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

Abstract

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.

Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation

“…This could be due to the low MAP kinase activation achieved in stably transfected cells as compared to transiently transfected ones (this paper and M. McMahon, personal communication). We do not favor this hypothesis, since it has been shown in the case of the Raf family members, which act directly upstream of MEK, that a high MAP kinase activation is not required for long term e ects on cell proliferation, and in fact there is an inverse correlation between mitogenic properties and the ability to induce MAP kinase activation among the di erent Raf forms (Bosch et al, 1997). The role of MAP kinase activation in epithelial transformation has been explored in other cell systems.…”

The Transcriptional Response to Raf Activation Is Almost Completely Dependent on Mitogen-activated Protein Kinase Kinase Activity and Shows a Major Autocrine Component

“…For the control of apoptosis, it may be that basal levels of ERK activity are critical and these are less easily measured. The data with the Raf-1 FF mutant (Hü ser et al, 2001) need to be interpreted with caution because this mutant has been reported to be able to phosphorylate and activate MEK in a number of assays (Fabian et al, 1993;Bosch et al, 1997;Woods et al, 1997). These issues could be resolved by the generation of c-Raf-1 kinase dead mutant knockin mice.…”

The roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway; a potential route to radio- and chemo-sensitization of tumor cells resulting in the induction of apoptosis and loss of clonogenicity

“…All 'Raf' family members can phsophorylate and activate MEK1/2, although the relative ability of each member to catalyze this reaction varies (B-RAF Ͼ Raf-1 Ͼ A-RAF). 13,14 Seminal studies by the laboratories of Wolfman and Wigler demonstrated that the CR1 domain of Raf-1 could reversibly interact with the Ras proto-oncogene in the plasma membrane. 15,16 The ability of Raf-1 to associate with Ras was dependent upon the Ras molecule being in the GTP-bound state.…”