Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch

the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (R-I and R-II) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the R-I and (II) subunit isoforms on C subunit binding to phosphatidylcholine/ phosphatidylserine liposomes was examined. Only the combination of N-myristylation and R-II subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R-II subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the R-I and R-II subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit and the time-resolved emission anisotropy was determined. The interaction of the R-II subunit. but not the R-I subunit significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R-II subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate, R-II subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.