Synaptic Activity and Muscle Contraction Increases PDK1 and PKC?I Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction.

ABSTRACT: Conventional protein kinase C ?I (cPKC?I) 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. cPKC?I 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 cPKC?I phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with ?-conotoxin GIIIB (?-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 cPKC?I also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKC?I protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKC?I levels increase in concordance with total cPKC?I. 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 cPKC?I, which is involved in ACh release.

SUBMITTER: Hurtado E 

PROVIDER: S-EPMC5574929 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature