Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase.

Abstract

We tested 42 tetrapeptides for their ability to bind to the rat brain p2l' protein farnesyltransferase as estimated by their ability to compete with p2lH"-in a farnesyltransfer assay. Peptides with the highest affinity had the structure Cys-Al-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is-occupied by a COOHterminal methionine, serine, or phenylalanine. Charged residues reduced affinity slightly at the Al position and much more drastically at the A2 and X positions. Effective inhibitors included tetrapeptides corresponding to the COOH termini of all animal cell proteins known to be farnesylated. In contrast, the tetrapeptide Cys-Ala-Ile-Leu (CARL), which corresponds to the COOH termini of several neural guanine nucleotide binding (G) protein y subunits, did not compete in the farnesyl-transfer assay. Inasmuch as several of these proteins are geranylgeranylated, the data suggest that the two isoprenes (farnesyl and geranylgeranyl) are transferred by different enzymes. A biotinylated heptapeptide corresponding to the COOH terminus of p21K'-1B was farnesylated, suggesting that at least some of the peptides serve as substrates for the transferase. The data are consistent with a model in which a hydrophobic pocket in the protein farnesyltranferase recognizes tetrapeptides through interactions with the cysteine and the last two amino acids.A farnesyl residue is attached in thioether linkage to the COOH-terminal cysteine of a variety of intracellular membrane-associated proteins. The list includes cellular p21lS proteins (1), nuclear lamin B (2), and the 'y subunit of bovine transducin (3). This modification is also found on mating factors secreted by fungi (4,5). In each case the farnesylated cysteine is initially the fourth residue from the COOH terminus (for review, see ref. 6). Farnesylation is followed by proteolytic removal of the three terminal residues and carboxylmethylation of the cysteine. These reactions render the COOH termini hydrophobic, presumably facilitating the initial attachment of the proteins to cell membranes. In some instances the hydrophobicity is increased by palmitoylation of nearby cysteines. Inspection of the sequences of the known farnesylated proteins has defined a weak consensus sequence for farnesylation that consists of Cys-A1-A2-X, where positions Al and A2 are occupied by aliphatic amino acids and position X is occupied by an undefined amino acid (7, 8).The likely donor of farnesyl residues is farnesyl pyrophosphate (FPP), an intermediate in the synthesis of sterols and polyisoprenes in eukaryotic cells (9). Recently, we have isolated from rat brain an enzyme that transfers a farnesyl group from [3H]FPP to p2lHa-as protein (10,11). The purified enzyme preparation contains two proteins, each with an apparent molecular mass ofabout 50 kDa, that appear to form a heterodimer (10, 11). The enzyme recognizes sequences as short as four amino acids provided that cysteine is at the fourth position from the COOH terminus. Recognition was demonst...

LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

“…Commonly, proteins such as LKB1, were X is a glutamine residue, are farnesylated [15]. In general, A " and A # are aliphatic amino acids ; however, peptide studies have shown that most residues are tolerated in the A " position [31]. Other proteins, like LKB1, which have a basic residue in the A1 position and are known to be prenylated, include hepatitis delta virus large antigen (CRPQ) [32] and RhoB (CKVL) [33].…”

PostprenylationCAAXProcessing Is Required for Proper Localization of Ras but Not Rho GTPases

“…Nascent CAAX proteins are synthesized in the cytosol where they encounter one of two protein prenyltransferases that modify the CAAX cysteine by thioether linkage of a C-15 farnesyl or C-20 geranylgeranyl lipid (Clarke, 1992;Casey and Seabra, 1996). The substrate specificity for farnesyltransferase versus geranylgeranyltransferase type I (GGTase I) is determined by the residue in the X position of the CAAX motif: S or M specifies farnesylation, whereas L specifies geranylgeranylation (Moores et al, 1991;Reiss et al, 1991). Although Ras family proteins are farnesylated, most Rho family proteins are geranylgeranylated.…”

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: Implications in prevention of apoptosis

“…Ras prenylation is thought to facilitate membrane targeting and to be essential for Ras function (Kato et al, 1992). In addition, this modi®cation can have important consequences for protein-protein interactions (Glomset and Farnsworth, 1994;Reis et al, 1991;James et al, 1995). Prenylated H-Ras and NRas proteins can be further lipidated by palmitoylation.…”