Three linear sequences termed regions A, B, and C (residues 31–52, residues 76–101, and residues 116–145, respectively) target AKAP79 to the PM in HEK-293 cells and cortical neurons (29
). This study shows that AKAP79 is palmitoylated at cysteine 36 and 129 within the A and C basic regions. These palmitoylations are not necessary to localize AKAP79-YFP to the PM but instead promote association with lipid rafts. Treatment with the palmitoylation inhibitor, bromopalmitate as well as mutation of cysteines 36 and 129 decreased the amount of AKAP79-YFP isolated in lipid raft fractions. Mutation of either one of these cysteines affects the interaction of AKAP79 with insoluble membranes, although the mutation of both abolishes almost completely the recovery of AKAP79-YFP from lipid raft fractions. Note, however, that when the raft-targeted AC8 is overexpressed, significant amounts of AKAP79 can still be pulled down by AC8 (A
). This implies that AKAP79 can still associate with lipid rafts albeit indirectly via a binding partner that is expressed in rafts. As would have been anticipated from these findings, PKA is recruited to rafts only by the wild type AKAP79 and not by the nonpalmitoylatable mutants.
A more benign live cell microscopic approach was also adopted to study the consequences of AKAP79 palmitoylation. Using FRAP analysis, we compared the mobility of wild type and the C36S,C129S mutant AKAP79 and examined how this behavior was modulated in cells treated with MBCD, which disrupts lipid rafts by extracting membrane cholesterol. The results clearly show that MBCD treatment affects only the diffusion of the wild type AKAP79 and not the nonpalmitoylatable mutant. MBCD treatment reduced the diffusion rate of the raft resident wild type AKAP79. Although this counterintuitive effect of MBCD has been widely reported for other lipid raft proteins, it may be worth speculating that the reason may lie in the destruction of lipid rafts leading to enhanced encounters of AKAP79 with other membrane targets, thereby reducing its overall mobility. Indeed, two studies have reported that MBCD treatment also decreases the diffusion of non-raft proteins (42
). However, we find that MBCD treatment does not affect the diffusion of the nonpalmitoylatable AKAP79 mutant. The lack of effect on the diffusion of the nonpalmitoylatable mutant may be because this protein is not immersed in the lipid bilayer but only associated with the membrane by interaction with the negatively charged phospholipid headgroups.
In this regard, although we did not detect any difference in the diffusion coefficient of the nonpalmitoylatable AKAP79 versus
the wild type when modeled as simple Brownian motion, we did see an increased transport coefficient and evidence of anomalous diffusion (α < 1) of the wild type protein. This anomalous diffusion is largely determined by free diffusion coupled to transient trapping or binding, suggesting periods of temporary confinement of AKAP79 (41
), presumably in lipid rafts. It is conceivable that AKAP79 can experience multiple types of interactions both in lipid rafts as well as in the non-raft domains. The mutant AKAP79 may undergo more interactions with the non-raft phospholipid membrane and with proteins in this region that may decrease its diffusion rate (and hence reduce its transport coefficient relative to the wild type). However, this interaction does not promote an anomalous diffusion of the nonpalmitoylatable AKAP79, because the exponent α is not significantly different from 1.
PI4P and PI(4,5)P2
depletion experiments were aimed at getting a sense of the relative importance of palmitoylation versus
simple ionic attachments in the maintenance of AKAP79 at the PM. Although depletion of phosphoinositides produced only a small loss of wild type AKAP79 from the PM, the C36S,C129S mutant was rendered purely cytosolic. These data argue that palmitoylation is the predominant mechanism used to maintain AKAP79 at the PM. In terms of the association of AKAP79 with lipid rafts, it is known that PI(4,5)P2
can be sequestered within rafts as a result of association with polybasic stretches of amino acids found in raft-associated proteins (51
), similar to those found in AKAP79. Because mutation of the palmitoylatable cysteines clearly results in the loss of AKAP79 from the lipid rafts, it must be concluded that palmitoylation is also the key mechanism for maintenance of AKAP79 in rafts and that the residual association of the AKAP79 with the PM relies on the association with phosphoinositides.
The functional consequence of AKAP79 association with lipid rafts is of course its recruitment of PKA and phosphorylation of proteins associated with these microdomains. Multiple independent lines of evidence have demonstrated that β-adrenergic receptors at the cell surface may occupy rafts and non-raft membranes but co-localize with their G protein and adenylyl cyclase effectors when in lipid rafts (52
). In cardiac myocytes, the disruption of lipid rafts (or caveolae, in that case) converts the sarcolemma-confined cAMP signal associated with β-adrenergic receptor stimulation to a global signal (54
). In this context, our data showing phosphorylation in lipid rafts dependent upon wild type AKAP79 support the hypothesis that the co-localization of the β-adrenergic receptor with its effectors, in this case AKAP79 and PKA, promotes compartmentalization of the signaling.
A potentially even more acute target of PKA in lipid rafts is AC8. AC8 is highly compartmentalized (25
), and the regulation of AC8 by Ca2+
depends on its localization in rafts (28
). Recently, we have shown that AC8 binds AKAP79, and its responsiveness to SOCE is attenuated by this association (18
). This study strongly suggests that the residence of AKAP79 in lipid rafts is a key component of its ability to influence AC8 activity, because no modulation is seen by the nonpalmitoylatable AKAP79 mutant, and PKA-mediated phosphorylation of AC8 is only possible when the wild type AKAP79 is expressed.
Overall, we see that AKAP79 is a palmitoylated protein, whose palmitoylation dictates the targeting of AKAP79 to lipid rafts, to permit PKA-mediated phosphorylation and regulation of raft-associated targets, such as AC8. It would seem reasonable to predict that this propensity of AKAP79 to be palmitoylated could be exploited in its role to promote the selective phosphorylation of other target proteins that reside in rafts or in post-synaptic densities (which share the lipid composition of rafts). Although acylation of proteins is reportedly involved in targeting of proteins to lipid rafts, palmitoylation is a unique reversible lipid modification that can be regulated by specific extracellular signals. In mammalian systems, palmitoylation is mediated by a recently identified family of the transmembrane Asp-His-His-Cys motif protein acyltransferases and palmitoyl protein thioesterases (56
). Recent studies have shown that palmitoylation/depalmitoylation cycles regulate the localization of specific proteins. In fact, even the subcellular localization of protein acyltransferases is regulated (59
). Palmitoylation cycles can therefore be expected to regulate the affinity of AKAP79 for lipid rafts and in this way regulate the interaction of AKAP79 with proteins in these microdomains. Our data demonstrate that the palmitoylation/depalmitoylation cycle of a key regulatory protein, such as AKAP79, can have important consequences for fine-tuning signaling transduction pathways mediated by key effector proteins that bind AKAP79, such as the adenylyl cyclases or numerous ion channels (1