Papin C, Denouel-Galy A, Laugier D, Calothy G, Eychène A
Unité Mixte de Recherche 146 du CNRS, Institut Curie, Centre Universitaire, Laboratoire 110, 91405 Orsay Cédex, France.
J Biol Chem. 1998 Sep 18;273(38):24939-47. doi: 10.1074/jbc.273.38.24939.
Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.
raf癌基因家族的成员编码丝氨酸/苏氨酸蛋白激酶，这些激酶通过直接相互作用和磷酸化激活丝裂原活化蛋白激酶激酶MEK（丝裂原活化蛋白激酶或细胞外信号调节激酶）。最近的几项研究揭示了该家族的两个成员Raf-1和B-Raf在激活、调节和激酶活性方面存在有趣的差异。特别是，已表明B-Raf比Raf-1表现出更高的MEK激酶活性。通过使用双杂交分析和免疫共沉淀实验，我们在此证明B-Raf与Raf-1的另一个显著差异在于其对MEK具有更高的亲和力。我们之前报道过，B-raf基因编码多种蛋白质异构体，这些异构体是由位于B-Raf激酶结构域上游的两个外显子（外显子8b和10）的复杂可变剪接产生的。在本研究中，我们表明蛋白质序列中的这些天然修饰显著调节了B-Raf的生化特性和致癌特性。外显子10序列的存在增强了对MEK的亲和力、基础激酶活性以及全长B-Raf的促有丝分裂和转化特性，而外显子8b序列的存在似乎具有相反的作用。因此，可变剪接代表了Raf家族蛋白质的一种新型调节机制。
