Biomedical subjects

D Bottorff

Publications and source records attributed to D Bottorff.

4 recordsLinked to original sources

PubMed1996

ras effector loop mutations that dissociate p120GAP and neurofibromin interactions.

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions. Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP-related domain of neurofibromin (NF1-GRD). The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1-GRD. Tyr32His was weakly transforming in Rat2 cells. Tyr32His ras was primarily GDP-bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor. These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.

Animals↗

PubMed1995

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase. The mutant MEK1 is coupled to the epidermal growth factor pathway but exhibits decreased physical interaction with RAF. When overexpressed, the MEK1 mutation is transforming and causes hyperphosphorylation of RAF. Signalling from RAS to MEK1 may be mediated by something other than RAF alone, but signalling through MEK1 is probably sufficient for RAS transformation.

Amino Acid Sequence↗

PubMed1993

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.

Amino Acid Sequence↗

PubMed1992

The murine leukemia inhibition factor gene (Lif) is located on proximal chromosome 11, not chromosome 13.

Lif, the murine gene encoding leukemia inhibition factor (LIF), has been previously localized to proximal Chromosome (Chr) 11. Hilda, the murine gene encoding "human interleukin in DA cells" (HILDA) has been localized to Chr 13. Since these two growth factors are identical, the proposal for two different structural loci is intriguing. To address this issue, blot hybridization methods have been used to establish the position of the structural gene sequence unambiguously. DNAs from somatic cell hybrids, recombinant inbred mice, and backcross mice have been probed with a sequence that encodes LIF/HILDA. The results support the assignment of this sequence to proximal Chr 11. These studies also establish a synteny group, including Lif and Tcn-2, the structural gene for transcobalamin 2, that is conserved between man and mouse.

Animals↗