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

Guy A Perkins 1 Lin Wang 0 1 Lily Jun-shen Huang 0 1 Kenneth Humphries 0 1 Virginia J Yao 1 Maryann Martone 1 Thomas J Deerinck 1 David M Barraclough 0 1 Jonathan D Violin 1 Donelson Smith 1 Alexandra Newton 1 John D Scott 1 Susan S Taylor 0 1 Mark H Ellisman 1 0 Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California , San Diego, La Jolla, CA 92093-0654 , USA , 3University of Texas, MD Anderson Cancer Center, Dept. of Genitourinary Medical Oncology Box 427 , 1515 Holcombe Blvd., Houston, TX 77030-4009 , USA , 4Department of Pharmacology, University of California , San Diego, La Jolla, CA 92093- 0640 , USA and 5Howard Hughes Medical Institute, Vollum Institute , Portland, OR 97201-3098 , USA 1 Address: 1Department of Neurosciences and the National Center for Microscopy and Imaging Research, University of California , San Diego, La Jolla, CA 92093-0608 , USA 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 in addition to the RII subunits. Results: Immunohistochemistry and confocal microscopy were used here to determine that DAKAP1 colocalizes with RI at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RI 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 and RII) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKC 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 and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RI in these regions. Likewise, gravin may be an anchor of PKC at the NMJ. - Background The cell's response to hormones is a dynamic process that requires the efficient transmission of signals from the extracellular environment to specific intracellular sites. For PKA and PKC, the primary signal is generated at the plasma membrane and proceeds through intermediary G-proteins that stimulate adenylyl cyclase [1] or phospholipases [2] to generate the second messengers cAMP and diacylglycerol, respectively. The diffusion of these soluble second messengers leads to the activation of PKA and PKC at specific subcellular locations [3,4]. The two families of the regulatory subunits of PKA, RI and RII, have distinct cAMP binding properties and phosphorylation states [4,5]. Each family has two different isoforms, a and (3, resulting in four distinct types, RI, RI, RII, RII. The different R isoforms have distinct tissue and subcellular distributions suggesting that they may be selectively targeted by scaffold proteins, e.g., AKAPs anchored through different anchoring proteins [6,7]. PKC has at least 11 isoforms [8]. The physiological role of the individual PKCs has not been elucidated because most cells express multiple isoforms having nearly identical ligand binding properties and substrate specificity. Compartmentation of PKC isoforms has been suggested as a mechanism for targeting selectivity near their physiological substrates [9,10]. Consistent with this theme of selectivity, work with isoform-specific antibodies demonstrated that PKC isoforms have distinct subcellular locations [11]. It has been known for the past decade that compartmentalization of PKA can occur through association with Akinase anchoring proteins (AKAPs). This association ensures specificity in signal transduction by placing kinases close to their appropriate substrates where they can swiftly respond to second messengers [6]. Another advantage of compartmentation to specific subcellular locations is to restrict accessibility to certain substrates. Selected AKAPs have been identified that can interact with both PKA and PKC [12,13]. For example, it was shown that AKAP 79 maintains PKA, PKC, and protein phosphatase 2B at the postsynaptic density of mammalian synapses [14,12]. Likewise, gravin, a second PKA/PKC anchoring protein, targets these kinases to the membrane cytoskeleton [13]. AKAP79, gravin, and its mouse homolog clone 72 thus belong to the recently identified class of scaffolding proteins that coordinate multiple kinase activity by bringing them to one site. For PKA binding, it was thought for a long time that only RII subunits bind to AKAPs. However, a new class of AKAPs was discovered when two dual-specific AKAPs (D-AKAP1 and D-AKAP2) were cloned using a fusion protein of the RI as the bait in a yeast two-hybrid screen [15,16]). Therefore, the contrasted distribution pattern Only recently has the subcellular distribution of PKA at neuromuscular junctions (NMJs) and the adjacent muscle been systematically studied [17,18]. Imaizumi-Scherrer and colleagues demonstrated that RI is localized at the NMJ [19], but whether RI associates with a D-AKAP in this region of muscle is not known. However, a recent study showing the colocalization of D-AKAP1 with RI at the NMJ supports the prediction that localization of the R subunit there requires interaction with an AKAP [17]. RII has been shown to be associated with mAKAP in heart and skeletal muscle [20,21]; however, little is known regarding the expression patterns of RII and AKAPs in the perijunctional regions. More information has accumulated concerning PKC expression associated with the NMJ and skeletal muscle. PKC was shown to localize to the presynaptic terminals of NMJs [22,23], whereas PKC plays a postsynaptic role at the NMJ [24]. In skeletal muscle, PKC was found in human muscle fibers [25] and with T-tubules in rabbit skeletal muscle [26]. However, it is unknown whether tethering to a PKC-anchoring protein mediates the localization to these compartments. The work reported here was undertaken to further understand the compartmentalization of PKA and PKC through association with AKAPs. Because RI was observed in close association with the NMJ postsynaptic membrane [19], we probed for D-AKAP expression and possible colocalization with this and other regulatory subunits. We also investigated the distribution of gravin at the NMJ because this AKAP is an autoantigen for myasthenia g (...truncated)