DEVELOPMENTAL REGULATION OF A PROTEIN-KINASE-C ISOFORM LOCALIZED IN THE NEUROMUSCULAR-JUNCTION

Protein kinase C (PKC) is a family of protein serine/threonine kinases consisting of multiple isoforms whose distinct physiological roles wi thin cells are unknown. The message encoding the nPKC theta isoform, a member of the novel calcium-independent class of PKCs, has recently b een shown to be abundant in mouse skeletal muscle. The message for cPK C alpha, a calcium-dependent isoform, was also found to be highly expr essed in this tissue. In an effort to distinguish between the physiolo gical roles of these two isoforms of PKC in rat skeletal muscle, we ex amined their subcellular distribution, developmental expression and in tracellular localization. We generated an isotype-specific antiserum d irected against a peptide sequence unique to nPKC theta. This antiseru m recognized a 79 kDa protein highly enriched in rat skeletal muscle, which is likely to be nPKC theta. cPKC alpha was also readily detectab le in skeletal muscle, using another isotype-specific antibody, but it appeared to be ubiquitously expressed in all of the tissues we examin ed. Together these results suggest that nPKC theta, rather than cPKC a lpha, is involved in physiological functions that are specific for ske letal muscle. The immunoreactivity for nPRC theta was highest in the m embrane subcellular fraction compared to the cytosolic fraction of ske letal muscle. In contrast, cPKC alpha was found to be predominantly di stributed in the cytosolic rather than the membrane fraction. nPKC the ta appeared to be developmentally regulated post-natally in rat skelet al muscle, with a 4-fold increase in expression occurring exclusively in the membrane fraction during postnatal days 3 through 21. This time coursecoincides with the period in rat development associated with ma turation of neuromuscular junctions. Expression of nPKC theta in rat s pleen, another tissue expressing detectable levels of this isoform, wa s not found to be developmentally regulated during this time. cPKC alp ha expression was found to increase slightly from postnatal days 3 thr ough 11 and no developmental increase in expression of this isoform wa s 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 immuno-cytochemistry. nPKC theta was detected in association with the sarcolemma of skeletal muscle and was found to be localized in the neuromuscular junction. Enhanced stainin g for nPKC theta in the neuromuscular junction appeared as early as po stnatal day 4 during development. Staining for nPKC theta in the neuro muscular junction persisted after prolonged denervation, suggesting th at the enzyme is distributed postsynaptically. In contrast, in adult r ats, the most intense cPKC alpha immunoreactivity appeared as a puncta te stain in the cytosol as well as associated with the sarcolemma. Whi le cPKC a was also detected in the neuromuscular junction, the stronge st staining signal was not found to be localized in this synapse. Take n together, these data suggest that nPKC theta may play a specific rol e in skeletal muscle signal transduction in both the developing and th e mature neuromuscular synapse.