Dislodgment and Accelerated Degradation of Ras

Abstract

Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.

Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation

“…In accordance with several studies in other cell systems (Haklai et al, 1998;Kim et al, 1997;Nagase et al, 1996), treatment of the IBEC with Rasinhibitors inhibited FGF-2 induced cell proliferation. Our data suggest an obligatory signaling cascade to IBEC proliferation that starts with FGFR-1 activation, phosphorylation of the adaptor proteins Shc and FRS2 and subsequent activation of Ras followed by the linear Raf1/MEK/MAPK pathway.…”

“…To determine if FGFR-1 signaling in cells cultured on ®bronectin or collagen was dependent on monomeric GTP-binding proteins, we utilized two di erent inhibitors; the farnesylation inhibitor manumycin (Hara et al, 1993) and the Ras-dislodging antagonist FTS (Haklai et al, 1998). These inhibitors are known to a ect Ras function since membrane association is a prerequisite for Ras activity (Howe et al, 1992;Leevers and Marshall, 1992); the e ect of manumycin on geranylgeranylated Ras-like proteins such as Rac and RhoA and C remain to be determined (Cox and Der, 1997).…”

“…On the next day, medium was changed to Ham's F12 medium containing 2% FBS with either vehicle (DMSO) or inhibitor (Ras inhibitors manumycin (a kind gift of Dr Mitsunobu Hara, Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd, Tokyo, Japan; ®nal concentration 1 mM) (Hara et al, 1993) or S-trans, trans-farnesylthiosalicylic acid (FTS; 50 mM ®nal concentration) (Haklai et al, 1998). Twenty minutes later, 1 ng/ml FGF-2 was added as indicated and the culture was continued for 3 days.…”

Farnesylthiosalicylic acid induces caspase activation and apoptosis in glioblastoma cells

“…33 In addition, FTA seems to affect mainly Ras proteins in their activated state. 24 This specific action of FTA is claimed to be responsible for the lack of cytotoxicity in vivo.…”

“…34 Similarly, FTA did not affect blood counts (erythrocytes, leukocytes, or platelets), creatine, liver enzymes, or creatine kinase (all isoforms). 33 In another study, FTA inhibited melanoma tumor growth by 90% with no observable (15,30,60 min, 3 and 6 h) for the activation of MAPK and Akt were determined first. EGF was added to the cells for 15 min, followed by FTA for 45 min and MAPK phosphorylation was determine by Western blotting histopathologic toxic effect in lung, liver, brain, kidney, or small intestine.…”