Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein–membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Myristoylation is a major type of posttranslational modification that occurs at the N terminus of a myriad of proteins (1–4). Depending on the target, myristoylation can contribute to the structure, stability, protein–protein interactions, and subcellular localization of the modified proteins (2–4). In particular, myristoylation often facilitates protein association with the membrane. However, it is thought that, with an acyl chain of only 14 carbons, myristate confers insufficient energy for stable association of a protein with the membrane (5, 6). Subsequent studies have shown that a second membrane-affinity motif, such as a stretch of basic residues or a second lipid modification, is required for the membrane association of several myristoylated proteins (reviewed in refs. 2–4). When the second membrane-affinity motif is removed or neutralized, either physiologically or via mutagenesis, the membrane localization of the protein is disrupted. Thus, the canonical view is that myristoylation alone is not sufficient to provide a functionally significant association of a protein with the plasma membrane, even though myristoylation has been observed to be associated with reconstituted lipid bilayers (7).Myristoylation was first discovered in the catalytic subunit of protein kinase A (PKA) (1, 8), which is a primary mediator of the second messenger cAMP that plays diverse essential roles in nearly all organisms, from bacteria to humans. At rest, PKA is a tetrameric protein that consists of two regulatory subunits (PKA-Rs) and two catalytic subunits (PKA-Cs). PKA holoenzymes are anchored to specific subcellular locations via the binding of PKA-R with A-Kinase anchoring proteins (9–12). In the presence of cAMP, PKA-C is released from PKA-R and becomes an active kinase (8, 13–16).Despite being myristoylated, PKA-C is thought to function as a cytosolic protein because of its high solubility (14, 15). Consistently, a PKA-C mutant with disrupted myristoylation has been shown to support the phosphorylation of certain substrates and to maintain several PKA functions in heterologous cells (17). Structural studies found that the PKA-C myristoylation is folded into a hydrophobic pocket, and it was proposed that this myristoylation serves a structural role (18–20). This view has started to shift based on recent reports showing that activated PKA-C can associate with the membrane in a myristoylation-dependent manner (16, 21, 22), including in neurons. However, the extent to which PKA-C associates with the plasma membrane in living cells and its functional significance are not known. Furthermore, as discussed above, myristolyation-mediated membrane association is thought to require a second-membrane motif. The identity of this second membrane-affinity motif has not been determined. Therefore, we set out to address these questions.