Staddon J M, Chanter N, Lax A J, Higgins T E, Rozengurt E
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
J Biol Chem. 1990 Jul 15;265(20):11841-8.
Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.
多杀巴斯德菌毒素，无论是天然的还是重组的（rPMT），都是瑞士3T3细胞极为有效的促细胞分裂剂，且在皮摩尔浓度下起作用（罗曾古特，E.，希金斯，T. E.，钱特，N.，拉克斯，A. J.，以及斯塔登，J. M.（1990年）《美国国家科学院院刊》87，123 - 127）。在此，我们表明相似浓度的rPMT能显著刺激[32P]Pi标记的瑞士3T3细胞中一种80 kDa酸性蛋白的磷酸化。在一维及二维凝胶上的共迁移以及磷酸肽分析表明，这种磷蛋白与80K无法区分，80K是一种已知的蛋白激酶C底物。在平行培养中，rPMT对80K磷酸化的刺激作用（5 - 10倍）与蛙皮素或佛波酯（PBt2）诱导的相当。然而，与PBt2或蛙皮素相对即时的刺激作用不同，rPMT引起的磷酸化增加在明显的延迟期（1 - 3小时，取决于rPMT的浓度）后才出现。早期（而非晚期）添加PMT抗血清或溶酶体促营养剂甲胺能选择性抑制rPMT诱导的80K磷酸化。去除游离毒素后80K磷酸化仍持续存在，且不受放线菌酮抑制。看来rPMT通过内吞途径进入细胞，以启动并维持导致80K磷酸化的事件。rPMT与PBt2一样，也能刺激瑞士3T3细胞中87 kDa和33 kDa蛋白的磷酸化。在缺乏蛋白激酶C的细胞中，rPMT或PBt2对80K和87 kDa蛋白的磷酸化作用大幅减弱。相反，在缺乏蛋白激酶C的情况下，rPMT对33 kDa蛋白的磷酸化作用持续存在，而PBt2则不然。rPMT与蛙皮素一样，能使蛋白激酶C转位至细胞颗粒部分。该毒素增加了二酰基甘油的细胞含量。rPMT还会导致125I - 表皮生长因子与其受体的结合呈时间和剂量依赖性降低，这种降低被甲胺阻断，且仅部分依赖于蛋白激酶C的存在。我们得出结论，rPMT能刺激瑞士3T3细胞中依赖和不依赖蛋白激酶C的蛋白磷酸化。
