The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.
Bibliographical noteFunding Information:
The authors would like to acknowledge Susan Taylor for many helpful discussions. This work was supported by the National Institutes of Health ( GM072701 and HL080081 to G.V. and T32DE007288 to L.R.M.) and the American Heart Association ( 09PRE2080017 to E.M.). NMR data were collected at the University of Minnesota NMR Facility (National Science Foundation BIR-961477 ).
- NMR spectroscopy
- lipid bicelles
- peripheral membrane proteins
- protein kinase A