Wang X D, Kiang J G, Smallridge R C
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307, USA.
Thyroid. 1995 Apr;5(2):137-40. doi: 10.1089/thy.1995.5.137.
Protein kinase C (PKC) has been implicated as an important regulator of signal transduction in the FRTL-5 thyroid cell line, but little is known about its isoforms in this cell line. In the present investigation, we characterized the activation of PKC by measuring the enzyme activity and identifying its isoforms in both cytosol and membrane fractions. Phorbol 12-myristate 13-acetate (PMA) was used as a PKC activator in this study. PKC activity assay revealed that PMA (300 nM) induced a rapid translocation from cytosol to membrane within 1 min and led to an almost complete translocation within 15 min. Multiple PKC isoforms were examined by Western blot analysis with specific antibodies against alpha, beta, gamma, delta, epsilon, and zeta isoforms. PKC alpha, delta, epsilon, and zeta were identified in this cell line, but PKC beta and gamma were not. Exposure of the cells to PMA (300 nM) for 5 to 30 min led to the translocation of PKC alpha, delta, and epsilon from the cytosol to the membrane fraction, while PKC zeta was not affected. Treatment with PMA (300 nM) for 24 h resulted in the down-regulation of PKC alpha, delta, and epsilon, but not PKC zeta. This study demonstrates for the first time direct evidence for the activation of PKC, and expression and distribution of its isoforms in FRTL-5 thyroid cells.
蛋白激酶C（PKC）被认为是FRTL-5甲状腺细胞系信号转导的重要调节因子，但对该细胞系中其亚型的了解甚少。在本研究中，我们通过测量酶活性并鉴定其在胞质溶胶和膜组分中的亚型，对PKC的激活进行了表征。在本研究中，佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）被用作PKC激活剂。PKC活性测定表明，PMA（300 nM）在1分钟内诱导从胞质溶胶到膜的快速转位，并在15分钟内导致几乎完全转位。用针对α、β、γ、δ、ε和ζ亚型的特异性抗体通过蛋白质印迹分析检测多种PKC亚型。在该细胞系中鉴定出了PKCα、δ、ε和ζ，但未鉴定出PKCβ和γ。将细胞暴露于PMA（300 nM）5至30分钟导致PKCα、δ和ε从胞质溶胶转位至膜组分，而PKCζ不受影响。用PMA（300 nM）处理24小时导致PKCα、δ和ε下调，但PKCζ未下调。本研究首次证明了PKC激活及其亚型在FRTL-5甲状腺细胞中的表达和分布的直接证据。
