Troppmair J, Bruder J T, App H, Cai H, Liptak L, Szeberényi J, Cooper G M, Rapp U R
Viral Pathology Section, National Cancer Institute, Frederick, Maryland 21702-1201.
Oncogene. 1992 Sep;7(9):1867-73.
A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.
Ras的显性负性突变体M17 Ras被用于研究Ras在Raf-1和B-Raf蛋白丝氨酸/苏氨酸激酶（PSK）受体偶联中的作用。我们发现，突变型Ras可阻断NIH3T3细胞中血清和12-O-十四烷酰佛波醇-13-乙酸酯诱导的Raf-1激酶激活，以及PC12嗜铬细胞瘤细胞中神经生长因子（NGF）对Raf-1和B-Raf PSK的刺激。通过测定Raf的过度磷酸化以及对外源底物的活性来检测有丝分裂原对Raf激酶的刺激，在表达M17 Ras的细胞中，这两个事件均受到抑制。相比之下，活化的酪氨酸激酶受体的直接底物磷脂酶C-γ1（PLC-γ1）的酪氨酸磷酸化未受影响。这些数据表明，PLC-γ1的酪氨酸磷酸化不足以诱导NIH3T3细胞生长，并且Ras介导了从活化的膜受体到胞质溶胶中Raf激酶的信号传递。由于活化的Raf可诱导表达M17 Ras的PC12细胞分化，我们得出结论，Raf激酶激活可能足以解释NGF功能的这一方面。
