title: Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme.
abstract: 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 RIIβ holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ 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:RIIβ holoenzyme since the RIIβ holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβp2: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'-(β,γ-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 pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIβ signaling cycle, we show that release of pRIIβ 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 RIIβ 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.
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, University of Kassel, 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, United States of America; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Lalima G
authors.initials: LG
authors.lastname: Ahuja
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Malik M
authors.initials: MM
authors.lastname: Keshwani
authors.affiliation: Department of Pediatrics, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Charles C
authors.initials: CC
authors.lastname: King
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California, United States of America; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Mira
authors.initials: M
authors.lastname: Sastri
authors.affiliation: Department of Biochemistry, University of Kassel, Kassel, Germany.
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla, California, United States of America; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, United States of America.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D000242:Cyclic AMP / Q000378:metabolism; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000378:metabolism*; D004926:Escherichia coli; D006367:HeLa Cells; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats
chemical_list: D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D020035:Holoenzymes; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D002118:Calcium