Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system

Gutiérrez, L.; Magee, Anthony I.

doi:10.1016/0167-4838(91)99003-b

Cited by 74 publications

(42 citation statements)

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“…Other PAT activities have also been found in membrane fractions, e.g. the PAT that palmitoylates viral glycoproteins and H-Ras (31,35). Whether these PAT activities correspond to identical or different enzymatic activities is not known at the present.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

Berthiaume

Resh

1995

Journal of Biological Chemistry

146

103

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Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates "myrGlyCys" containing members of the Src family of protein tyrosine kinases (PTKs Modification of proteins by fatty acids greatly alters their structure, function, and subcellular localization and has recently been shown to be involved in several aspects of cellular signaling (see Refs. 1 and 2 for recent reviews). More than 200 proteins are known to be fatty acylated, including viral and cellular proteins (1-3). Roles for protein fatty acylation range from anchoring proteins to membranes (3, 4) and stabilizing protein-protein interactions (5) to regulating enzymatic activities in mitochondria (6). In general, mutations that prevent fatty acylation abolish or greatly alter the biological function of these proteins.Protein fatty acylation can be divided into two categories: myristoylation and palmitoylation. N-Myristoylation involves the cotranslational attachment of the 14-carbon fatty acid myristate onto an N-terminal glycine residue of a protein via an amide linkage. Due to the high stability of this amide bond, myristoylation is irreversible, with some exceptions (7). The enzymology of N-myristoylation has been well characterized (Ref. 8 and references therein). A methionyl aminopeptidase first removes the initiator methionine. N-Myristoyl transferase (NMT) 1 then catalyzes the transfer of myristate to the glycine residue in position two. This glycine residue is an essential element of the substrate recognition sequence, since its substitution by any other amino acid within a protein prevents myristoylation.Many proteins contain the 16-carbon fatty acid palmitate attached to specific cysteine residues. In contrast to myristoylation, palmitoylation occurs post-translationally and is readily reversible. The enzymology of palmitoylation is not well characterized, and a palmitoyl acyltransferase (PAT) has not yet been isolated. No apparent consensus sequence has been found at the palmitoylation site, suggesting the presence of more than one type of PAT. However, the N termini of 7 of 9 members of the Src family of PTKs and several of the ␣ subunits of the heterotrimeric G proteins contain the sequence myrGlyCys, where Cys-3 is palmitoylated (2, 3). In most of these cases, myristoylation has been shown to be a prerequisite for palmitoylation to occur (9, 10).Many signal-transducing proteins translocate reversibly between plasma membrane and cytosol (1, 2). Several have been shown to be reversibly palmitoylated, such as ␣ subunits of G proteins and nitric oxide synthase. These proteins undergo agonist-stimulated palmitate turnover (11,12), suggesting that dynamic palmitoylation of proteins can regulate signal transduction.Little is known about the...

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Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

Berthiaume

Resh

1995

Journal of Biological Chemistry

146

103

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“…As an alternative, we chose to study porcine brain tubulin that was palmitoylated in a cell-free system (Caron, 1997). In support of this decision, studies of other proteins have shown that sites of palmitoylation in vivo and in vitro are equivalent (Bizzozero et al, 1987;Gutierrez and Magee, 1991;Stanley et al, 1994). Different methodologies for protein structure analysis have been used to identify palmitoylated amino acids (Papac et al, 1992;Weimbs and Stoffel, 1992;Bizzozero et al, 1994a;Hackett et al, 1994 (Ozols, 1984), necessitating the use of gel filtration on Sephadex LH60.…”

Section: Proteinsmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Ozols

Caron

1997

MBoC

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As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3Hlpalmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of a-tubulin. 3H-labeled palmitoylated a-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to Veptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in a-tubulin. The palmitoylated residues in a-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.

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“…Despite the accumulation of evidence showing that the palmitoylation of various proteins, including RAS, is critical for their biologic functions, the enzymes involved in this process are still poorly understood. Several groups have sought to purify PAT enzymes (Gutierrez and Magee, 1991;Liu et al, 1996;Das et al, 1997;Ueno and Suzuki, 1997;Hiol et al, 2003), unfortunately none have provided any examples of bona fide human Pat enzymes.…”

Section: Introductionmentioning

confidence: 99%

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase

et al. 2004

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Protein palmitoyltransferases (PATs) represent an exciting new target for anticancer drug design due to their pivotal roles in the subcellular localization of a number of oncogenes. We show that the Huntingtin interacting protein 14 (HIP14) is a PAT with a preference for the farnesyl-dependent palmitoylation motif found in H-and N-RAS. Characterization of HIP14 in mouse cells has revealed that it has the ability to induce colony formation in cell culture, anchorage-independent growth, and tumors in mice. Activity of the enzyme and its ability to transform cells is dependent on critical residues in the active site of the enzyme.

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Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system

Cited by 74 publications

References 0 publications

Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

Biochemical Characterization of a Palmitoyl Acyltransferase Activity That Palmitoylates Myristoylated Proteins

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase

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