Féliers D, Dang P M, Haye B, Pavlovic-Hournac M
INSERM U96, Le Kremlin-Bicêtre, France.
Cell Signal. 1994 Jul;6(5):513-22. doi: 10.1016/0898-6568(94)90005-1.
In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands. The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period. Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures. In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca. 200%) in the two compartments. This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction. In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate. Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma. When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.
在猪甲状腺细胞培养物中，磷脂依赖性蛋白激酶（PKCs）具有与完整腺体相同的特征。在两天的培养期内，PKC的总体活性、PKC同工酶的存在、色谱模式和内源性底物特异性均未改变。通过免疫印迹分析在两个亚细胞组分（胞质溶胶和颗粒提取物）中鉴定出三种PKC同工酶（cPKCε、nPKCε和aPKCζ），无论是在完整腺体还是两天龄的培养物中。在存在促甲状腺激素（TSH，0.1 mU/ml）的情况下培养两天的细胞中，两个区室的PKC总体活性均受到刺激（约200%）。这种刺激与三种PKC同工型的蛋白质表达增加平行（如免疫印迹分析所示），并伴随着cPKCα和nPKCε向颗粒部分的重新分布。在TSH处理的细胞中，胞质PKC的羟基磷灰石色谱显示在195 mM磷酸钾处洗脱有一个额外的PKC活性峰。其洗脱摩尔浓度与任何已知的PKC同工酶的摩尔浓度均不对应，并且它与针对cPKC同工酶（α、β或γ）的抗体不发生交叉反应。当用福斯可林（10^(-5) M）替代TSH时，获得了相同的定量和定性修饰，这表明在甲状腺细胞中，环磷酸腺苷依赖性调节级联可能参与TSH对PKC同工型表达的控制。
