The complete coding sequence of the human A-<i>raf</i>-1 oncogene and transforming activity of a human A-<i>raf</i>carrying retrovirus

Beck, Thomas W.; Huleihel, M; Gunnell, M.; Bonner, Tom I.; Rapp, Ulf R.

doi:10.1093/nar/15.2.595

Cited by 137 publications

(69 citation statements)

References 44 publications

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“…Our isolation of Nterminal-truncated A-Raf-1 recapitulates a previous report that truncated forms of RAF proteins that could render TF-1 cells cytokine independent (McCubrey et al, 1998). Retroviral infection with a gag-A-RAF-1 fusion protein induced the development of sarcomas and erythroleukemias in mice (Beck et al, 1987). This specificity of transforming the erythroid lineage is again reminiscent of the predominant erythroid expansion in PV.…”

Section: Discussionsupporting

confidence: 83%

Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation

2003

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Section: Discussionsupporting

confidence: 83%

Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation

2003

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“…HA-Raf-1 inserts were subcloned into pcDNA3 (Invitrogen) using the HindIII and Xba restriction enzymes. A triple HA-tag was fused in-frame to the C-terminus of PUC19-A-Raf (Beck et al, 1987). ARaf-HA was subcloned into pcDNA3 (Invitrogen) using the appropriate restriction sites.…”

Section: Construction Of Plasmid Dnasmentioning

confidence: 99%

“…In nonvertebrates, such as Caenorhabiditis elegans or Drosophila melanogaster, only one homolog of Raf appears to be expressed, suggesting a linear mechanism of signal ampli®cation (Dickson and Hafen, 1994). In mammals three isoforms of Raf, c-Raf-1, A-Raf and BRaf, are encoded suggesting that the speci®city of Ras signaling in those cells which express the di erent isozymes of Raf is governed by a more complex mechanism (Rapp et al, 1988;Sithanandam et al, 1990;Beck et al, 1987). Virtually all members of the Ras family of proteins share the capability of interacting with all three Raf isozymes, although what regulates the speci®city of this interaction is largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes

et al. 2000

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In the mitogenic signaling cascade interaction of Ras with Raf represents a critical step for the regulation of cell growth and di erentiation. The major e ector of Ras, the serine/threonine kinase Raf exists as three isoforms with di erent tissue distributions. We demonstrate that transient transfection of oncogenic Ha-Ras leads to a preferential activation of endogenous c-Raf-1 in HEK 293 cells as opposed to A-Raf. In vitro binding studies using puri®ed Ras binding domains of Raf as well as in vivo bindings tests with full length molecules reveals signi®cantly lower binding a nities of A-Raf to Ha-Ras as compared to other Raf isoforms. The Ras-binding interface of c-Raf di ers from A-Raf by a conservative Arg to Lys exchange at residue 59 or 22 respectively. Mutational analysis reveals that this residue represents a point of isozyme discrimination: c-Raf-R59K binds HaRas weaker than the wildtype, likewise A-Raf-K22R increases its a nity to Ha-Ras in vivo and in vitro. Di erential binding a nities are re¯ected in downstream signaling. Immunecomplex kinase assays reveal that HaRas mediated Raf activation is decreased for c-Raf-R59K and increased for A-Raf-K22R when compared to the respective wildtype forms. Thus our observations introduce a new level of isoform discrimination in Ras/ Raf signaling as a functional consequence of a conservative amino acid exchange in the Ras binding domains.

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“…The CR1 region encodes the Ras binding domain of the protein and a`cysteine ®nger' Finney et al, 1993;Koide et al, 1993;Moodie et al, 1993;Papin et al, 1996;Van Aelst et al, 1993;Vojtek et al, 1993;Warne et al, 1993;Zhang et al, 1993). Although CR1 and CR2 contain regulatory sites of phosphorylation, it appears that the CR2 region is the major negative regulator of protein kinase activity as a deletion encompassing this region in Raf-1 is constitutively activated and capable of promoting oncogenic transformation of several cell types (Beck et al, 1987;Ikawa et al, 1988;Marx et al, 1988;Stanton et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1

et al. 1997

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The catalytic domains of the Raf family of protein kinases (DRaf) dier in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to signi®cantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these dierences in amino acid sequence in¯uence the relative catalytic activity of the Raf kinase domains we constructed forms of DA-Raf, DB-Raf and DRaf-1 that encode either aspartic acid forms of the DRaf-1:ER and DA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of DA-Raf:ER and DRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the DB-Raf:ER proteins were highly transforming and DBRaf catalytic activity was largely unaected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of DA-Raf, DB-Raf and DRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.

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The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-rafcarrying retrovirus

Cited by 137 publications

References 44 publications

Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation

Genetic complementation of cytokine signaling identifies central role of kinases in hematopoietic cell proliferation

Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1

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