghardt, Nicole C.G, Zimmermann, Bastian, Taylor, Susan S, Herberg, Friedrich W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.10.2015
• Subjects: Amino Acid Sequence; Binding Sites; Calcium - chemistry; Calcium - pharmacology; Cations, Divalent - chemistry; Cations, Divalent - pharmacology; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Activation - drug effects; Gene Expression Regulation, Enzymologic - drug effects; Ions; Magnesium - chemistry; Magnesium - pharmacology; Models, Biological; Molecular Sequence Data; Sequence Alignment
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/acschembio.5b00271

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 Ca2+, 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 Ca2+, in contrast to Mg2+, does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca2+ 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 Mg2+ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg2+, Ca2+ apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca2+ is not as efficient as Mg2+ in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg2+ easily releases the phospho-substrate while Ca2+ traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca2+. Furthermore, Ca2+ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg2+. We therefore hypothesize that the physiological metal ions Mg2+ and Ca2+ both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca2+, our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.