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 ([V12]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 mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21H-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.
Ricketts
M H
MH
Department of Psychiatry, UMDNJ, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Durrheim
G A
GA
North
H M
HM
van der Merwe
M J
MJ
Levinson
A D
AD
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D010649:Phenylalanine; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D051381:Rats; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D010649:Phenylalanine; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)