%0 Journal Article %1 knape2015divalent %A Knape, M. %A Ahuja, L. %A Bertinetti, D. %A Burghardt, N. %A Zimmermann, B. %A Taylor, S. S. %A Herberg, F. W. %D 2015 %J ACS Chemical Biology %K herberg myown %N 10 %P 2303-2315 %R 10.1021/acschembio.5b00271 %T Divalent metal ions Mg2+ and Ca2+ have distinct effects on protein kinase A activity and regulation %U http://dx.doi.org/10.1021/acschembio.5b00271 %V 10 %X 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.

@article{knape2015divalent, 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 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.}, added-at = {2015-07-20T10:12:22.000+0200}, author = {Knape, M. and Ahuja, L. and Bertinetti, D. and Burghardt, N. and Zimmermann, B. and Taylor, S. S. and Herberg, F. W.}, biburl = {https://www.bibsonomy.org/bibtex/2bb51045395768cd2eeafa6e483b1bdf2/biochemie}, doi = {10.1021/acschembio.5b00271}, interhash = {bc740b646879971d36f957806685f65d}, intrahash = {bb51045395768cd2eeafa6e483b1bdf2}, journal = {ACS Chemical Biology}, keywords = {herberg myown}, number = 10, pages = {2303-2315}, timestamp = {2016-11-07T14:10:25.000+0100}, title = {Divalent metal ions Mg2+ and Ca2+ have distinct effects on protein kinase A activity and regulation}, url = {http://dx.doi.org/10.1021/acschembio.5b00271}, volume = 10, year = 2015 }