Abstract
We have previously shown that both Smg GDP dissociation stimulator (GDS) and mammalian Cdc25 (mCdc25) stimulate the GDP/GTP exchange reaction of Ki-Ras and that Smg GDS is active only on the post-translationally lipid-modified form of Ki-Ras, whereas mCdc25 is active on both the lipid-modified and unmodified forms but is more active on the lipid-modified form. In the present study, we compared more detailed kinetic properties of Smg GDS and mCdc25 by use of the lipid-modified form of Ki-Ras as a common substrate. Both Smg GDS and mCdc25 stimulated the dissociation of GDP from Ki-Ras and formed the stable binary complex with Ki-Ras. In the presence of guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), the stable ternary complex of Smg GDS-GTP gamma S-Ki-Ras was produced, whereas GTP gamma S induced the dissociation of mCdc25 from mCdc25-Ki-Ras complex, yielding GTP gamma S-Ki-Ras. mCdc25 stimulated the dissociation of GDP from both the membrane-bound and soluble forms of Ki-Ras, whereas Smg GDS was far less active on the membrane-bound form than on the soluble form. Moreover, Smg GDS translocated the GTP gamma S-bound form of membrane-bound Ki-Ras to the soluble fraction as the stable ternary complex of Smg GDS-GTP gamma S-Ki-Ras, whereas mCdc25 did not show this activity. These results suggest that Smg GDS and mCdc25 play different roles in the regulation of Ki-Ras.
Highlights
Ki-Ras and that Smg GDS is active only on the post- cysteine residue ispalmitoylated
The GTPasereaction complex, yielding GTP@-Ki-Ras. mCdc25 stimulated tihs eregulated by Ras GAP' and ne~ofibromin[6, 8].The GDP/ dissociationof GDP from boththe membrane-bound andGTP exchange reaction is regulated by GEPs [7].Three GEPs soluble forms of Ki-Ras, whereas Smg GDS was far less have far beefnound for mammali~nRus: mCdc25, &os, active on thmeembrane-boundform than on the soluble and SmgGDS
GTPyS-Ki-Ras, whereas mCdcfl5 did not shotwhis activ- tor for Rus anda downstream molecule for the tyrosine kinase ity. These results suggest that Smg GDS andmCdc25 receptor, Sevenless, in Drosophila melunogaster
Summary
Sf9 cells as previously described [18]. The anti-Ras antibody (RASK4) trifugation in the Absence of Guanine Nucleotides-When the was kindly supplied from Dr H. MCdc was purified as a [3H]GDP-bound form of Ki-Ras, Smg GDS, or mCdc was separately subjected tocontinuous sucrose densitygradient ultracentrifugation in the absence of guanine nucleotides in the centrifugation buffer, Ki-Ras appeared asa single peak at the position with a M , of about 160,000 (Fig.IA). Briefly,the [3HlGDP-bound previously [18].Smg GDS and mCdc appeared at the posior [35S]GTPyS-bound formof Ki-Ras (30 pmol6,-8 x lo3cpdpmol) was tion witha M , of about 60,000 These values are similar to those prepared as described elsewhere [18,21]and incubated with Smg GDS or mCdc25(150 pmol each) for 5 min at 4 "C in a reaction mixture (300 pl) containing 25 mM Tris/Cl, pH 7.5,lO mM MgCl,, 2.5 mM EDTA, 1 mM dithiothreitol, 240 p~ L-a-dimyristoylphosphatidylcholine0,.06% 3-[(3cholamidopropyl)-dimethyl-ammoniol-l-propanesulfonatea,nd 1 p~. Incubation was carried out at 4 "C for 5 min in areaction centrifugation buffer, Ki-Ras appeared at the sameposition as
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
