Single Turnover Autophosphorylation Cycle of the PKA RII[Beta] Holoenzyme: e1002192

To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RII[Beta] holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RII[Beta] subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RII[Beta] holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RII[Beta] holoenzyme since the RII[Beta] holoenzyme is located close to ion channels. The 2.8Å structure of an RII[Beta]p2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-([Beta],[gamma]-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRII[Beta] subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RII[Beta] signaling cycle, we show that release of pRII[Beta] in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RII[Beta] holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.