Frantz C N, Doherty K, Gelsomino N, Cervoni M, Rust L
J Cyclic Nucleotide Protein Phosphor Res. 1986;11(3):217-31.
In order to identify common mechanisms of action by which both the platelet-derived growth factor (PDGF) and the tumor promoter tetradecanoyl phorbol acetate (TPA) initiate cell growth, the effects of PDGF and TPA on phosphorylation of cellular proteins were examined in density-inhibited Balb/c-3T3 cells. Cultures were incubated with 32Pi and growth factor, and 32P-labeled cellular proteins were examined after separation by SDS-polyacrylamide gel electrophoresis and autoradiography. TPA and PDGF each induced phosphorylation of a major cytosol protein of approximately 75,000 molecular weight (pp75). Phosphorylation of this protein was not induced by either epidermal growth factor or insulin, neither of which initiate 3T3 cell growth but enhance growth later in the 3T3 cell cycle. pp75 was a single band under reduced and non-reduced conditions, and a single spot was seen on two-dimensional gels. Phosphorylation did not occur at 4 degrees C. Phosphorylation of the protein was observed within 3 min and reached a maximum in 10-30 min. Submitogenic doses of TPA and PDGF induced submaximal phosphorylation. The phosphoprotein was labeled only on serine. Cell free phosphorylation of pp75 occurred at 4 degrees C in the presence of Mg++ and Ca2+. Homogenates from cultures pretreated with TPA phosphorylated pp75 in the presence or absence of Ca2+. Phosphorylation of this protein may possibly be related to activation of the Ca2+-dependent, phospholipid sensitive protein kinase C.
为了确定血小板衍生生长因子（PDGF）和肿瘤启动子十四酰佛波醇乙酸酯（TPA）启动细胞生长的共同作用机制，研究了PDGF和TPA对密度抑制的Balb/c-3T3细胞中细胞蛋白磷酸化的影响。将培养物与³²Pᵢ和生长因子一起孵育，通过SDS-聚丙烯酰胺凝胶电泳和放射自显影分离后检查³²P标记的细胞蛋白。TPA和PDGF各自诱导一种分子量约为75,000的主要胞质蛋白（pp75）的磷酸化。表皮生长因子或胰岛素均未诱导该蛋白的磷酸化，这两种因子均不能启动3T3细胞生长，但在3T3细胞周期后期可促进生长。在还原和非还原条件下，pp75均为单一条带，在二维凝胶上可见单个斑点。在4℃时不发生磷酸化。在3分钟内观察到该蛋白的磷酸化，并在10 - 30分钟内达到最大值。亚致有丝分裂剂量的TPA和PDGF诱导亚最大磷酸化。磷蛋白仅在丝氨酸上被标记。在Mg²⁺和Ca²⁺存在下，pp75在4℃时发生无细胞磷酸化。用TPA预处理的培养物的匀浆在有或无Ca²⁺的情况下使pp75磷酸化。该蛋白的磷酸化可能与Ca²⁺依赖性、磷脂敏感的蛋白激酶C的激活有关。
