A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

Abstract

Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in-trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C-tail of the substrate kinase onto the N-lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1-interacting sites, we discovered a second AGC-conserved motif in the C-tail that interacts with PDK1. Since this motif [FD(X) 1-2 Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1-interacting site in Akt and PRK2, and we predict it will be a PDK1-interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation ''switch'' that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C-tail serves as a polyvalent element that transregulates PDK1 for catalysis. Modeling of the PKA C-tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C-helix, which is positioned by the HF motif. Together, they demonstrate substrate-assisted catalysis involving two kinases that have co-evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C-tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.

Dynamic architecture of a protein kinase

“…The phosphate provides a multivalent electrostatic link between ComC and ComF, and the community maps suggest that the allosteric range reaches to ComA, ComB, and ComD as well, consistent with the fact that dephosphorylation leads to changes in catalytic activity, stability, global H/D exchange, and conformation, as reflected in a very open crystal structure, where the R-spine is broken and the activation loop and C-tail are disordered (3,60). The community maps also generate the same partitioning of the C-tail that was previously revealed by deep Bayesian analysis of the evolution of the C-tail (20) and by alanine scanning (61,62), as well as by detailed characterization of particular sites (36,(62)(63)(64). The myristyl binding pocket is in a region of the protein, whose community assignment is particularly sensitive to conditions, such as the presence or absence of bound ATP and Mg, and which comprises residues from multiple other communities.…”

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

“…Thus, recent peptide array data indicated that peptides derived from the CT of PKA interacted with PDK1 only when they were phosphorylated at the turn-motif site (35). In this respect, it should be mentioned that the TM site of PKA is thought to have a different structure and function than that of the Z/TM site of other AGC kinases (12).…”

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

“…A small N-terminal lobe (N-lobe) and a larger carboxyl-terminal helical lobe (C-lobe) create a cleft between the two lobes where the ATP substrate binds. All EPKs have an activation loop in the C-lobe that typically harbors a conserved activation loop phosphorylation site, which is a major regulatory site (2)(3)(4). Other kinase-specific phosphorylation sites may be auto-or trans-phosphorylated by heterologous kinases.…”

“…1A) (3). The turn motif and HM sites wrap around the N-lobe and position it for catalysis (3,4). Most AGC kinases also have one or more N-or C-terminal domains associated with the kinase domain that regulate kinase activity and/or subcellular localization (1).…”

“…In the C-subunit, Glu 333 replaces the conventional turn motif phosphate and positions the N-lobe for catalysis ( Fig. 1 B-D) (4). Unlike most PKs, which are transiently activated by phosphorylation of the activation loop phosphate, the C-subunit of PKA is assembled as a stable, fully phosphorylated holoenzyme that is typically anchored to a scaffold and has activity dependent on the second messenger, cAMP.…”