The Mouse B-raf Gene Encodes Multiple Protein Isoforms with Tissue-specific Expression

Abstract

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 NH 2 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 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.Proto-oncogenes of the mil/raf gene family encode serine/ threonine protein kinases, which act as signal transducers from membrane-associated receptors to nuclear transcription factors (1, 2). Members of the Raf family play a major role in the regulation of cell proliferation and are also required for the determination of cell fate during embryogenesis (3, 4). The p72/74 Raf-1 protein encoded by the c-raf/c-mil gene is ubiquitously expressed in embryonic and adult mouse tissues. This protein has been shown to act downstream of Ras and upstream of MAP kinase kinase (Mek-1) in mammalian and invertebrate cells (1). Raf-1 associates with the GTP-bound Ras protein, the activated form of p21 ras . Binding to Ras does not directly activate Raf-1 but rather serves to recruit this protein to the membrane. This step is required for Raf-1 activation by an unknown mechanism and subsequent activation of Mek-1 by Raf-1 (5-11). A-raf, the second member of this family (12) encodes a 67.5-kDa protein and is expressed predominantly in the epididymis and ovary (13). Its contribution to the signaling pathway has not yet been investigated.The third member of this family was identified as the c-Rmil gene in avian and B-raf in mammalian species (14,15). Like the Raf-1 protein, the B-Raf protein can be activated by growth factors, such as ...

B‐Raf‐mediated signaling pathway regulates T cell development

“…Although the individual Raf genes have some apparent functional redundancies with each other in ERK regulation, the different phenotypes of each Raf kinase-deficient mouse suggest that they have specific and non-redundant functions [12,[16][17][18][19], which seem to be due to their distinct expression patterns [20] and differences in the thresholds for activation [15,21,22].…”

A New Constitutively Active Brain PAK3 Isoform Displays Modified Specificities toward Rac and Cdc42 GTPases

“…Immunoprecipitation and in Vitro Kinase Assay-Transfected cells were lysed as previously described (31). In some experiments, in order to activate the kinase, cleared cell extracts from PAK3-transfected cells were incubated with 5 g of recombinant Cdc42V12 in the presence of 25 M ATP during 30 min at room temperature.…”

“…Expression Analysis by RT-PCR-RNAs from human brain or from adult mouse tissues were prepared and reverse transcribed as previously described (31). Expression of the new PAK3b isoform was characterized from mouse or human brain cDNA using an exon b-specific sense primer and a species-specific reverse primer, leading to a mouse set (primer set 2: 5Ј-CCAGATCTCTATGGCTCACAG-3Ј and 5Ј-GGAG-GAGCCAAAGGAGGTTC-3Ј) or a human set (primer set 3: 5Ј-CCAGA-TCTCTATGGCTCACAG-3Ј and 5Ј-GGAGGGGCCAATGGAGGC-3Ј), giving rise to a 275-bp fragment.…”

B-Raf Contributes to Sustained Extracellular Signal-regulated Kinase Activation Associated with Interleukin-2 Production Stimulated through the T Cell Receptor

“…Whereas Raf-1 is ubiquitously expressed, B-Raf shows a more restricted expression pattern (18,19). Mice deficient in the different Raf isoforms exhibit different developmental defects, suggesting the nonredundant function(s) of each Raf isoform (20).…”