Oncogene database

369

ARAF

A-Raf proto-oncogene, serine/threonine kinase;ARAF;A-Raf proto-oncogene, serine/threonine kinase

A-Raf proto-oncogene serine/threonine-protein kinase|Oncogene ARAF1|Ras-binding protein DA-Raf|proto-oncogene A-Raf-1|proto-oncogene Pks|serine/threonine-protein kinase A-Raf|v-raf murine sarcoma 3611 viral oncogene homolog 1

Xp11.4-p11.2

protein-coding

We have determined the expression pattern of the A-raf proto-oncogene in the embryonic and adult mouse. Western blot analysis of protein lysates from tissues of adult mice show that p69A-raf is ubiquitously expressed, but that levels of expression vary among different tissues. To determine the cell-specific expression pattern of A-raf, we generated transgenic mice expressing the beta-galactosidase reporter gene from the A-raf promoter. We show that A-raf expression is highly specific within a given tissue, and we identify cell types expressing this gene in the adult testis, epididymis, vas deferens, seminal vesicle, ovary, oviduct, bladder, kidney, intestine, heart, spleen, thymus, and cerebellum. In the embryo, ubiquitous expression of the reporter gene is observed, but the highest levels of expression are specifically detected in the embryonic heart at stages 9.5-11.5 days post-coitum.

Previous studies have indicated an important role for the Raf family of protein kinases in controlling cellular responses to extracellular stimuli and activated oncogenes, through their ability to activate the MEK/ERKs. To investigate the specific role of A-Raf in this process we generated A-Raf deficient mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells by gene targeting and characterized their ability to undergo proliferation, differentiation, apoptosis, ERK activation, and transformation by oncogenic Ras and Src. The A-Raf deficient cells are not disrupted for any of these processes, despite the fact that this protein is normally expressed at high levels in both cell types. This implies either that A-Raf plays no role in MEK/ERK activation, that its function is fully compensated by other Raf proteins or MEK kinases or that its role in MEK/ERK activation is highly tissue-specific. Interestingly, B-Raf and Raf-1 activity towards MEK as measured by the immunoprecipitation kinase cascade assay are both significantly increased in the A-Raf deficient MEFs.

The transcribed murine A-raf proto-oncogene has been localized to the proximal region of the mouse X chromosome, within the context of four other active genes in this region which together constitute a conserved linkage group between mouse and man. This localization has been accomplished using species-specific restriction fragment length variation and DNAs from a previously defined informative subset of progeny representative of a set of 100 progeny from an interspecific backcross between inbred C57BL/6JRos and wild-derived Mus spretus. This new data regionally orders the mouse A-raf locus relative to the 24 X-linked markers previously examined in this backcross. We find that A-raf co-localizes with two other active genes, tissue inhibitor of metalloproteinases (Timp) and synapsin (Syn-1), 4.0 +/- 2.0 cM distal to the Otc gene at the proximal end of the mouse X chromosome, for a partial gene order in this region of: centromere-Cybb-Otc-Timp/A-raf/Syn-1-Xlr-1-Hprt.

The complete 606 amino acid sequence of the human A-raf oncogene has been deduced from the 2453 nucleotide sequence of a human T cell cDNA. A cysteine-rich region located near the amino terminus, which is highly conserved in A-raf and c-raf, shows significant homology with protein kinase C. A 5 deleted fragment of the cDNA has been incorporated into a murine retrovirus which endows the virus with the ability to transform cells in vivo and in vitro. Functionally, human A-raf is similar to v-raf and v-mos in that transformation is independent of ras gene function.

The localization of the A-raf cellular oncogene on the mouse X chromosome has been determined using Xbal-restricted DNAs prepared from progeny of an interspecies backcross between the B6.CBA.R1 and the Spe/Pas mouse strains. This localization to the proximal part of the mouse X chromosome has been confirmed by the use of somatic cell hybrids, carrying partially deleted X chromosomes and suggests that the A-raf oncogene localizes to a region lying some 10-17 centimorgans proximal to the hypoxanthine phosphoribosyltransferase (Hprt) gene between the locus DXPas4 and the locus DXPas7 defined by the cross-reacting human X chromosome-specific probe DXS32 (M2C). This localization on the mouse X chromosome is compatible with the presence of the A-raf oncogene on the short arm of the human X chromosome between the centromere and Xp21.

A 1.6-kilobase cDNA (A-raf) has been isolated from a murine spleen cDNA library which encodes part of a protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of 100 amino acids. In contrast to raf, A-raf shows a highly restricted tissue distribution of expression, with highest levels observed in epididymis, followed by intestine. When incorporated into a retrovirus, the resulting gag-A-raf fusion gene causes transformation in vitro and induces tumors in newborn mice. Thus, A-raf represents a new proto-oncogene. Transformation by A-raf is independent of ras gene function, as is the case for raf and mos but not other oncogenes.

Murine and human cDNAs, related to but distinct from c-raf-1, have been isolated and designated mA-raf and hA-raf, respectively. The mA-raf and hA-raf cDNAs detect the same murine and human fragments in Southern blots of restriction enzyme-cleaved murine and human cellular DNA. The murine restriction enzyme fragments homologous to mA-raf cDNA cosegregate with mouse chromosome X in a panel of Chinese hamster-mouse hybrid cells, thus localizing the mA-raf locus to mouse chromosome X. Two independently segregating loci, detected by the hA-raf cDNA (or mA-raf cDNA), hA-raf-1 and hA-raf-2, are located on human chromosomes X and 7, respectively. The mA-raf locus and the hA-raf-1 locus are actively transcribed in several mouse and human cell lines.

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of ALL three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.

The DRE/DREF system plays an important role in transcription of DNA replication genes such as those encoding the 180 and 73 kDa subunits of DNA polymerase alpha as well as that for encoding PCNA. In this study, we found two sequences homologous to DRE (5 -TATCGATA-3 ) in the 5 -flanking region (-370 to -357 with respect to the transcription initiation site) of the D-raf gene and confirmed transcriptional activity through gel mobility shift assays, transient CAT assays, and spatial patterns of lacZ expression in transgenic larval tissues carrying D-raf and lacZ fusion genes. Further, we demonstrated that the D-raf gene is another target of the Zerknullt (Zen) protein with observation of D-raf repression by Zen protein in cultured cells and its ectopic expression in the dorsal region of the homozygous zen mutant embryo. The evidence of DRE/DREF involvement in regulation of the D-raf gene obtained in this study strongly supports the idea that the DRE/DREF system is responsible for the coordinated regulation of cell proliferation-related genes in Drosophila.

It has previously been shown that maximal activation of Raf-1 is produced by synergistic signals from oncogenic Ras and activated tyrosine kinases. This synergy arises because Ras-GTP translocates Raf-1 to the plasma membrane where it becomes phosphorylated on tyrosine residues 340 and 341 by membrane-bound tyrosine kinases (Marais, R., Light, Y., Paterson, H. F., and Marshall, C. J. (1995) EMBO J. 14, 3136-3145). We have examined whether the other two members of the Raf family, A-Raf and B-Raf, are regulated in a similar way to Raf-1. A-Raf behaves like Raf-1, being weakly activated by oncogenic Ras more strongly activated by oncogenic Src, and these signals synergize to give maximal activation. B-Raf by contrast is strongly activated by oncogenic Ras alone and is not activated by oncogenic Src. These results show that maximal activation of B-Raf merely requires signals that generate Ras-GTP, whereas activation of Raf-1 and A-Raf requires Ras-GTP together with signals that lead to their tyrosine phosphorylation. B-Raf may therefore be the primary target of oncogenic Ras.