Janssen R A, Veenstra K G, Jonasch P, Jonasch E, Mier J W
Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Massachusetts, Boston 02115, USA.
J Biol Chem. 1998 Nov 27;273(48):32182-6. doi: 10.1074/jbc.273.48.32182.
Transformation is accompanied by the down-regulation of the high molecular weight isoforms of non-muscle tropomyosin. Several lines of evidence suggest that tropomyosin down-regulation may be essential for ras-induced tumorigenicity. It is unclear which of the many signaling pathways downstream of Ras are involved in tropomyosin down-regulation. Here we demonstrate that Raf activation induces tropomyosin down-regulation comparable to that induced by Ras. Expression of the effector-domain mutant Ras-G12V,Y40C, which is unable to bind Raf, induced only modest down-modulation of tropomyosin. Treatment with the MEK-specific inhibitor PD98059 had little effect on tropomyosin levels in ras- or raf-transformed cells. In contrast, a mutant form of MEK-1, MEK-1-S218A,S222A, restored tropomyosin levels in ras-transformed NIH3T3 cells almost to the levels observed in non-transformed cells. MEK-1-S218A,S222A does not inhibit MEK phosphorylation and is a poor inhibitor of ERK phosphorylation. These data suggest that this mutant form of MEK-1 interferes with a yet uncharacterized pathway controlled by Raf. We conclude that the ras-induced down-modulation of tropomyosin is predominantly Raf-mediated, but MEK-independent, and that a novel pathway exists downstream of Raf which may play an important role in regulation of the cytoskeleton.
细胞转化伴随着非肌肉原肌球蛋白高分子量异构体的下调。多项证据表明，原肌球蛋白下调可能对Ras诱导的致瘤性至关重要。目前尚不清楚Ras下游众多信号通路中的哪一条参与了原肌球蛋白的下调。在此我们证明，Raf激活诱导的原肌球蛋白下调与Ras诱导的相当。效应结构域突变体Ras-G12V、Y40C无法结合Raf，其表达仅诱导原肌球蛋白适度下调。用MEK特异性抑制剂PD98059处理对ras或raf转化细胞中的原肌球蛋白水平影响很小。相反，MEK-1的突变形式MEK-1-S218A、S222A可将ras转化的NIH3T3细胞中的原肌球蛋白水平恢复到几乎与未转化细胞中观察到的水平相同。MEK-1-S218A、S222A不抑制MEK磷酸化，且对ERK磷酸化的抑制作用较弱。这些数据表明，这种MEK-1突变形式干扰了由Raf控制的一条尚未明确的信号通路。我们得出结论，ras诱导的原肌球蛋白下调主要由Raf介导，但不依赖MEK，并且在Raf下游存在一条新的信号通路，其可能在细胞骨架调节中发挥重要作用。
