POSITIVE AND NEGATIVE MODULATION OF H-RAS TRANSFORMING POTENTIAL BY MUTATIONS OF PHENYLALANINE-28

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V -12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Non-conservative muta tions of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transfor ming potential of [V-12]p21. The conservative mutation of phenylalanin e-28 to tryptophan ([(VW28)-W-12]p21) was also still transforming. Sig nificantly, in the absence of the valine-12 activating mutation, trypt ophan-28-ras ([W-28]p21) was weakly transforming while, in contrast, [ (VD28)-D-12]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to nor mal and mutated p21 expressed in Escherichia coli showed that [(VD28)- D-12]p21 and [D-28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W-28]p21 is increased, suggesting a mechanism fo r its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p2 1(H-ras). The mutations described could be valuable tools in investiga tions of cellular signal transduction involving small GTP-binding prot eins.