Differences in the interaction of p21c-Ha-ras-GMP-PNP with full-length neurofibromin and GTPase-activating protein.
Neurofibromin, the product of the neurofibromatosis type 1 gene, was found to form a stable complex with immobilized p21c-Ha-ras-GMP-PNP (a non-hydrolyzable GTP analog). This complex, detectable as early as 30 min after addition of crude brain extract, is extremely stable, with less than 50% dissociating after 5 h at 4 degrees C. We interpret this to suggest that the dissociation of full-length neurofibromin from p21c-Ha-ras-GMP-PNP is tightly linked to the hydrolysis of GTP to GDP. Failure to remove a significant proportion of the bound neurofibromin in the presence of EDTA and GDP implies that the binding of neurofibromin to p21c-Ha-ras-GMP-PNP results in the ras protein becoming resistant to guanine nucleotide exchange. Under conditions in which neurofibromin quantitatively binds to p21c-Ha-ras-GMP-PNP, we were unable to detect a complex between p21c-Ha-ras and GAP (GTPase-activating protein). The failure to detect GAP binding to immobilized p21c-Ha-ras-GMP-PNP cannot be explained by the known differences in affinities of the GAP-related domain of neurofibromin and GAP for p21c-Ha-ras-GTP. GAP is, however, able to interact biochemically with immobilized p21c-Ha-ras, suggesting a difference in the interaction between GAP and neurofibromin with p21c-Ha-ras-GMP-PNP.
DiBattiste
D
D
Department of Vascular Cell Biology and Atherosclerosis, Cleveland Clinic Foundation, Ohio 44195.
Golubic
M
M
Stacey
D
D
Wolfman
A
A
D000818:Animals; D001923:Brain Chemistry; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D025542:Neurofibromin 1; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D051381:Rats; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)