Molecular cloning and characterization of an activated human c-raf-1 gene.

Abstract

Results of previous studies have shown that a raf-related transforming DNA sequence is present in NIH 3T3 transformants that are derived from GL-5-JCK human glioblastoma DNA transfection. The transforming DNA was molecularly cloned by using cosmid vector pJB8 to determine its structure and origin. Analyses of selected clones revealed that the transforming DNA consisted of three portions of human DNA sequences, with the 3' half of the c-raf-1 gene as its middle portion. This raf region was about 20 kilobases long and contained exons 8 to 17 and the poly(A) addition site. RNA blot analysis showed that the raf-related transforming DNA was transcribed into 5.3-, 4.8-, and 2.5-kilobase mRNAs; the 2.5-kilobase transcript was thought to be the major transcript. Immunoprecipitation analyses revealed that a 44-kilodalton raf-related protein was specifically expressed in the NIH 3T3 transformants. The raf-related transforming DNA was considered to be activated when its amino-terminal sequence was truncated and the DNA was coupled with a foreign promoter sequence. On hybridization analysis of the original GL-5-JCK glioblastoma DNA, no rearrangement of c-raf-1 was detectable in the tumor DNA. The rearrangement of c-raf-1 may have occurred during transfection or may have been present in a small population of the original tumor cells as a result of tumor progression.We previously detected a transforming DNA sequence in NIH 3T3 cells that were transformed by GL-5-JCK human glioblastoma DNA and showed that the transforming DNA was a human homolog of the retroviral oncogene v-raf (8). Among the transforming DNA sequences so far detected by NIH 3T3 transformation assay, a few were found to be related to retroviral oncogenes. Besides the transforming raf gene mentioned above, a transforming Ha-ras gene (21, 25) and a transforming Ki-ras gene (7) were identified as cellular homologs of the oncogenes of Harvey murine sarcoma virus and Kirsten murine sarcoma virus, respectively. A transforming N-ras gene (9, 29) and a human melanoma transforming gene, mel (20), showed homology to v-Ha-ras and v-Ki-ras. In addition, a transforming gene of rat neuroblastomas and glioblastomas, neu, showed homology to v-erbB (27). The transforming ras genes Ha-ras, Ki-ras, and N-ras have been well characterized. These three transforming ras genes and the two viral ras genes were demonstrated to have a similar coding sequence and point mutations that are responsible for their transforming activity (22,33,36,39). The v-raf gene was originally detected in a murine sarcoma virus, 3611-MSV (22). A functional human gene homologous to v-raf was cloned and termed c-raf-1. This c-raf-1 gene contained 17 exons, 9 of which were homologous to v-raf (4). The v-raf gene was a truncated gene containing only the 3' portion of the cellular homolog, and its translational products were identified as gag-fused proteins (22). Activation of raf-related transforming DNA derived from GL-5-JCK glioblastoma DNA might occur in the same way as activation of v-raf. In this study we...

P21Cip1 induced by Raf is associated with increased Cdk4 activity in hematopoietic cells

“…One signal transduction pathway that can be activated by such cytokines in hematopoietic cells is the Ras/Raf/MEK/ERK pathway (Dent et al, 1998;Hibi and Hirano, 1998;Blalock et al, 1999;Frank, 1999;Lee, 1999;Hoyle et al, 2000;McCubrey et al, 2000;Moye et al, 2000). So far, three Raf genes have been identi®ed in mammalian cells; they are Raf-1, ARaf and B-Raf (Fukui et al, 1987;Sithanandam et al, 1990;Daum et al, 1994;Lee et al, 1994). The Raf proteins have three conserved domains: CR1, CR2 and CR3.…”

B-raf, a new member of the raf family, is activated by DNA rearrangement.

“…The v-raf protein of 3611 murine sarcoma virus (26) and the v-mil protein of avian sarcoma virus MH2 (29) were generated by fusion of viral gag protein with the carboxyl-terminal half of c-raf-1 protein. In most other activated c-raf s, the polypeptides in the amino-terminal portion are replaced by foreign polypeptides (8,16,28). Also, viral LTR insertion between exon 5 and 6 of c-raf-1 also activated the gene (25).…”

Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

“…In each of the activated raf genes which has been characterized, there was a deletion at the 5' end of the normal cellular gene. In two cases, the 5' deletion extends to intron 5 (29,31); in one case (v-mil), the deletion extends to exon 7 (4, 40); in seven cases, the deletion extends to intron 7 (14,19,37,41); in one case (v-raJ), the deletion extends to exon 9 (4); and in one case (B-raJ), the deletion extends to the equivalent of intron 9 (16). These results have suggested the hypothesis that the rafamino-terminal sequences encode a regulatory domain, the deletion of which results in constitutive activity of the carboxy-terminal kinase domain.…”