Hilgenberg L, Miles K
Department of Anatomy and Cell Biology, State University of New York Health Science Center at Brooklyn 11203, USA.
J Cell Sci. 1995 Jan;108 ( Pt 1):51-61. doi: 10.1242/jcs.108.1.51.
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)
蛋白激酶C（PKC）是一类蛋白丝氨酸/苏氨酸激酶家族，由多种亚型组成，其在细胞内不同的生理作用尚不清楚。编码新型PKCθ亚型（一种新型钙非依赖性PKC成员）的信息最近显示在小鼠骨骼肌中含量丰富。还发现钙依赖性亚型cPKCα的信息在该组织中高度表达。为了区分这两种PKC亚型在大鼠骨骼肌中的生理作用，我们研究了它们的亚细胞分布、发育表达和细胞内定位。我们针对PKCθ特有的肽序列产生了一种亚型特异性抗血清。这种抗血清识别出一种在大鼠骨骼肌中高度富集的79 kDa蛋白，可能是PKCθ。使用另一种亚型特异性抗体也很容易在骨骼肌中检测到cPKCα，但它似乎在我们检查的所有组织中普遍表达。这些结果共同表明，PKCθ而非cPKCα参与了骨骼肌特有的生理功能。与骨骼肌的胞质部分相比，PKCθ在膜亚细胞部分的免疫反应性最高。相反，发现cPKCα主要分布在胞质部分而非膜部分。PKCθ在大鼠骨骼肌中似乎在出生后受到发育调节，在出生后第3天至21天期间，其表达仅在膜部分增加了4倍。这个时间进程与大鼠发育中与神经肌肉接头成熟相关的时期一致。在这段时间内，未发现PKCθ在大鼠脾脏（另一个表达可检测水平该亚型的组织）中的表达受到发育调节。发现cPKCα的表达从出生后第3天至11天略有增加，在出生后第11天至21天期间，未观察到该亚型在骨骼肌中的表达有发育性增加。通过免疫细胞化学检查了PKCθ和α亚型在大鼠骨骼肌中的细胞内定位。在骨骼肌的肌膜上检测到PKCθ，并发现其定位于神经肌肉接头。在发育过程中，早在出生后第4天，神经肌肉接头中PKCθ的染色就增强。长时间去神经后，神经肌肉接头中PKCθ的染色持续存在，表明该酶分布在突触后。（摘要截至于400字）
