Developmental regulation of a protein kinase C isoform localized in the neuromuscular junction

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 theta 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 alpha, 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 theta. This antiserum recognized a 79 kDa protein highly enriched in rat skeletal muscle, which is likely to be nPKC theta. cPKC alpha 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 theta, rather than cPKC alpha, is involved in physiological functions that are specific for skeletal muscle. The immunoreactivity for nPKC theta was highest in the membrane subcellular fraction compared to the cytosolic fraction of skeletal muscle. In contrast, cPKC alpha was found to be predominantly distributed in the cytosolic rather than the membrane fraction. nPKC theta 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 theta in rat spleen, another tissue expressing detectable levels of this isoform, was not found to be developmentally regulated during this time. cPKC alpha 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 theta and alpha isoforms in rat skeletal muscle was examined by immunocytochemistry. nPKC theta was detected in association with the sarcolemma of skeletal muscle and was found to be localized in the neuromuscular junction. Enhanced staining for nPKC theta in the neuromuscular junction appeared as early as postnatal day 4 during development. Staining for nPKC theta in the neuromuscular junction persisted after prolonged denervation, suggesting that the enzyme is distributed postsynaptically.(ABSTRACT TRUNCATED AT 400 WORDS)