Emkey R, Kahn C R
Research Division, Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1997 Dec 5;272(49):31172-81. doi: 10.1074/jbc.272.49.31172.
The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), acutely stimulates the tyrosine phosphorylation of proteins of approximately 190, 120, and 70 kDa in the well differentiated Fao rat hepatoma cell line. This phosphorylation is dependent on protein kinase C (PKC) and is abolished by down-regulation of PKC or pretreatment with a PKC inhibitor. Purification of the 190-kDa tyrosine-phosphorylated protein revealed that it consists of both ErbB2 and ErbB3. Following PMA-induced tyrosine phosphorylation, ErbB2 and ErbB3 were able to associate with the SH2 domains of several signaling proteins including the p85alpha subunit of phosphatidylinositol 3-kinase, Syp, and Grb2. The 120-kDa protein phosphorylated in response to PMA consists of at least two proteins: focal adhesion kinase that exhibits a minor increase in tyrosine phosphorylation following treatment with PMA, and a major 120-kDa tyrosine-phosphorylated species in PMA-stimulated Fao cells which as yet is unidentified. Similarly, the 70-kDa tyrosine-phosphorylated protein also appears to represent more than one protein, including paxillin and a second protein of similar mobility which appears to be the major tyrosine phosphorylation in response to PMA. Both ErbB2 and paxillin also exhibit reduced migration on SDS-polyacrylamide gel electrophoresis following PMA treatment, suggesting that they are also phosphorylated on serine/threonine residues. The mobility shift of both of these proteins is abolished by treatment with inhibitors of PKC or mitogen-activated protein kinase/extracellular signal-related kinase kinase. These results suggest a novel mechanism of cross-talk between the serine/threonine kinase PKC and tyrosine phosphorylation pathways. The activation of ErbB2 and ErbB3 that is initiated by PMA may contribute to the tumor promoting activity of these compounds.
促肿瘤佛波酯，佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA），能在分化良好的Fao大鼠肝癌细胞系中急性刺激分子量约为190、120和70 kDa的蛋白质发生酪氨酸磷酸化。这种磷酸化依赖于蛋白激酶C（PKC），通过下调PKC或用PKC抑制剂预处理可消除。对190 kDa酪氨酸磷酸化蛋白的纯化显示，它由ErbB2和ErbB3组成。PMA诱导酪氨酸磷酸化后，ErbB2和ErbB3能够与几种信号蛋白的SH2结构域结合，包括磷脂酰肌醇3激酶的p85α亚基、Syp和Grb2。响应PMA而磷酸化的120 kDa蛋白至少由两种蛋白组成：粘着斑激酶在用PMA处理后酪氨酸磷酸化略有增加，以及在PMA刺激的Fao细胞中一种主要的120 kDa酪氨酸磷酸化蛋白，目前尚未鉴定。同样，70 kDa酪氨酸磷酸化蛋白似乎也代表不止一种蛋白，包括桩蛋白和另一种迁移率相似的蛋白，后者似乎是对PMA响应的主要酪氨酸磷酸化蛋白。PMA处理后，ErbB2和桩蛋白在SDS -聚丙烯酰胺凝胶电泳上的迁移率也降低，表明它们在丝氨酸/苏氨酸残基上也发生了磷酸化。用PKC或丝裂原活化蛋白激酶/细胞外信号相关激酶激酶抑制剂处理可消除这两种蛋白的迁移率变化。这些结果提示了丝氨酸/苏氨酸激酶PKC与酪氨酸磷酸化途径之间一种新的相互作用机制。由PMA引发的ErbB2和ErbB3的激活可能有助于这些化合物的促肿瘤活性。
