Protein Kinase A Phosphorylates a Conformationally High-energy State of Raf Kinase Inhibitory Protein

Protein kinase A (PKA) is found in all mammalian tissues and plays a critical role in mediating many signaling processes. The structure and phosphorylation mechanism of the catalytic subunit of PKA (PKA-C) have been extensively studied in interaction with linear pseudosubstrate inhibitors, peptide substrates, or protein fragments. To date, however, there have been only a few studies examining how PKA recognizes full-length, folded proteins. Here, we utilized solution NMR spectroscopy in combination with replica-averaged restrained molecular dynamics (RARMD) calculations to investigate the interactions between PKA-C and Raf Kinase Inhibitory Protein (RKIP). RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family, which regulates important kinase pathways such as the Raf/MAPK pathway and the β-adrenergic receptor/cAMP-dependent PKA signaling cascade. The X-ray structure of RKIP reveals a compact fold, with the phosphorylation consensus sequence well-structured and essentially inaccessible to the kinase. Using Carr-Purcell-Meiboom-Gill (CPMG) and chemical exchange saturation transfer (CEST) experiments, we discovered that RKIP undergoes a conformational equilibrium between a compact fold and a more open conformation, where the C-terminal helix splays away from the RKIP core and exposes its phosphorylation sequence, allowing the kinase to bind and phosphorylate this substrate. The dynamic interplay between the kinase and the substrate may represent a common mechanism for kinases to recognizes well-folded substrates.