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Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.

1992; Elsevier BV; Volume: 267; Issue: 25 Linguagem: Inglês

10.1016/s0021-9258(19)37170-4

1083-351X

Paul A. Randazzo, John K. Northup, Richard Kahn,

Trace Elements in Health

Proteins Are Not Activated Directly by N D K 18183 been examined.Thus, NDK has been shown to catalyze the transfer of phosphate from ATP to GDP that had been bound to ras (19), EF-al (19), tubulin (20, 21), G, (15), G, (19), and, most recently (lS), ADP-ribosylation factor (ARF), a 21-kDa GTP-binding protein that regulates protein traffic in eukaryotes.Second, NDK has been shown to activate GTP-binding proteins in vitro.For example, ATPrS-dependent activation of adenylate cyclase in platelet membranes (22), acetylcholine-activated K+ channels in cardiac myocytes ( 23), and NADPH-oxidase in HL-60 membranes (24) were all reported t o be NDK-mediated.ADP-ribosylation factor activity (18) and the GTPase activity of G, (15) have been reported to increase with added NDK and ATP.However, in very few of these studies has an attempt been made to document that the GDP is actually bound to the GTP-binding protein when phosphorylated by NDK.This is a critical distinction as addition or production of GTP in solution, in the absence of receptor and agonist, is known to have little or no effect on any GTP-binding protein-mediated effector system.A clear demonstration of a GTP-binding protein being activated by NDK absolutely requires proof that the phosphorylation of GDP occurs while bound to the regulatory protein.We believe that this condition has not been met by any published report t o date.Omission or improperly controlled tests of this condition may have led to erroneous conclusions regarding the actions of NDK.In this report, we have examined GDP bound to ARF, Gt, and Ha-ras p21 as potential substrates for NDK, specifically testing whether phosphate is transferred to the bound GDP.We were unable to demonstrate such a transfer under conditions in which phosphate was efficiently transferred to free nucleoside diphosphate.Several artifacts are described and documented which we believe account for all of the published data that supported the conclusion that a GTP-binding protein is activated by NDK when, in fact, such an event may not occur. MATERIALS AND METHODS ReagentsRecombinant mARFlp (25), nm23-lp (18), and GPlyZ2 were puril'ied from bacteria or Sf9 cells as previously described.nm23-Hlp and nm23-H2p were expressed in bacteria and purified by the same procedure previously described for nm23-lp (18).Recombinant Harm p21 and (G12V) Ha-ras p21, purified from bacteria as described (27), were the generous gifts from Dr. Richard Michitsch (Oncogene Science Inc., Manhasset, NY) and Drs.Douglas Lowy and Alex Papageorge (NCI, Bethesda, MD).G,, G,a, rod outer segment discs, and urea-washed discs containing rhodopsin were purified from bovine retina as described (28, 29).Bovine liver nucleoside diphosphokinase (catalog no.N-2635), ATP, GTP, L-a-dimyristoyl phosphatidylcholine, and Sephadex G-25 were purchased from Sigma.[a-32P] GTP, made by Du Pont-New England Nuclear, was used to prepare [tu-:"P]GDP as described in Hamel and Lin (30).PEI-cellulose thin layer plates were purchased from J. T. Baker Chemical Co.BA-85 nitrocellulose filters (25 mm) were obtained from Schleicher & Schuell.Loading Proteins with fa-"PIGDP ARF-ARF was loaded with [a-"PIGDP as described in Ref. 18. Ras-Between 0.5 and 3 r g of recombinant ras p21 was incubated in 25 mM HEPES, pH 7.4, containing 2.5 mM MgCl,, 1 mM dithiothreitol, and approximately 10 pCi of [o-~'P]GDP (1000-6000 Ci/ mmol) in a volume of 100 pl for 2 h a t 37 "C.The solution was cooled to 4 "C, and free nucleotide was removed and buffer was exchanged on a Sephadex G-25 column equilibrated in 25 mM HEPES, pH 7.4, containing 1 mM EDTA, 2 mM MgC12, 100 mM NaC1, 1 mM dithiothreitol, and 0.25% sodium cholate.G,a and C,-Gta (1 p M ) was added to a solution containing purified '