Purification of Rad-GAP from rat liver. GST-Rad-Sepharose was loaded with [α-³²P]GTP and was incubated with or without GAP preparations as described in Experimental Procedures. The beads were washed, and the bound nucleotides were eluted in 1% SDS and 50 mM EDTA at 65°C for 5 min, were separated by PEI-TLC, and were quantitated on a PhosphorImager (Molecular Dynamics). The data are expressed as the percent conversion of [α-³²P]GTP to [α-³²P]GDP. (A) [α-³²P]GTP-bound GST-Rad was incubated in the absence or presence of 10 μg cytosol for the time between 0 and 20 min. (B) After buffer exchange, the active fractions from Yellow 2 column were subjected to Mono S separation. Elution was carried out with step-and-linear gradient of NaCl between 0 and 1 M. The upper panel shows the salt gradient, and the lower panel shows the Rad GAP activity (closed circle, solid line) and UV absorbance (open circle, dashed line). (C) GAP preparations from each purification step were resolved by 10% SDS/PAGE followed by silver stain. (D) Resolution of the Mono S active fractions by 12% SDS/PAGE and silver stain.

NM23 as the source of Rad-GAP activity and its association with Rad in vivo. (A) Human skeletal muscle extracts were prepared as described in Experimental Procedures and were subjected to two rounds of immunoprecipitation (IP) with an anti-human nm23 antibody. The immunocomplex and the cytosol before and after nm23-depletion were used for the GAP assay as described in Fig. 1. (B) C2C12 myotubules overexpressing Rad were solubilized and subjected to immunoprecipitation for 2 hr with anti-Rad (1:100) and anti-nm23 (1:50) antibodies, then were subjected to SDS/PAGE and immunoblotting with both antibodies. Starting from the left, lanes 1 and 2 represent an IP with anti-Rad antibody; lanes 3 and 4 with anti-nm23 antibody; and lanes 5 and 6 represent straight blotting.

NM23 is a Rad-specific GAP. (A) GST-Rad (1 μg) and GST-nm23 (10 ng), alone or together, were incubated with [α-³²P]GTP at 25°C for 0–10 min. The reaction was quenched with addition of 10-fold cold buffer, and 1 μl was spotted onto a PEI-TLC plate as described above. (B) One microgram of GST-Rad, GST-Ral, GST-Ran, GST-Ras, and GST-Rho were loaded with 3 μCi of [α-³²P]GTP, were washed extensively, and then were incubated in the absence or presence of 0.1 μg of GST-nm23 at 25°C for 5 min. The bound nucleotides were eluted and resolved by TLC. The upper panel shows a representative TLC profile; the lower panel depicts the percent conversion of GTP to GDP quantitated by using a PhosphorImager and represents the average of two separate assays. (C) One microgram of GST-Rad and GST-Gem on GSH-Sepharose beads were loaded with 3 μCi of [α-³²P]GTP, were washed and incubated with or without 1 μg of GST-nm23 at 14°C or 25°C for 5 min. The bound radioactivity was eluted, and nucleotides were detected as described above. (D) Wild-type GST-Rad and GST-S105NRad were incubated with 3 μCi of [α-³²P]GTP in the absence or presence of 50 ng GST-nm23 at 25°C for 5 min. The nucleotides were resolved by TLC. (E) One microgram of GST-Rad was incubated in the DTT-free buffer with 3 μCi of 8-azido-[γ-³²P]GTP at 25°C, in the dark, for 5 min. Samples were UV irradiated with UV at 6 cm for the time indicated. The samples were transferred to DTT-containing buffer, and nucleotides were rebound to GSH-Sepharose beads. After 3 washes, the labeled GST-Rad was incubated with or without 1 μg of GST-nm23 and was washed again, and the proteins were resolved by 12% SDS/PAGE.

Mutations of NM23 impair its GAP activity. (A) Wild-type and nm23(Pro96) (50 ng) were incubated with 3 μCi of [α-³²P]GTP in the absence or presence of 1 μg of GST-Rad at 25°C for 5 min. The reaction was stopped by the addition of cold buffer, and the nucleotides were resolved as above. (B) [α-³²P]GTP-loaded Rad was incubated in the absence or presence of one of the following: 60 ng/1 μl of Mono S fraction 20, 0.1 μg each of wild-type nm23, H118F-nm23, and P96S-nm23. The Rad-bound nucleotides were eluted and resolved by TLC.

NM23 is a functional GEF for Rad, Gem, and Ras. (A) [α-³²P]GDP was prepared as described in Experimental Procedures. GST-Rad (1 μg each) was loaded with this [α-³²P]GDP (3 μCi each) at 37°C for 30 min, followed by incubation in the absence or presence of 1 μg of GST-nm23 and 10 mM ATP at 25°C for 5 min. The bound nucleotides were eluted and resolved by TLC. (B) One microgram each of GST-Rad, GST-Gem, and GST-Ras were loaded with 10 mM nonradioactive GDP at 37°C for 30 min, followed by UV irradiation. The protein was rebound to the GSH-Sepharose beads and was washed 3 times. The GDP-bound Rad was incubated in the presence of 10 μCi [γ-³²P]ATP and 1 μg of GST-nm23 at 25°C for 5 min, then was resolved by 12% SDS/PAGE. (C) One microgram of GST-Rad, GST-Gem, and GST-Ras were incubated with 3 μCi of [α-³²P]GDP prepared as described above at 37°C for 30 min. Samples were UV-irradiated for 30 min and were resolved by 12% SDS/PAGE. (D) GST-Rad-Sepharose was incubated with 10 mM nonradioactive GDP at 37°C for 30 min followed by 3 washes. Aliquots were incubated with 10 μCi [γ-³²P]ATP with or without 0.1 μg of GST-nm23 at 25°C for the time indicated. The beads were washed, and the nucleotides were eluted and resolved by TLC, and the amount of [γ-³²P]GTP was quantitated by using a PhosphorImager.

Rad is a regulator of NM23. (A) GST-nm23 (50 ng) was incubated with 3 μCi each of [α-³²P]GTP, [α-³²P]CTP, [α-³²P]TTP, and [α-³²P]ATP in the absence or presence of 1 μg GST-Rad. After 5 min at 25°C, the reaction was stopped with 10-fold cold buffer, and the nucleotides were resolved by PEI-TLC [mobile phase = 0.1 M LiCl and 0.06 M (NH₄)SO₄] and were quantitated by PhosphorImager analysis, and the percent conversion was calculated as the ratio of NDP/(NDP + NTP) × 100. (B) Wild-type and Pro96 GST-nm23 (0.5 μg) was incubated with 5 μCi of [γ-³²P]ATP in the absence or presence of 1 μg of GST-Rad at 25°C for 5 min. After addition of 2 × SDS/PAGE sample buffer (pH 8.8), the mixture was incubated at 25°C for 10 min, and proteins were resolved by 10% SDS/PAGE.

The in vivo significance of Rad and NM23 interaction. (A) Effects of Rad and nm23 on serum stimulation of thymidine incorporation. K-1735 TK melanoma cells were stimulated with serum as indicated for 16 hr, were pulsed with 2 μCi of [³H]thymidine in 500 μl of sample per well for 1 hr, and were lysed and assayed for incorporation of radioactivity into trichloroacetic acid-precipitated DNA. Data are presented as fold induction by serum relative to the medium control for each cell line. Values are the mean of three independent experiments, each in triplicate; error bars indicate the standard error of the mean. (B) Model of a coordinate, bidirectional, and bimolecular regulation of Rad and NM23. (Left) (i) Rad-bound GTP is hydrolyzed by Rad GTPase activity stimulated by nm23 to Rad-bound GDP in situ. (ii) γ-phosphate of NTPs are transferred to the histidine residue of nm23 (intermediate), and NDPs are formed. The phosphate can be transferred to NDPs to form new NTPs. (iii) The dotted lines denote that GTP may dissociate from Rad and be converted to GDP by nm23, which is then rebound to Rad. (Right) (i) Rad-bound GDP is transphosphorylated to form Rad-bound GTP in situ. (ii) GDP is phosphorylated by nm23 to form GTP at the expense of ATP in solution. (iii) the dotted lines denote that GDP may dissociate from Rad and be converted to GTP by nm23, which is then rebound to Rad. The balance between GTP⋅Rad and GDP⋅Rad may be maintained through interactions with nm23 and other yet-determined molecules.