p21 with a phenylalanine 28----leucine mutation reacts normally with the GTPase activating protein GAP but nevertheless has transforming properties.
The H-ras gene product p21H has been mutated at Phe-28, which makes a hydrophobic interaction with the guanine base of bound GDP/GTP. The mutation Phe-28----Leu drastically increases nucleotide dissociation rates without affecting association rates. This is due to a perturbed binding of base, alpha- and beta-phosphate, and Mg2+, as evidenced from 31P NMR and fluorescence measurements. The region around the gamma-phosphate appears normal. The affinity of Mg2+ for both the di- and the triphosphate conformation of the mutant was also measured by fluorescence. The association constant is 3.5 x 10(7) M-1 for the Gpp(NH)p complex, 500 times higher than for the GDP form. The mutation does not change appreciably the intrinsic or the GTPase activating protein (GAP)-stimulated GTPase. The mutated protein induces neurite differentiation however when pressure-loaded into PC12 cells, which is equivalent to transformation of NIH 3T3 cells. This shows that p21 (F28L) is converted to the GDP bound form by GAP but is transforming because the high dissociation rate for nucleotides leads to a protein predominantly in the active GTP bound form.
Reinstein
J
J
Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Federal Republic of Germany.
Schlichting
I
I
Frech
M
M
Goody
R S
RS
Wittinghofer
A
A
D000818:Animals; D002471:Cell Transformation, Neoplastic; D002942:Circular Dichroism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014407:Tumor Cells, Cultured; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D010649:Phenylalanine; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D007930:Leucine