PrKX Is a Novel Catalytic Subunit of the cAMP-dependent Protein Kinase Regulated by the Regulatory Subunit Type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 μmol/(min·mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RI α ) of cAMP-dependent protein kinase ( K D = 10 n m ) and the heat-stable protein kinase inhibitor ( K D = 15 n m ), but not with the type II regulatory subunit (RII α , K D = 2.3 μ m ) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RI α subunit and by protein kinase inhibitor in vitro , but only weakly by the RII α subunit. The inhibition by the RI α subunit is reversed by addition of nanomolar concentrations of cAMP ( K a = 40 n m ), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the C α subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RII α subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RI α subunit and by phosphorylating the RII α subunit in the absence of cAMP.