PKA, PKC, and AKAP localization in and around the neuromuscular junction

Background: One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RI alpha in addition to the RII subunits. Results: Immunohistochemistry and confocal microscopy were used here to determine that DAKAP1 colocalizes with RI alpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIa and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RII alpha and RII beta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKC beta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. Conclusions: The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RI alpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RI alpha in these regions. Likewise, gravin may be an anchor of PKC beta at the NMJ.