Vol. 3, 461-469, July 1992 Cell Growth & Differentiation 461 Research Capsule Protein Prenylation: Key to Ras Function and Cancer Intervention?’ Roya Khosravi-Far,2 Adrienne D. Cox,2 Kiyoko Kato,3 and Channing J. Der2 La lolla Cancer Research Foundation, La lolla, California 92037 Ihe frequent association of mutated, oncogenic forms of cellular ras proteins with a broad spectrum of human malignancies has prompted intensive investigations into identifying their role in normal cellular physiology and into establishing the contribution of aberrant ras function to human tumorigenesis (1-4). Despite considerable knowledge of the structural and biochemical properties of ras proteins, we remain ignorant of the function of ras proteins. The recent discovery that ras transforming activity is critically dependent on modification (prenylation) by a farnesyl isoprenoid, an essential intermediate in cholesterol biosynthesis, has unveiled potentially important clues to ras function and identified novel prospects for cancer therapy (5-8). In this research capsule, we will summarize our present understanding of the role of protein prenylation in ras biological activity, and we will assess the promises and problems of pharmacological approaches to antagonizing prenylation. Finally, understanding the role of protein prenylation in ras function will provide a foundation for defining the significance of this previously obscure protein modification for the functions of a very diverse and growing number of isoprenylated proteins (9, 10). CXXX-signaled Processing and Membrane Association Are Critical for ras Transforming Activity The three human ras genes (H-, K-, and N-ras) encode four structurally related proteins of 188-189 amino acids whose functions as molecular switches are dictated by interaction with guanine nucleotides (11) (Fig. 1). The normal protein cycles between an active, GTP-com- plexed state, and an inactive, GDP-complexed state. Regulatory factors have been identified which stimulate guanine nucleotide dissociation (GDSs5) to promote ras- GIP formation (12-1 5) or which act as GIPase-activating proteins (ras GAP, NFl) to negatively mediate ras activity (16-21). Oncogenic ras proteins contain single amino acid substitutions at residues 12, 13, or 61 (1-4), are defective in GDP/GTP cycling, persist constitutively in Received 4/22/92. n Our research studies were supported by grants from the NIH (CA42978, CA52072. CA55008, and CA08791), the American Cancer Society (FRA 57817), and the Tobacco Related Disease Research Program (2FT0039(. 2 Present address: Department of Pharmacology. University of North Carolina, Chapel Hill, NC 27599. 3 Present address: Kyushu University, Beppu, Oita, lapan. 4 To whom requests for reprints should be addressed, at Department of Pharmacology, University of North Carolina. Chapel Hill, NC 27599. S The abbreviations used are: GDS, guanine nucleotide dissociation slim- ulator; PDE, phosphodiesterase; HMG, hydroxymethylglutaryl. the active, GTP-bound form, and chronically stimulate an as yet unidentified growth-stimulatory pathway(s) to promote the uncontrolled growth of the tumor cell (22, 23). In addition to guanine nucleotide interactions, the second critical requirement for ras function is association with the inner face of the plasma membrane (6-8). ras proteins lack the conventional transmembrane or hydro- phobic sequences associated with other membrane-as- sociated proteins and are initially synthesized as soluble, cytoplasmic proteins (24). Their membrane association (25) is triggered by a series of closely linked posttransla- tional processing steps that are signaled by the conserved COOH-terminal CXXX motif present in all ras proteins (26-28) (Fig. 2). The first modification is the addition, via a thioether bond to the cysteine residue of the CXXX, of a Cn5 farnesyl isoprenoid moiety (29-31). The farnesyl moiety is a product of mevalonate (Fig. 3), the essential precursor of all cellular isoprenoids, including cholesterol (32). The other modifications to ras proteins are proteo- lytic removal of the three terminal XXX residues (33, 34) and, finally, carboxyl methylation of the now terminal farnesyl-cysteine residue (27, 33) (Fig. 2). The critical contributions of CXXX-signaled processing to ras function have been demonstrated by two comple- mentary lines of experimental evidence. First, COOH- terminal structural mutants of ras proteins that lack either the cysteine or XXX residues of the CXXX motif do not undergo any of the CXXX-signaled processing steps, do not associate with the plasma membrane, and are com- pletely nontransforming (29, 35, 36). Second, inhibitors of ras isoprenylation (e.g., lovastatin) result in the accu- mulation of unprocessed, cytosolic proteins and also prevent ras biological activity (30, 37). Thus the CXXX- signaled modifications are clearly critical in the trafficking of ras proteins to the plasma membrane, and membrane association is apparently essential to trigger ras transformation. Farnesyl Addition Is the Critical Modification for ras Membrane Association and Transforming Activity Although the three linked CXXX-signaled modifications impart a hydrophobic nature to the protein and are critical for ras function, the precise contribution and functional role of each modification are presently not clear. Isoprenylation provides the greatest contribution to increased protein hydrophobicity; the proteolytic re- moval of the XXX residues may then enhance membrane association by allowing a better interaction of the prenyl group either with the lipid bilayer or with a possible membrane receptor(s). Carboxyl methylation may pro- vide further enhancement to the hydrophobicity, and/or alter the conformation or ionic charge, of ras proteins. Whereas farnesylation is apparently a stable addition (31, 38), carboxyl methylation is reversible (27, 39, 40) and may have an additional function in regulating association