Molecular basis for isoform-specific autoregulation of protein kinase A

• Published in: Cellular signalling Vol. 19; no. 10; pp. 2024 - 2034
• Main Authors: Diskar, Mandy, Zenn, Hans-Michael, Kaupisch, Alexandra, Prinz, Anke, Herberg, Friedrich W.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.10.2007
• Subjects: Adenosine Triphosphate - metabolism; Animals; Autoinhibitory site; Bioluminescence Resonance Energy Transfer; cAMP; Catalytic Domain; Cercopithecus aethiops; COS Cells; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism; Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit - metabolism; Cyclic AMP-Dependent Protein Kinases - metabolism; Holoenzymes - metabolism; Homeostasis; Humans; Isoenzymes - metabolism; Kinetics; PKA; PrKX; Protein Subunits - metabolism; Surface Plasmon Resonance; BRET; Surface Plasmon Resonance; Bioluminescence Resonance Energy Transfer; R; C; SPR; IBMX; PKA; PrKX; Autoinhibitory site; cAMP
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2007.05.012

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P0-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 P0-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P0, whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P0 from serine to alanine. In contrast, PKA-Cα forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P0: 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.