Journal article
Divalent Metal Ions Mg2+ and Ca2+ Have Distinct Effects on Protein Kinase A Activity and Regulation
Publication Details
Authors: | Knape, M.; Ahuja, L.; Bertinetti, D.; Burghardt, N.; Zimmermann, B.; Taylor, S.; Herberg, F. |
Publisher: | AMER CHEMICAL SOC |
Publication year: | 2015 |
Journal: | ACS Chemical Biology |
Pages range : | 2303-2315 |
Volume number: | 10 |
Start page: | 2303 |
End page: | 2315 |
Number of pages: | 13 |
ISSN: | 1554-8929 |
eISSN: | 1554-8937 |
DOI-Link der Erstveröffentlichung: |
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 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 PICA 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 MeATP. 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.
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 PICA 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 MeATP. 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.
