Evidence for a High Affinity, Saturable, Prenylation-dependent p21 Binding Site in Plasma Membranes

Abstract

Oncogenic p21ras proteins can only exert their stimulation of cellular proliferation when plasma membraneassociated. This membrane association has an absolute requirement for post-translational modification with isoprenoids. The mechanism by which isoprenoids participate in the specific association of p21 ras with plasma membranes is the subject of this report. We present in vitro evidence for a plasma membrane binding protein for p21ras that can recognize the isoprenoid substituent and, therefore, may facilitate the localization of p21 ras .Post-translational modification with isoprenoids results in considerable structural diversity at the carboxyl terminus of many proteins. Four such carboxyl-terminal structural motifs have been identified (for review see Ref. 1). These are farnesylated-methylated cysteine, geranylgeranylated-methylated cysteine, digeranylgeranylated vicinal cysteines of the -CC rab proteins, and digeranylgeranylated residue-interrupted cysteines of the -CXC rab proteins. The carboxyl-terminal cysteine of the -CXC rabs is methylated, whereas the carboxyl-terminal cysteine of the -CC rabs is not. Since these modifications always increase the hydrophobic character of the substituted proteins, it is generally assumed that they are involved in the association of proteins so modified with lipid bilayer membranes. This assumption is supported by the observation that most prenylated proteins are bound to cellular membranes, at least under some physiological conditions. A more recent hypothesis for the function of protein prenylation that is more in keeping with the observed structural diversity and membrane specificity is that these lipid modifications also serve to mediate protein-protein interactions (2). A well studied example of this function for protein prenylation is the heterodimeric association of rab proteins with GDP dissociation inhibitor molecules to form soluble complexes that appear to be dependent on the digeranylgeranylation of the rab proteins (3). Another recent example from our laboratory is the mechanism of endoproteolytic cleavage of the farnesylated prelamin A molecule, which is mediated by an enzyme that possesses a specific farnesyl binding site (4).It has been noted that it is possible that even membraneassociated prenylated proteins may utilize a polyisoprenoid-dependent protein-protein interaction for membrane binding (2). Recognition of the polyisoprenoid and other structural elements of the protein by membrane receptors could confer the necessary localization of particular prenylated proteins to particular subcellular compartments. Sequestration of the polyisoprenoid would be important in preventing nonspecific association of the protein with inappropriate membranes.Farnesylated proteins are found in a number of cellular compartments (1) including plasma membrane (p21 ras , ␥-transducin), peroxisomes (PxF) and nuclei (lamin B, prelamin A). For the naturally occurring forms of N, H, and K p21 ras , it is clear that plasma membrane localization shows an absolute requirement...

Structure and function of the C-terminal hypervariable region of K-Ras4B in plasma membrane targetting and transformation

“…This does not exclude the possibility, however, that a speci®c sequence (and/or a speci®c lipid) might target proteins to particular subdomains or docking proteins within the plasma membrane, as has been suggested by several authors (Niv et al, 1999;Roy et al, 1999;Siddiqui et al, 1998) Detailed studies of the pathways involved in posttranslational modi®cations of Ras proteins have shown that, initially, Ras is farnesylated in the cytosol and binds to the ER membrane. After cleavage of the terminal three amino acids of the CAAX box the farnesylated cysteine is methylated.…”

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice

“…Both wild-type (wt) H-Ras and activated H-Ras were found to be localized in such membrane domains, although ®broblasts transformed by activated H-Ras appear to have lost their caveolae (Koleske et al, 1995). FTS may exert its action by interfering with the interaction of Ras with speci®c lipids and proteins present in caveolae or in analogous membrane domains (Lisanti et al, 1994;Simons and Ikonen, 1997), or by interfering with the interaction of Ras with a recently described Ras-binding protein (Siddiqui et al, 1998). Distinction between these and other mechanisms should become possible given the progress in our understanding of the mode of interactions of Ras and of other proteins with membrane domains.…”

“…It inhibits the growth of Ras-transformed and ErbB2-transformed fibroblasts, but not of v-Raf-transformed cells . Its mechanism of action is likely to be associated primarily with the dislodgment of the mature protein from membrane domains that interact with Ras (Siddiqui et al, 1998) and with the subsequent accelerated degradation of the dislodged Ras proteins . These e ects of FTS are manifested by a decrease in the amount of cellular Ras accompanied by interruption of the Ras-dependent Raf-1-extracellular signal-regulated kinase (ERK) signaling cascade (Gana-Weisz et al, 1997).…”

Regulation of Methylbenzoate Emission after Pollination in Snapdragon and Petunia Flowers

“…The involvement of post-translational modifications in the regulation of enzyme activities has been shown for several other plant enzymes (Khayat et al, 1993;Chang and Gallie, 1997;Rudrabhatla and Rajasekharan, 2002;Smith et al, 2002). Protein modifications, such as phosphorylation, methylation, carboxylation, glucosylation, acetylation, and prenylation, often contribute to the regulation of enzyme activities (Lillo et al, 1997;Siddiqui et al, 1998;Tanase et al, 2002). Future experiments involving sequencing of the post-translationally modified protein will show the nature of the modification that occurs for BAMT.…”