Direct Demonstration of Geranylgeranylation and Farnesylation of Ki-Ras in Vivo

Abstract

It has recently been reported that Ki-Ras protein can be modified in vitro by farnesylation or geranylgeranylation. However, a previous analysis of Ki-Ras prenylation in vivo found only farnesylated Ki-Ras. In this report it is shown that under normal conditions, Ki-Ras is farnesylated in vivo and when cells are treated with the farnesyl transferase inhibitors B956 or B957, farnesylation is inhibited and Ki-Ras becomes geranylgeranylated in a dose dependent manner. These results have strong implications in the design of anticancer drugs based on inhibition of prenylation.Post-translational modifications of small molecular weight GTP-binding proteins have been the subject of intense studies since the first reports of prenylation appeared (1-3). Many of these proteins, and specifically the Ras proteins, are involved in tumorigenesis. Since prenylation is necessary for the transformation potential of Ras proteins, a number of groups have been dedicated to the design of inhibitors of prenylation as a strategy to inhibit tumor formation (4).Two types of prenyl modifications have been described: farnesylation and geranylgeranylation. The enzymes FTase 1 and GGTase I recognize a tetra peptide sequence at the C terminus of a protein (5, 6). This sequence has been designated the CAAX box, and it determines whether a protein will be prenylated (7). In this sequence, C stands for cysteine and A for any amino acid. The amino acid X specifies whether the protein will be farnesylated (methionine or serine) or geranylgeranylated (leucine or phenylalanine) by the appropriate enzyme. This rule is followed in the majority of cases. However, recent reports have shown that Ki-Ras, whose C terminus CVIM predicts farnesylation, can also be geranylgeranylated in vitro, (8) and that its prenylation in cells is inhibited by a GGTase I inhibitor (9). Since activated Ki-Ras is the type of Ras most frequently found in human cancers, it is very important to determine how it is modified in vivo and whether this modification can be inhibited. Cell labeling with [ 3 H]mevalonate in the presence of inhibitors of hydroxymethylglutaryl-CoA reductase such as lovastatin allows the radioactive labeling of cholesterol and its intermediate isoprenoids. However, a major drawback of this approach is the low level of [ 3 H]mevalonate incorporation into cells. The fortuitous identification of the gene for a mevalonate transport membrane protein (pMev) has provided a tool that makes the identification of isoprenoid and non-steroidal isoprenoid intermediates easier (10, 11). Transfection of cells expressing either Ha-Ras or Ki-Ras oncogenes with the mevalonate transporter and growth of the cells under conditions in which the endogenous pools of farnesyl diphosphate and geranylgeranyl diphosphate are radioactively labeled enabled us to demonstrate that both Ha-Ras and Ki-Ras are farnesylated under normal conditions. In the presence of the farnesyl transferase inhibitor B956, farnesylation of both Ha-Ras and Ki-Ras was inhibited in a dose-dependent manner. Ho...

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts

“…We have subsequently designed an inhibitor to the closely related enzyme geranylgeranyltransferase I (GGTase I), which transfers a C 20 geranylgeranyl group to the cysteine of CAAX sequences that have a leucine at the carboxyl terminus, and shown that K B -Ras processing and oncogenic signaling was sensitive to this inhibitor (Lerner et al, 1995b). This was consistent with the work of which showed that K B -Ras can be both farnesylated and geranylgeranylated in vitro as well as with the more recent work of Whyte et al (1997) and Rowell et al (1997) in intact human tumor cell lines. The above studies, however, were carried out in cells transfected with an exogenous K B -Ras oncogene and whether or not inhibition of the processing of K-Ras in human tumors requires both FTase and GGTase I inhibitors has not been investigated.…”

“…We have therefore made GGTase I inhibitors and used these compounds to give further support for this notion (Lerner et al, 1995b and Figure 1). More recently, K-and N-Ras have been shown to be geranylgeranylated in cells treated with FTase inhibitors (Whyte et al, 1997;Rowell et al, 1997). Although the e ects of FTase inhibitors on normal and malignant cells have been well studied, only recently have the e ects of GGTase I inhibitors been investigated.…”

“…Each data point is an average of eight tumors. Statistical signi®cance between control and treated groups were evaluated by using Student's t-test (P50.05) FTI-276 and GGTI-297 antitumor activity and K-Ras prenylation J Sun et al Whyte et al, 1997;Rowell et al, 1997). In A-549 cells, co-treatment with FTI-277 and GGTI-298 did not inhibit K B -Ras prenylation.…”

Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line

“…Recent studies have shown that while K-Ras and N-Ras can be geranylgeranylated in the absence of farnesylation, H-Ras cannot (Whyte et al, 1997;Rowell et al, 1997). Thus, H-Ras is only activated in the presence of FOL and not in the presence of GGOL, making it a strong candidate for mediating FOL-induced spreading of lovastatin-treated TRAMP cells.…”

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter

“…For example, there was no correlation between the Ras mutation status and reversal of transformation as some cancer cells that do not express oncogenic Ras were sensitive to FTI, and inversely, some cancer cells containing activated Ras were resistant to FTI (Cox and Der, 1997;Gibbs and Oliff, 1997;Sebti and Hamilton, 2000;Sebti and Der, 2003). In addition, in vitro and in vivo experiments have shown that K-Ras was geranylgeranylated in cells treated with FTIs (Rowell et al, 1997;Whyte et al, 1997;Sun et al, 1998). Thus, both FTase and GGTase I need to be inhibited to fully block K-Ras isoprenylation (Lerner et al, 1997).…”