Role of N-Myristoylation of Camp-Dependent Protein Kinase a in Recognition and Phosphorylation of Membrane-Bound Substrates

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The catalytic subunit of cAMP-dependent protein kinase A (PKA-C), controls the activation and deactivation of over 100 unique cytosolic and membrane bound substrates. Nearly all the PKA-C in mammals is subjected to myristoylation and myristoylation can be dependent on cell/tissue type. Although the function of myristoylation is not known, it has been hypothesized to mediate PKA-C/membrane and PKA-C/membrane protein interactions. Here, we developed an in vitro myristoylation method to obtain homogeneously myristoylated PKA-C (myrPKA-C) in quantities suitable for NMR studies. Using NMR spectroscopy, the interactions of membrane mimicking isotropic bicelles (DMPC/DHPC) with 15N-labeled myrPKA-C or 15N-labeled non-myristoylated PKA-C was monitored by [1H-15N]-TROSY-HSQC spectroscopy. Significant chemical shift perturbations were detected for the N-terminus and the myristate pocket of myrPKA-C, while both perturbations and line broadening was detected in remote regions far away from the myristoyl binding pocket. Such changes were not observed for the non-myristoylated form, suggesting that myristoylation drives the enzyme towards the membrane and may act allosterically on the enzyme. Membrane localization was further supported by intrinsic tryptophan fluorescence measurements which showed drastically different patterns between myrPKA-C and PKA-C. Finally, the insertion of the myristate group into lipid membranes was confirmed using 2H solid-state NMR spectroscopy on myrPKA-C with a deuterated myristoyl group. We propose that the myristoyl group of myrPKA-C steers the enzyme towards the membrane, even in the absence of its regulatory subunit or A-kinase anchoring proteins (AKAPs). These results provide an additional mechanism for PKA localization and recognition of membrane bound substrates. Preliminary data on PKA recognition of the integral membrane protein, phospholamban, are also presented.