B-RAF PROTEIN KINASE--STRUCTURE, EXPRESSION AND ACTIVATION IN VIVO

到目前为止，已经在MAN中发现了三种Raf激酶的同工型：Raf-1， A-RAF和B-RAF。B-RAF在胎儿和成人脑中的水平最高 大脑。除了常见的10kb和13kb的转录本 表达B-RAF的组织，2.6和4.5kb的交替大小转录本 在睾丸里发现的。以前的工作建立了部分B-RAF序列。至 获得全长cDNA，使用B-RAF特有的42聚体寡核苷酸 目的：筛选人睾丸cDNA文库。获得了一个2.3kb的克隆 包含一个1950个碱基对的开放阅读框，可能编码 全长B-RAF蛋白。 序列分析表明有三个保守区，即CR1、CR2和CR3， 典型的RAF蛋白激酶。在CR1区域中，包含 推测的锌指区，B-RAF与A-RAF的同源性为75%，与A-RAF的同源性为65% 英国皇家空军一号。富含丝氨酸/苏氨酸的CR2区与两者有47.6%的同源性 A-RAF和RAF-1，CR3与A-RAF的同源性为76.4%，同源性为79% 与Raf-1结合，它对应于激活域。 在大肠杆菌T7表达系统中表达了B-RAF蛋白。这个 B-RAF融合蛋白与B-RAF特异性抗体反应 针对C-末端多肽和映射到 激酶结构域(接近于三肽保守序列基序) 家族蛋白激酶位点)，预测与所有RAF反应 家庭成员。与RNA表达数据一致，预测 72.5kodalton的B-RAF蛋白在小鼠脑内表达。

Three isotypes of Raf kinases have been identified to date in man: raf-1, A-raf and B-raf. B-raf has the highest levels in fetal brain and adult cerebrum. In addition to the 10- and 13-kilobase (kb) transcripts common to B-raf-expressing tissues, alternate-size transcripts of 2.6 and 4.5 kb were found in testes. Previous work established a partial B-raf sequence. To obtain a full-length cDNA, a 42mer oligonucleotide unique to B-raf was used to screen a human testes cDNA library. A 2.3-kb clone was obtained that contains an open reading frame of 1950 base pairs, presumably encoding full-length B-raf protein. Sequence analysis indicates three conserved regions, CR1, CR2 and CR3, typical of raf protein kinases. In the CR1 region, which encompasses the putative zinc finger region, B-raf shows a 75% homology to A-raf and 65% to raf-1. The serine/threonine-rich CR2 region has a 47.6% homology with both A-raf and raf-1, and CR3 has a 76.4% homology with A-raf and 79% homology with raf-1, which corresponds to the kinase domain. B-raf protein has been expressed in the E. coli T7 expression system. The B-raf fusion protein reacts with antibodies specific for B-raf raised against the C-terminal peptide and to monoclonal antibodies which map into the kinase domain (near a tripeptide conserved sequence motif for all Src family protein kinase sites) which are predicted to react with all raf family members. Consistent with the RNA expression data, the predicted 72.5-kiloDalton B-raf protein is expressed in mouse brain.