Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.
The expression of nucleoside diphosphate kinase (NDK) genes has been implicated as a negative regulator of murine and human tumor metastases and is critical to proper development in Drosophila melanogaster. Molecular mechanisms for the role(s) of NDK in these complex processes have not yet been elucidated, but several reports have suggested that these and many other signal transduction pathways may be activated by NDK acting directly on a regulatory GTP-binding protein(s). To test this hypothesis, we examined the ability of NDK to catalyze the phosphorylation of the GDP bound to the following three members of the superfamily of regulatory GTP-binding proteins: Gt, Ha-ras p21, and ARF. We have found no evidence to support the hypothesis that NDK can directly activate any GTP-binding protein. Rather, evidence is presented which clearly shows that all of the GTP formed upon incubation of GTP-binding proteins with NDK is the result of NDK utilizing free GDP as substrate. The GDP bound to the regulatory proteins is not a substrate for NDK under conditions in which free nucleotides are rapidly and efficiently phosphorylated. The importance of appropriate controls for dissociation of GDP from the regulatory proteins both during the NDK reaction and during the analysis of product is demonstrated. We believe there is currently no experimental evidence to support the hypothesis that NDK can directly activate a regulatory GTP-binding protein.
Randazzo
P A
PA
Laboratory of Biological Chemistry, National Cancer Institute, National Institute of Mental Health, Bethesda, Maryland 20892.
Northup
J K
JK
Kahn
R A
RA
D020727:ADP-Ribosylation Factors; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D016283:Proto-Oncogene Proteins p21(ras)