Lee H W, Smith L, Pettit G R, Bingham Smith J
Department of Pharmacology and Toxicology, School of Medicine, University of Alabama at Birmingham 35294, USA.
Am J Physiol. 1996 Jul;271(1 Pt 1):C304-11. doi: 10.1152/ajpcell.1996.271.1.C304.
We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.
我们发现苔藓抑素1（Bryo）能在肾上皮细胞的LLC-MK2系中迅速产生一种无活性、无功能的76 kDa形式的蛋白激酶C-α（PKC-α）。与佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）一样，Bryo能急性激活PKC，这可通过PKC-α的自磷酸化和转位来表明，PKC-α是细胞中主要的对PMA敏感的同工型。Bryo通过自磷酸化同时增加了80 kDa PKC-α的32P标记，并产生了一种缺乏可检测到的32P的76 kDa形式的PKC-α。76 kDa形式存在于颗粒部分而非胞质部分，这表明它是由活化的激酶产生的。对免疫沉淀的PKC-α进行碱性磷酸酶处理可将80 kDa形式转化为76 kDa，但对76 kDa形式的迁移率没有影响，这表明它没有被磷酸化。用[35S]甲硫氨酸/半胱氨酸对PKC-α进行脉冲追踪标记表明，80 kDa和76 kDa形式之间分别存在前体-产物关系。蛋白质合成的抑制对Bryo产生76 kDa PKC-α没有影响。PMA也产生76 kDa PKC-α，但效力和效果均不如Bryo。与PMA相比，Bryo能使80 kDa PKC-α蛋白以及总的钙和磷脂依赖性PKC活性更快地丧失。76 kDa形式无活性且无功能，因为在能强烈自磷酸化80 kDa形式的条件下，它缺乏可检测到的32P。我们认为去磷酸化使PKC易于被蛋白水解，并且76 kDa形式的更多产生解释了与PMA相比，Bryo对该激酶的下调更有效。
