Biochemical and biological consequences of changing the specificity of p21ras from guanosine to xanthosine nucleotides.
The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.
Schmidt
G
G
Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Dortmund, Germany.
Lenzen
C
C
Simon
I
I
Deuter
R
R
Cool
R H
RH
Goody
R S
RS
Wittinghofer
A
A
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D012680:Sensitivity and Specificity; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D012265:Ribonucleotides; D020666:ras-GRF1; D006153:Guanosine Diphosphate; C065326:xanthosine 5'-triphosphate; D006160:Guanosine Triphosphate; D010749:Phosphoprotein Phosphatases; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)