Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells.

Abstract

The ras gene product (p21) is a GTP-binding protein and is thought to play an important role in signal transduction of growth and differentiation in many types of mammalian cells. The p2FGTP complex is an active conformation, as described previously for polypeptide chain elongation factors (EF-Tu and EF-G) and heterotrimeric GTPbinding proteins (G proteins). In the study reported here, we measured the amounts ofp21-bound guanine nucleotides under various conditions in the G54 cell line, a derivative of Swiss 3T3 cells that overexpresses normal c-Ha-ras. More p21GTP complexes were present in growing cells than in quiescent cells. When quiescent cells were stimulated with fetal bovine serum to promote DNA synthesis, p2l-GTP increased --fold. Among a number of purified growth factors, platelet-derived growth factor enhanced the formation of p2lFGTP, whereas the combination of bombesin and insulin, which also induces DNA synthesis, did not. These results strongly suggest that p21 is a transducer of the growth signal from the platelet-derived growth factor receptor in Swiss 3T3 cells and that the signal is transmitted through a p2FGTP complex.The ras protooncogene product (p21) is a GTP-binding protein involved in the transduction of signals controlling cellular proliferation and differentiation (1). The activity of a GTPbinding protein is regulated by the bound guanine nucleotide, GDP or GTP. The protein-GDP complex is an inactive conformation and nucleotide exchange from GDP to GTP activates the protein. The active protein-GTP complex specifically interacts with an effector molecule, which transduces the signal downstream. Hydrolysis of the bound GTP to GDP and Pi converts the protein-guanine nucleotide complex to an inactive form. Thus, the activity of a GTPbinding protein is regulated at two steps-i.e., the GDP/GTP

Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells

“…We found that the activation state of Rap 1 was *13% in parental HL-60 cells in a serum-starved nonstimulated state which is very similar to the 14% Rap 1B activation reported in serum-starved nonstimulated PC-12 cells transfected with poly-histidinetagged Rap 1B and metabolically labeled with 32 PO 4 (Vossler et al, 1997). This degree of Rap 1 activation is higher than we (Scheele et al, 1995) and others (Satoh et al, 1990) have found for basal activation of wild type non-mutated Ras and may be attributed to the threonine in position 61 of Rap 1 which decreases the intrinsic GTPase activity of Rap 1 10-fold compared to Ras (Kitayama et al, 1989;Hart and Marshall, 1990). We found the activation of endogenous Rap 1 in HL-60 cells to be similar to the activation of transfected Rap 1A in BHK cells; to avoid saturation of endogenous RapGEFs and RapGAPs, the transfected Rap 1A was expressed at relatively low levels in the BHK cells (as determined by immunoblotting (data not shown) and by comparing total nucleotide-bound Rap1 in HL-60 cells to that of transfected Rap 1A in BHK cells (Tables 1 and 2)).…”

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia

“…Assuming that c-Crk is linking insulin-and PDGF-induced signaling pathways to Ras activation, our data demonstrate that insulin and PDGF dependent pathways di er in the requirement for MAP kinase activation. Work from several laboratories has indicated that the level of GTP-bound Ras increases following PDGF stimulation of cells (Gibbs et al, 1990;Satoh et al, 1990;Nanberg and Westermark, 1993;Maruta and Burgess, 1994). In cells expressing wild-type PDGF receptor, PDGF stimulation resulted in an approximate twofold increase in the GTP content of Ras, with only 5% of GTP versus 95% of GDP following immunoprecipitation with anti-Ras antibodies .…”

VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells

“…Details of procedures were described elsewhere (Satoh et al, 1990a). Brie¯y, SE cells were labeled for 6 h with 0.1 mCi/ml 32 P-orthophosphate in phosphate-free DMEM.…”