Patrick J. Casey

Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides

We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified approximately 60,000-fold from rat brain cytosol through use of a chromatography step based on the enzymes ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced approximately 50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.

Evidence that direct binding of Gβγ to the GIRK1 G protein-gated inwardly rectifying K+ channel is important for channel activation

Abstract Activation of G protein-gated K + channels by G protein-coupled receptors contributes to parasympathetic regulation of heart rate in the atrium and inhibitory postsynaptic potentials in the peripheral and central nervous system. Having found that G βγ activates the cloned GIRK1 channel, we now report evidence for direct binding of G βγ to both the N-terminal hydrophilic domain and amino acids 273–462 of the C-terminal domain of GIRK1. These direct interactions are physiologically important because synthetic peptides derived from either domain reduce the G βγ binding as well as the G βγ activation of the channel. Moreover, the N-terminal domain may also bind trimeric G αβγ , raising the possibility that physical association of G protein-coupled receptors, G proteins, and K + channels partially accounts for their compartmentalization and hence rapid and specific channel activation by receptors.

G proteins control diverse pathways of transmembrane signaling.

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interact directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide‐binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellularly oriented receptors to membrane‐bound effectors. Traffic in these pathways is regulated by a GTP (on)‐GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.— Freissmuth, M.; Casey, P. J.; Gilman, A. G. G proteins control diverse pathways of transmembrane signaling. FASEB J. 3: 2125‐2131; 1989.

Protein farnesyltransferase and geranylgeranyltransferase share a common α subunit

Abstract Mammalian farnesyltransferase, which attaches a 15 carbon isoprenoid, farnesyl, to a cysteine in p21 ras proteins, contains two subunits, α and β. The β subunit is known to bind p21 ras proteins. We show here that the α subunit is shared with another prenyltransferase that attaches 20 carbon geranylgeranyl to Ras-related proteins. Farnesyltransferase and geranylgeranyltransferase have similar molecular weights on gel filtration, but are separated by ion exchange chromatography. Both enzymes are precipitated and immunoblotted by multiple antibodies directed against the a subunit of farnesyltransferase. The two transferases have different specificities for the protein acceptor; farnesyltransferase prefers methionine or serine at the COOH-terminus and geranylgeranyltransferase prefers leucine. The current data indicate that both prenyltransferases are heterodimers that share a common a subunit with different β subunits.

Post-prenylation-processing enzymes as new targets in oncogenesis

RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps — particularly that of ICMT-catalysed methylation — might provide a better approach to the control of cancer-cell proliferation.

Farnesyltransferase Inhibitors Alter the Prenylation and Growth-stimulating Function of RhoB

Protein farnesyltransferase inhibitors (FTIs) inhibit Ras transformation and Ras-dependent tumor cell growth, but the biological mechanisms underlying these activities is unclear. In previous work, we presented support for the hypothesis that the anti-transforming effects of FTIs depend upon alterations in the function of RhoB, a member of the Rho family of proteins that regulate cytoskeletal actin, cell adhesion, and cell growth. A significant question that needed to be addressed was whether FTIs could directly alter the prenylation as well as the function of RhoB in cells. This issue is complex because farnesylated and geranylgeranylated forms of RhoB (RhoB-F and RhoB-GG) both exist in cells. Here, we show that RhoB farnesylation in vitro can be catalyzed by protein farnesyltransferase and that the peptidomimetic FTIl-739,749 inhibits the farnesylation of RhoB both in vitro and in intact cells. In drug-treated cells, the level of RhoB-GG increased in parallel with the decrease in RhoB-F. In addition to altering RhoB prenylation, l-739,749 suppressed RhoB-dependent cell growth. Taken together, the results suggest that the inhibitory effects of FTIs on RhoB function can be mediated by a relative loss of RhoB-F, a gain of RhoB-GG, or both. Our findings strengthen the causal link between RhoB inhibition and the anti-transforming effects of FTIs and indicate that differently prenylated forms of RhoB may have unique functions.

Lipid modifications of G proteins

Covalent attachment of lipids is a near-universal mechanism through which eukaryotic cells direct and, in some cases, control membrane localization of G proteins. Studies conducted over the past year have substantially advanced our understanding of both the molecular mechanisms and the functional consequences of these modifications. Of particular note are the processes of palmitoylation of the alpha-subunits of heterotrimeric G proteins, and prenylation of members of the Ras superfamily of monomeric G proteins, where recent findings point to unexpected roles for lipid modifications in signaling through these proteins.

Disruption of the Mouse Rce1 Gene Results in Defective Ras Processing and Mislocalization of Ras within Cells

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1in mammalian Ras processing, we generated and analyzedRce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling ofRce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes fromRce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Gγ1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate thatRce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.

Site-specific analysis of protein S-acylation by resin-assisted capture

Protein S-acylation is a major posttranslational modification whereby a cysteine thiol is converted to a thioester. A prototype is S-palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modification is a well-recognized determinant of protein function and localization, current techniques to study cellular S-acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S-nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S-acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S-acylation. Acyl-RAC should therefore find general applicability in studies of both global and individual protein S-acylation in mammalian cells.

Cloning and Characterization of a Mammalian Prenyl Protein-specific Protease

Proteins containing C-terminal “CAAX” sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Gγ1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.