Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

Abstract

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

The Impact of Kinases in Amyotrophic Lateral Sclerosis at the Neuromuscular Synapse: Insights into BDNF/TrkB and PKC Signaling

“…However, before PKCs can be functional, they need to be phosphorylated by PDK1 [77][78][79][80][81]. PDK1 activates multiple signaling pathways and seems to be constitutively active at the NMJ [82]. PDK1-induced PKC phosphorylation is enhanced by synaptic activity but is not directly linked to BDNF/TrkB signaling, which is, contrarily, involved in PKC activation once the isoforms are phosphorylated and mature [36].…”

“…In skeletal muscles, nerve-induced stimulation, high Ca 2+ concentrations and muscarinic signaling couple PKC to result in neurotransmitter release at the adult NMJ [84,87,88]. In particular, nPKCε maintains ACh release and modulates muscarinic signaling [86], while cPKCβI enhances neurotransmission [82] at the NMJ. Furthermore, evidence supports the importance of neurotrophic signaling via TrkB activity to regulate neuromuscular ACh release at short times and to maintain synaptic function and structural integrity in adult rodent NMJs long term [28,36,42,89,90].…”

Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways

“…Because of these uncertainties, we induced muscle paralysis with μ-CgTx-GIIIB ( Garcia et al, 2013 ; Tomàs et al, 2014 ; Obis et al, 2015 ; Santafé et al, 2015 ; Hurtado et al, 2017a , b ), a specific inhibitor of the sodium channel of the muscle cells which preserves NMJ function ( Garcia et al, 2013 ) and its safety factor. This experimental condition mimics the physiological conditions of this synapse in the living animals except for the absence of the contraction-dependent retrograde influence of the muscle cells ( Besalduch et al, 2011 ; Hurtado et al, 2017a , b ). We observed that 25 μM adenosine reduced (50%) the quantal content of ACh release in agreement with other authors ( Ginsborg and Hirst, 1972 ; Ribeiro and Walker, 1975 ).…”

Stra8 may inhibit apoptosis during mouse spermatogenesis via the AKT signaling pathway

“…The AGC protein kinase family includes PDK1, cyclic AMP-dependent protein kinase, PKB/AKT and Ca2 + -activated protein e kinase (22,26). Among the DEGs, the present study found that PDK1, a key upstream gene of AKT, activates ~23 protein kinases in the AGC family, including PKB/AKT, which is a key signaling intermediate involved in growth and l survival (27). Therefore, the levels of these genes were analyzed and it was found that the mRNA and protein expression levels of AKT and PDK1 were significantly increased in the Stra8-GC1 cells compared with those in the Control-GC1 cells.…”

“…Based on the above results, the present study further examined the function of AKT-associated genes and pathways. It has (22,26,27). According to associated studies in the literature, AKT can act as a survival factor which balances pro-survival and pro-death signals, including activating genes in the Bcl-2 family and interacting with MAPK gene families, which eventually suppresses escaspases (22,26).…”