title: Divalent Metal Ions Mg²⁺ and Ca²⁺ Have Distinct Effects on Protein Kinase A Activity and Regulation.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Kassel , 34132 Kassel, Germany.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Knape
authors.affiliation: Department of Pharmacology, University of California at San Diego , La Jolla, California 92093, United States.
authors.forename: Lalima G
authors.initials: LG
authors.lastname: Ahuja
authors.affiliation: Department of Biochemistry, University of Kassel , 34132 Kassel, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bertinetti
authors.affiliation: Department of Biochemistry, University of Kassel , 34132 Kassel, Germany.
authors.forename: Nicole C G
authors.initials: NC
authors.lastname: Burghardt
authors.affiliation: Biaffin GmbH and Co KG , 34132 Kassel, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Zimmermann
authors.affiliation: Department of Pharmacology, University of California at San Diego , La Jolla, California 92093, United States.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.affiliation: Department of Biochemistry, University of Kassel , 34132 Kassel, Germany.
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002413:Cations, Divalent / Q000737:chemistry / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007477:Ions; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D002413:Cations, Divalent; D007477:Ions; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium; D002118:Calcium