The work presented here adds to the emerging evidence that PKA and PKC signaling at the NMJ and adjacent intercostal muscle is compartmentalized. Differential compartmentation of these signaling molecules in cells and tissues is being recognized as a mechanism to regulate their specificity [reviewed recently by ]. A Kinase Anchoring Proteins (AKAPs) can direct their subcellular localization by binding directly to regulatory subunits of PKA or to the catalytic core of PKC. Many AKAPs have been identified, although information about their subcellular distribution in different tissues is largely lacking. Because we produced antibodies to two novel dual-specific AKAPs (D-AKAP1 and D-AKAP2), and gravin, we were uniquely positioned to use immunofluorescence to investigate their possible roles in kinase signaling in and around the NMJ. Our interest in PKA and PKC activity in the NMJ derives from: (1) a report that RIα not only is enriched there, but also appears to be tethered to the postsynaptic membrane , (2) the rich literature describing selective PKC isoform activity and functional compartmentalization in the NMJ and skeletal muscle, (3) our discoveries that D-AKAP1 and D-AKAP2 bind to and appear to colocalize with RIα, and often mitochondria [15, 16, 28]; and (4) our finding that gravin binds to both PKA and PKC .
We found that D-AKAP1 appears to colocalize with RIα at the postsynaptic membrane of the NMJ, which houses the nAChR, but not in the presynaptic region. This observation is supported by previous work that demonstrated RIα codistribution with nAChR  and D-AKAP1  suggesting that this AKAP is the anchoring protein recruiting RIα to this membrane. This putative association is strengthened by finding for the first time that targeting of RIα requires the AKAP binding surface . Yotiao is another AKAP enriched at the NMJ, but appears to bind only RII . PKA phosphorylates the nAChR when stimulated at the synapse by the neuropeptide CGRP and by adenosine from muscle responding to acetylcholine [reviewed by ]. This phosphorylation may regulate the degradation of the nAChR . Consistent with the action at the presynapse, it was shown that PKA facilitation of acetylcholine release is dependent on nerve endplate calcium concentration . Based on our observation of colocalization with synaptophysin at presynaptic vesicles, RIα may be the isotype that influences this release of neurotransmitter.
We found that RIα occupies the same region as mitochondria in intercostal muscle. The presence of RIα in soleus muscle was shown with Western blots by Hoover and coworkers , but was not seen with immunofluorescence of tibialis anterior muscle [17, 19]. Reinitz and coworkers  provided evidence suggesting that RIα associates with membrane fractions in cardiac muscle when it is not associated with the catalytic subunit. The same may be true for intercostal muscle, in which case, association with the sarcolemma, mitochondria or other organelles is possible. Furthermore, RIα was shown to bind to cytochrome c oxidase subunit Vb (a mitochondrial inner membrane protein), which faces the matrix, in a cAMP-dependent manner . How RIα passes across the mitochondrial inner membrane is not known. Previously, we observed that D-AKAP1 mRNA and protein expressions are high in muscle . In situ hybridization showed consistency of D-AKAP1 and RIα staining in tongue muscle, indicating that in vitro association can indeed predict in situ interaction for these molecules. Consistent with the observed mitochondrial staining by anti-D-AKAP1, it was shown previously that one of the two alternative NH2-terminal motifs of this AKAP is necessary and sufficient for targeting to mitochondria . Interestingly, another AKAP governs voltage-dependent potentiation of L-type calcium channels through anchoring of PKA in transverse tubules . Because this potentiation is not dependent on RIIα, it is thought that RIα is capable of this AKAP-directed modulation of calcium channel activity .
The labeling pattern of D-AKAP2 is diffuse in the NMJ, yet localized to the actin region in muscle. In our previous immunofluorescence work examining D-AKAP2 subcellular localization, the protein displayed a diffuse background in addition to mitochondrial staining . It was thought that the combination of specific and diffuse labeling indicated the presence of more than one pool of D-AKAP2 inside the cells examined. Whereas, D-AKAP1 appears to colocalize with mitochondria in muscle fibers, D-AKAP2 shows no such association. This observation is not surprising because it is not expected that their targets need to be in the same organelle or even subcellular region. Whether D-AKAP2 binds specifically to actin remains to be determined.
RIIα localized quite well to mitochondria in both NMJ and muscle regions. PKA association with mitochondria has been well documented [38–42]. In mammalian sperm, it was found that nearly all RII-specific labeling was with mitochondria . Furthermore, in bovine heart, PKA activity was detected in the matrix or the matrix side of the mitochondrial inner membrane [44, 45]. However, the work of Halestrap and coworkers provided little evidence for intramitochondrial protein phosphorylation by PKA, but gave some evidence for outer membrane phosphorylation, both integral and loosely bound proteins [46–48]. The localization of RIIα to the postsynaptic region beyond the postsynaptic membrane is consistent with part of the immunostaining of NMJs in the soleus muscle , yet differs in part because there is no overlap with the bungarotoxin label; however, this is in agreement with another study .
PKCβ colocalizes with gravin at the postsynaptic membrane where the gravin staining is strongest. PKC has been suggested to play a role in synapse stability and signal transduction at the NMJ . When calcium influx through the nAChR occurs, acetylcholine appears to activate PKC phosphorylation of this receptor [reviewed by ]. Phorbol ester activation of PKC causes loss of synapses and nAChR . Because the presynaptic PKCβ fluorescence band parallels the nAChR band (see fig. 7), it may be labeling the presynaptic membrane, consistent with reports of PKCβ localization to the presynaptic terminals of NMJs [22, 23]. PKCβ initially associates with the cytoskeleton . Phosphorylation at two places on the kinase core is required first to activate PKC, and second to release it from its cytoskeletal anchorage. Our immunofluorescence results may be the visualization of two pools of PKCβ. The observed bands may represent kinase anchored directly to the cytoskeleton, perhaps on the presynaptic side, where little gravin staining was found, or anchored to gravin, perhaps on the postsynaptic side. The more diffuse staining found in both pre- and postsynaptic regions may represent released, hence activated kinase.
As with PKCβ, gravin was seen to be directed to the postsynaptic membrane. Strong labeling was also seen at the plasma membrane-cytoskeleton (see fig. 8), consistent with the report of Nauert and coworkers . Interestingly, a homology search of a database revealed a MARCKS homology actin-binding domain in gravin. In addition to acting as a scaffolding protein for kinases and phosphatases, gravin forms a dynamic complex with beta-adrenergic receptors . The same report also showed that the association of beta-adrenergic receptors with PKA could be blocked by the Ht31 peptide, which blocks AKAP-kinase binding. In addition to a PKC binding site, gravin contains a PKA binding site. Hence, the beta-adrenergic receptor may be tethered to PKA via gravin. PKC may bind to this anchoring protein through displacement of the pseudosubstrate, thus inhibiting the kinase activity, by analogy with a functionally similar scaffolding AKAP, AKAP79 .