Misra-Press A, Fields A P, Samols D, Goldthwait D A
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Glia. 1992;6(3):188-97. doi: 10.1002/glia.440060306.
Protein kinase C (PKC), an enzyme involved in signal transduction, responds to diacyl glycerol and also to phorbol ester, a ligand analogous to diacyl glycerol. We have studied the expression of the major isoforms (alpha, beta I, beta II, and gamma) in eight human glioblastoma cell lines. In all eight lines, PKC-alpha mRNA and protein were expressed. In none of the eight did a probe for PKC-beta I and -beta II mRNA give positive results nor were Western blots for PKC-beta II positive. The half-life for PKC alpha mRNA was approximately 16 h and levels of the mRNA were increased slightly following addition of phorbol myristate acetate (PMA) or transforming growth factor-beta (TGF beta). PKC-gamma was present in most of the glioblastomas. In cell line A172, 82% of the PKC-alpha was present in the cytosol with the remainder evenly divided between plasma membrane and nucleus. Thirty minutes after addition of PMA, 33% of the total original protein was in the plasma membrane and 48% in the nuclear fraction. By 21 h, no PKC-alpha was recovered from any fraction. PKC-gamma was also down-regulated in the presence of PMA, but there was no evidence for translocation to the plasma membrane or nuclear fraction. In a more detailed study, translocation of PKC-alpha in the presence of PMA was complete by 10 min, and a major decrease in the PKC translocated to the plasma-membrane fraction occurred some time between 2 and 4 h after PMA addition, while a major decrease in the translocated nuclear fraction occurred some time after 6 h. cAMP alone had no effect on the PKC alpha protein level or distribution, nor did it alter the translocation and down-regulation due to PMA exposure. In these studies the level of PKC-alpha mRNA in tumors was similar to that in normal glial cells.
蛋白激酶C（PKC）是一种参与信号转导的酶，对二酰甘油以及佛波酯（一种类似于二酰甘油的配体）有反应。我们研究了8种人胶质母细胞瘤细胞系中主要同工型（α、βI、βII和γ）的表达情况。在所有8种细胞系中，PKC-α的mRNA和蛋白均有表达。在这8种细胞系中，PKC-βI和-βII mRNA的探针均未给出阳性结果，PKC-βII的蛋白质印迹也均为阴性。PKCα mRNA的半衰期约为16小时，添加佛波醇肉豆蔻酸酯乙酸酯（PMA）或转化生长因子-β（TGF-β）后，mRNA水平略有升高。大多数胶质母细胞瘤中存在PKC-γ。在细胞系A172中，82%的PKC-α存在于细胞质中，其余部分均匀分布在质膜和细胞核之间。添加PMA 30分钟后，总原始蛋白的33%存在于质膜中，48%存在于核组分中。到21小时时，任何组分中均未检测到PKC-α。在PMA存在的情况下，PKC-γ也被下调，但没有证据表明其易位到质膜或核组分。在一项更详细的研究中，PMA存在时PKC-α的易位在10分钟时完成，添加PMA后2至4小时之间，易位到质膜组分的PKC大幅减少，而在6小时后，易位到核组分的PKC大幅减少。单独的cAMP对PKCα蛋白水平或分布没有影响，也不会改变由于PMA暴露引起的易位和下调。在这些研究中，肿瘤中PKC-α mRNA的水平与正常神经胶质细胞中的水平相似。
