Developmental regulation of a protein kinase C isoform localized in the neuromuscular junction
Authors: Hilgenberg Lutz , Miles Kathryn 
Published: April 2021

ABSTRACT
Protein kinase C (PKC) is a family of protein serine/threonine kinases consisting of multiple isoforms whose distinct physiological roles within cells are unknown. The message encoding the nPKC θ isoform, a member of the novel calcium-independent class of PKCs, has recently been shown to be abundant in mouse skeletal muscle. The message for cPKC α, a calcium-dependent isoform, was also found to be highly expressed in this tissue. In an effort to distinguish between the physiological roles of these two isoforms of PKC in rat skeletal muscle, we examined their subcellular distribution, developmental expression and intracellular localization.
We generated an isotype-specific antiserum directed against a peptide sequence unique to nPKC θ. This antiserum recognized a 79 kDa protein highly enriched in rat skeletal muscle, which is likely to be nPKC θ. cPKC α was also readily detectable in skeletal muscle, using another isotype-specific antibody, but it appeared to be ubiquitously expressed in all of the tissues we examined. Together these results suggest that nPKC θ, rather than cPKC α, is involved in physiological functions that are specific for skeletal muscle. The immunoreactivity for nPKC θ was highest in the membrane subcellular fraction compared to the cytosolic fraction of skeletal muscle. In contrast, cPKC α was found to be predominantly distributed in the cytosolic rather than the membrane fraction.
nPKC θ appeared to be developmentally regulated postnatally in rat skeletal muscle, with a 4-fold increase in expression occurring exclusively in the membrane fraction during postnatal days 3 through 21. This time course coincides with the period in rat development associated with maturation of neuromuscular junctions. Expression of nPKC θ in rat spleen, another tissue expressing detectable levels of this isoform, was not found to be developmentally regulated during this time. cPKC α expression was found to increase slightly from postnatal days 3 through 11 and no developmental increase in expression of this isoform was observed in skeletal muscle during postnatal days 11 through 21.
The intracellular localization of the PKC θ and α isoforms in rat skeletal muscle was examined by immunocytochemistry. nPKC θ was detected in association with the sarcolemma of skeletal muscle and was found to be localized in the neuromuscular junction. Enhanced staining for nPKC θ in the neuromuscular junction appeared as early as postnatal day 4 during development. Staining for nPKC θ in the neuromuscular junction persisted after prolonged denervation, suggesting that the enzyme is distributed postsynaptically. In contrast, in adult rats, the most intense cPKC α immunoreactivity appeared as a punctate stain in the cytosol as well as associated with the sarcolemma. While cPKC α was also detected in the neuromuscular junction, the strongest staining signal was not found to be localized in this synapse. Taken together, these data suggest that nPKC θ may play a specific role in skeletal muscle signal transduction in both the developing and the mature neuromuscular synapse.