Abstract
smg GDP dissociation stimulator (GDS) is a stimulatory GDP/GTP exchange protein for a group of ras p21-like small GTP-binding proteins (G proteins) including c-Ki-ras p21, smg p21A, smg p21B, and rhoA p21. smg GDS converts the GDP-bound inactive form to the GTP-bound active form of each small G protein by stimulating their GDP/GTP exchange reaction in a cell-free system. The point-mutated c-Ki-ras p21 (c-Ki-rasval12 p21) is known to strongly transform NIH/3T3 cells and to markedly stimulate the c-fos promoter/enhancer in this cell line, whereas the normal c-Ki-ras p21 is weak in these activities. In the present study, we examined the effect of smg GDS on these activities to explore its physiological function. Overexpression of both smg GDS and c-Ki-ras p21 strongly transformed NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly transformed the cells. Furthermore, overexpression of both smg GDS and c-Ki-ras p21 markedly stimulated the c-fos promoter/enhancer in NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly stimulated it. These results indicate that smg GDS transforms NIH/3T3 cells and stimulates the c-fos promoter/enhancer in this cell line in cooperation with c-Ki-ras p21.
Highlights
P21. srng GDP dissociation stimulator (GDS) converts the GDP-boundinactive form to the GTP-boundactive form ofeach small G protein by stimulating their GDP/GTP exchange reaction in a Kceillr-afsVr1e11e2 system
Overexpression of both smg GDS and c-Ki-ras p21 strongly transformed NIH/3T3 cells, whereas overexpression of either smg GDS or c-Ki-ras p21 alone weakly transformed the cells. Overexpression of both smg GDS and c-Ki-rasp21 markedly stimulated the cfos promoter/enhancer in NIH/3T3 cellsw, hereas overexpression of either smg GDS or c-Ki-rasp21 alone weakly stimulated it. These results indicate that smg GDS transformsNIH/3T3 cells and stimulates the c-fos promoter/enhancerin this cell line cinooperation with c-Ki-ras p21
3). smg GDS is originally found as a stimulatory GDP/GTP exchange protein for smg p21A and smg p21B [4, 5], but our recent studies have revealed that smg GDS is active on c-Ki-ras p21 andrhoA p21 [6]
Summary
Materials and Chemicals-pSRaneo and pCEV4 expression plasmids were donated from A. Focus-forming Assay-To assess the transforming activity of s m g GDS, c-Ki-ras p21, and c-Ki-rasVs"*p21, pSRa-GDS, pSRa-Ki-ras, or pSRa-Ki-ras'""2 (0.1, 1, or 10 pg) with pSRaneo (20 pg) was transfected into NIH/3T3 cells, seeded 24 h earliera t a density of 1.2 x lo5cells in 100-mm diameter dishes in DMEM supplemented with 10% calf serum [11]. Cells were trypsinized and counted using a hemacytometer (Burker-Turk) to ensure accurate plating At this time, the growth medium of the otherdishes was changed to DMEM supplemented with 0.5% calf serum. Anchorage-independent Growth Assay-To assess the anchorageindependent growth state of NIH/3T3 cell lines in soft agar, 1X 10' cells of each cell line were suspended in 2 ml of 0.3% agarose in DMEM supplemented with 10% calf serum and were overlaid above a layer of 4 mlof 0.5% agarose in the same medium in 60-mm diameter dishes as described [13]. Luciferase and CAT activities derived from the transfected cells were assayed as described [14, 15]
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