Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch.
Although 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 R(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.
Gangal
M
M
Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA.
Clifford
T
T
Deich
J
J
Cheng
X
X
Taylor
S S
SS
Johnson
D A
DA
D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes; D008958:Models, Molecular; D009154:Mutation; D019814:Myristic Acid / Q000737:chemistry*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D020033:Protein Isoforms; D019814:Myristic Acid; D017868:Cyclic AMP-Dependent Protein Kinases