Rushworth Linda K, Hindley Alison D, O'Neill Eric, Kolch Walter
Signalling and Proteomics Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, United Kingdom.
Mol Cell Biol. 2006 Mar;26(6):2262-72. doi: 10.1128/MCB.26.6.2262-2272.2006.
The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.
Ras-Raf-MEK-细胞外信号调节激酶（ERK）通路参与调控许多基本的细胞过程，包括增殖、存活和分化。该通路在高达30%的人类癌症中失调，这通常是由于Ras和B-Raf亚型的突变所致。Raf-1和B-Raf可形成异源二聚体，这可能对细胞转化很重要。在此，我们分析了Raf-1/B-Raf异源二聚体的生化和生物学特性。与各自的同源二聚体或单体相比，分离出的Raf-1/B-Raf异源二聚体具有显著增强的激酶活性。野生型Raf-1与激酶活性低或无激酶活性的B-Raf突变体之间的异源二聚体，以及野生型B-Raf与激酶阴性的Raf-1之间的异源二聚体，其激酶活性仍有所升高。相比之下，同时含有激酶阴性的Raf-1和激酶阴性的B-Raf的异源二聚体则完全无活性，这表明异源二聚体的激酶活性特异性地源自Raf，并且任何一种具有激酶活性的Raf亚型都足以赋予异源二聚体高催化活性。在细胞系中，Raf-1/B-Raf异源二聚体的水平较低。14-3-3蛋白和有丝分裂原可独立于ERK增强异源二聚化。然而，ERK诱导的B-Raf在T753位点的磷酸化促进了Raf异源二聚体的解离，而T753位点的突变则延长了生长因子诱导的异源二聚化。B-Raf T753A突变体增强了PC12细胞的分化，此前已证明这依赖于持续的ERK信号传导。通过肽阵列对相互作用位点进行精细定位表明，其相互作用模式复杂，涉及多个接触位点，其中B-Raf中包含T753的一个主要Raf-1结合位点。总之，我们的数据表明，Raf-1/B-Raf异源二聚化是生理激活过程的一部分，并且该异源二聚体具有独特的生化特性，这可能对某些生物学过程的调控很重要。
