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 RIIβp 2: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.

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP

“…ATP plays a different role in regulation of the RIIβ holoenzyme. In addition to being an inhibitor of the C-subunit activity, RIIβ is also a single-turnover substrate since in the RIIβ holoenzyme the P-site in the inhibitory sequence is phosphorylated when ATP is present (19). Using the FP assay, RIIβ displayed weaker affinity for the C-subunit in the presence of ATP (EC 50 = 10 nM), compared with the apo and ADP conditions (EC 50 ∼ 5 nM) ( Fig.…”

“…6B). When the RIIβ holoenzyme is phosphorylated, it is easier for cAMP to release the inhibitor sequence from the active-site cleft of the C-subunit (19). Since RIα and RIIβ localize differently and ATP concentrations vary in different cellular compartments, it is likely that the 2 holoenzymes have very distinct ATP-dependent roles (10).…”

“…The phosphorylation site (P-site) in this segment in the RI-subunits, like the cAMP-dependent protein kinase inhibitor, is either Gly or Ala so both proteins function as pseudosubstrates. In contrast, RII-subunits have a Ser at the P-site in the inhibitory sequence, and this site is autophosphorylated in the holoenzyme (1,19,20). The R-subunits and their corresponding holoenzymes also differ in their biochemical properties, localization, and cAMP sensitivities (3,10,21).…”

Anthrax Toxin as a Molecular Platform to Target Nociceptive Neurons and Modulate Pain

“…The generation of cAMP by adenylyl cyclases such as EF triggers the dissociation of PKA catalytic subunits from regulatory subunits during kinase activation. In immunological staining of fixed neurons, the phosphorylated inhibitory site of RII (pRII) becomes accessible to antibodies after the catalytic subunits dissociate (30,32). We thus identified sensory neurons by ubiquitin C-terminal hydrolase L1 (UCHL1, formerly PGP9.5), nociceptors by RIIβ, and PKA-II activation by pRII ( Fig.…”

Dynamic allostery-based molecular workings of kinase:peptide complexes

“…In contrast, the type II regulatory subunit inhibitor segment is actually a substrate that harbors a phosphorylation amicable serine residue at the P-site. An intense interplay between the kinase activity of the catalytic subunit and the cAMP binding properties of the regulatory subunit allow the type II holoenzymes to oscillate between phosphorylated and nonphosphorylated forms (47). The type II holoenzymes is a unique example of a single turnover reaction wherein the nonphosphorylated inhibitor segment of the regulatory subunit occupies the active site of the catalytic subunit as a substrate.…”