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 famil y of cAMP-dependent protein kinases, The catalytically active recombinant e nzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRA SLG) with a specific activity of 1.5 mu mol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulator y subunit type I (RIalpha) of cAMP-dependent protein kinase (K-D = 10 nM) a nd the heat-stable protein kinase inhibitor (K-D = 15 nM), but not with the type II regulatory subunit (RIIalpha, K-D = 2.3 mu M) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongl y inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro , but only weakly by the RIIalpha subunit. The inhibition by the RIalpha su bunit is reversed by addition of nanomolar concentrations of cAMP (K-alpha = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent pr otein kinase that is activated at lower cAMP concentrations than the holoen zyme with the C-alpha subunit of cAMP-dependent protein kinase. Microinject ion data clearly indicate that the type I R subunit but not type II binds t o PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate far PrKX an d is phosphorylated in vitro in a cAMP-independent manner, me discuss how P rKX can modulate the cAMP-mediated signal transduction pathway by preferent ial binding to the RIalpha subunit and by phosphorylating the RIIalpha subu nit in the absence of cAMP.