Article,
Regulation of cAMP-dependent protein kinases: the human Protein Kinase X (PrKX) reveals the role of the catalytic subunit alphaH-alphaI loop
J Biol Chem, 285 (46): 35910-8 (2010)
Abstract
cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human Protein Kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIalpha or RIbeta but not by the substrate inhibitors RIIalpha or RIIbeta. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied Bioluminescence Resonance Energy Transfer (BRET) and Surface Plasmon Resonance (SPR) in combination with site-directed mutagenesis. We observed that the conserved alphaH-alphaI loop residue Arg283 in PrKX is a crucial for its RI over RII preference, as a Arg283Leu mutant was able to form a holoenzyme complex with wildtype RII subunits. Changing the corresponding alphaH-alphaI loop residue in PKA Calpha (Leu277Arg), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of two to four, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.
