Barnier J V, Papin C, Eychène A, Lecoq O, Calothy G
Unité Mixte de Recherche 146 du CNRS, Institut Curie, Centre Universitaire, Orsay, France.
J Biol Chem. 1995 Oct 6;270(40):23381-9. doi: 10.1074/jbc.270.40.23381.
The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.
c-Rmil/B-raf原癌基因是mil/raf家族的成员，该家族编码丝氨酸/苏氨酸蛋白激酶，已证明其参与从细胞膜到细胞核的信号转导。我们从小鼠脑文库中分离出B-raf cDNA，其包含一个先前未鉴定的36个碱基对的选择性剪接外显子，位于外显子8和9之间，因此命名为外显子8b。人和小鼠的B-raf mRNA还包含先前在禽c-Rmil基因中描述的120个碱基对的选择性剪接外显子10。这两个位于保守区域2（CR2）和催化结构域（CR3）之间的外显子的独立剪接产生了可能编码四种不同蛋白质的mRNA。通过使用针对B-Raf不同部分产生的特异性血清，我们在成年小鼠组织中鉴定出至少10种蛋白质异构体。一些分子量在69-72 kDa范围内的异构体不被针对外显子1和2编码的肽的抗血清识别，这表明存在具有两种不同NH2末端的B-Raf蛋白。其他分子量在79-99 kDa范围内的异构体包含由外显子1和2、一个或两个选择性剪接外显子以及可能另一个未鉴定外显子编码的氨基酸。通过逆转录-聚合酶链反应分析B-raf mRNA表达以及对成年小鼠不同组织中的B-Raf蛋白进行免疫鉴定，结果显示B-Raf异构体的表达具有组织特异性模式。有趣的是，包含由外显子10编码的氨基酸的异构体在神经组织中特异性表达。综上所述，这些结果表明不同的B-Raf蛋白可能以组织特异性方式参与信号转导途径。
