Molecular basis for isofonn-specific autoregulation of protein kinase A

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P-0-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P-0-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme fort-nation only takes place with an alanine at position P-0, whereas RI subunits containing serine, phosphoscrine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P0 from serine to alanine. In contrast, PKA-C alpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type 11 or mutant PKA type I holoenzymes (P-0: Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells. (c) 2007 Elsevier Inc. All rights reserved.