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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1496-1499
doi: 10.1161/ATVBAHA.106.136226
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1496.)
© 2007 American Heart Association, Inc.

Editorials

Posttranslational Protein Palmitoylation

Promoting Platelet Purpose

Adam D. Munday; José A. López

From the Puget Sound Blood Center and Division of Hematology, Department of Medicine, University of Washington, Seattle.

Correspondence to José A. López, Puget Sound Blood Center, Research Division, 921 Terry Avenue, Seattle, WA 98104-1256. E-mail josel{at}psbc.org

Posttranslational modification of proteins is the foundation of intracellular signaling. Without the ability to reversibly modify proteins and lipids, cells would be unable to react to signals received from their environment. Posttranslational modification of proteins usually, but not always, occurs after a protein has arrived at the appropriate subcellular location. In certain instances, however, such modifications serve as addresses to correctly target the protein within the cell.

See page 1478

Phosphorylation is the most widely studied of the posttranslational modifications. Both proteins and lipids can be reversibly phosphorylated, with the modification thereby being able to act as an on/off switch for the propagation of intracellular signals. Proteins can also be modified by attachment of lipid moieties (lipidation), modifications which have in recent years gained attention for their significant roles in propagating intracellular signals.

Lipid modifications of proteins fall into several categories, of which 4 are predominant: (1) N-myristoylation, where myristate (C14) is cotranslationally attached to proteins at N-terminal glycine residues1–3; (2) Addition of glycosyl phosphatidylinositol (GPI) anchors, where GPI is attached to the C termini of proteins4; (3) S-prenylation, where a farnesyl (C15) or geranylgeranyl (C20) moiety is attached to a C-terminal cysteine within a CAAX motif (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid)5; and (4) S-acylation (also known as S-palmitoylation) where palmitate (C16) and, less frequently, other fatty acids are attached to cysteine residues.6,7

Of the known membrane-targeting lipid modifications, palmitoylation is unique in two respects: (1) cysteinyl residues to be palmitoylated are not specified by obvious consensus sequences6 and (2) the process is reversible,8,9 which renders palmitoylation/depalmitoylation capable of regulating cellular functions. Further, although palmitate is the preferred acyl chain transferred to proteins by the palmitoyl acyltransferases (PATs), other acyl chains can also be transferred, perhaps depending on availability and concentrations of the fatty acids.10–12 Acceptable substitutes include stearate (C18), oleate (C18:1), and even arachidonate (C20:4) moieties. Thus, saturated, monounsaturated, and polyunsaturated fatty acids can all be accommodated.

Attachment of lipid moieties can alter the properties and localization of proteins. For example, N-myristoylation and S-prenylation attach weak membrane anchors to proteins that target them to the cytoplasmic leaflet of the plasma membrane, GPI modification targets proteins to the outer leaflet of the plasma membrane, and S-acylation produces a strong membrane anchor that usually does not serve as the primary membrane targeting signal but does in many cases specify the membrane subdomain to which modified proteins are localized (more below).

Palmitoylation can occur on proteins that also undergo other lipid modifications and the effect on membrane targeting is a composite of the effects of the 2 moieties. The Src family tyrosine kinases such as Fyn and Lck are examples of proteins that are modified by both N-myristoylation and palmitoylation with the addition of both lipids being required to target the kinases to the plasma membrane.13–16 Proteins that undergo both S-prenylation and palmitoylation include H-Ras and N-Ras.17

Proteins can also be targeted to membranes primarily through their hydrophobic transmembrane domains, with subsequent palmitoylation serving to localize the proteins to subdomains within the membrane. An example of the latter is CD36, possessing 2 transmembrane domains and using palmitoylation as a means of lipid raft targeting.18 Thus, palmitoylation is rarely the primary modification necessary for membrane targeting but serves as a secondary modification to solidify incorporation into the membrane or to localize the protein within subdomains of the membrane such as lipid rafts.

Lipid rafts are areas of the plasma membrane enriched in cholesterol and glycosphingolipids19,20 that play a variety of roles in cellular homeostasis including cholesterol transport, endocytosis, and signal transduction. Indeed, rafts are proposed to act as platforms for the assembly of signaling complexes; disruption of lipid rafts abrogates cell signaling events.21–23

So how does palmitoylation occur? The reaction involves nucleophilic attack by a thiolate ion (–S–) from the target protein on the ester bond of palmitoyl coenzyme A (CoA). The result is the formation of a new thioester bond as the palmitate is transferred to the protein (see Figure). Protein palmitoylation can occur at the cytoplasmic face of membranes in the secretory pathway (endoplasmic reticulum and Golgi apparatus) and the plasma membrane.24–27 PATs catalyze the reaction. These enzymes belong to a family of proteins containing DHHC (Asp-His-His-Cys) cysteine-rich domains (CRD). PATs catalyze the addition of the palmitate to proteins, while palmitoyl thioesterases serve to depalmitoylate proteins. Two PAT activities have been observed.28 One type of PAT modifies the farnesyl-dependent palmitoylation motif found in H- and N-Ras, and the other type modifies the myristoyl-dependent palmitoylation motif found in the Src family of tyrosine kinases. It is unclear which PAT activity is responsible for palmitoylating transmembrane proteins that do not contain these motifs. To date several mammalian PATs have been identified, including GODZ, a Golgi apparatus-specific protein that plays a role in membrane protein trafficking,29 and Huntingtin-interacting protein 14 (HIP14), which localizes to the Golgi and to cytoplasmic vesicles and shows a preference for the farnesyl-dependent palmitoylation motif found in H- and N-Ras.29,30 Although the known PATs seem to be primarily associated with the Golgi, PAT activity has been detected in plasma membrane fractions.31


Figure 1
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  		Schematic representation of the transfer of the palmitoyl moiety from palmitoyl CoA to the thiolate side chain of the cysteine residue of the target protein.

Three palmitoyl thioesterases have been identified: protein palmitoyl thioesterases (PPT) 1 and 2 and acyl palmitoyl thioesterase 1 (APT1).32–34 PPT1 and PPT2 are lysosomal hydrolases that remove palmitate from proteins being degraded in lysosomes. PPT1 has been shown to depalmitoylate H-Ras and G{alpha} subunits, whereas the action of PPT2 is limited to hydrolysis of acyl-CoA. APT1 is the major thioesterase that depalmitoylates proteins. This enzyme is primarily cytosolic and can translocate to cellular membranes on cell stimulation.

Palmitoylation/depalmitoylation cycles may allow movement of proteins between cellular locations. In some cases, the rapidity of the exchange can enable a protein to shuttle between different subcellular locations over short time scales. For example, N-Ras palmitoylation has a half-life of less than 20 minutes,9,35,36 which may contribute to the observed cycling of Ras between the Golgi and the plasma membrane. In contrast, proteins such as the plasma membrane protein SNAP25, involved in vesicle docking to the plasma membrane, are stably palmitoylated37 and exhibit a more restricted cellular localization. This suggests that the palmitoyl thioesterases have differing specificities or access to palmitoylated proteins.

Protein palmitoylation can also be controlled spatially. The PATs act on sets of substrates; a protein to be palmitoylated may be a substrate for multiple PATs or only one. This suggests the possibility of multilevel regulation: first, to be palmitoylated, the target protein must be in the same location as a PAT that can facilitate its palmitoylation. Second, palmitoylation at different residues may affect the final location of a protein. For example, the receptor for {alpha}-amino-3-hydroxy-5-methyl-isoxazolepropanoic acid (AMPA) possesses 2 sites for palmitoylation that are palmitoylated by GODZ and sertoli cell gene with a zinc finger domain ß (SERZ-ß).38 Palmitoylation at one site by GODZ, located in the Golgi apparatus, results in the accumulation of AMPA in the Golgi with concomitant reduction in plasma membrane levels.39

Highlighting both the diversity of palmitoylation and its critical requirement for platelet function is the report by Sim et al40 in this issue of the journal. The report demonstrates that strategies that prevent protein palmitoylation in platelets inhibit both signaling events and platelet aggregation in vitro. To test the role of palmitoylation in platelet function in vivo, the authors infused mice with platelets treated with cerulenin, an inhibitor of protein palmitoylation. Cerulenin-treated platelets displayed a marked defect in their ability to accumulate in thrombi at sites of experimental blood vessel injury. The defect is very likely to be multifactorial, corresponding to defects in adhesion, signaling, and granule release, because each of these functions involves proteins that are palmitoylated, among them the platelet glycoprotein (GP) Ib-IX-V complex, which mediates the first step of platelet adhesion, G protein-{alpha} subunits involved in signaling, and SNARE proteins involved in granule secretion.

The authors also demonstrate, for the first time, that platelets possess at least 2 PATs, GODZ and HIP14, and the thioesterase APT1. Interestingly, whereas HIP14 and APT1 localize respectively to platelet membranes and cytosol, GODZ was found to be primarily cytosolic in the platelet. The latter finding is at odds with published data in other cells31,39 and may represent a platelet-specific localization of GODZ. However, the observed localization may also be a methodological artifact, because GODZ possesses 4 hydrophobic transmembrane domains and would therefore be expected to remain integrated into a membrane. Further work will be required to shed light on this issue. As shown in other cell types, APT1 translocates to the membrane/cytoskeleton on platelet activation. These data serve to emphasize the complexity of palmitate turnover and its effect on protein localization.

Platelet adhesion to an area of vascular injury is dependent on the interaction between the platelet receptor GP Ib-IX-V and matrix protein von Willebrand factor (vWF). This interaction is responsible for tethering the platelet to the site of injury and initiating intercellular signals. The result is the activation of the integrins {alpha}IIbß3 and {alpha}2ß1, which allow the platelets to adhere firmly to the subendothelial matrix. The GP Ibß subunit of the GPIb-IX-V complex is palmitoylated,41 and the GPIX subunit has been shown to be palmitoylated41 or myristoylated.42 Palmitoylation is required for raft association of the receptor complex.43 Indeed, raft localization is critical for receptor function. Cholesterol depletion from platelets eliminates GPIb-IX-V raft association. Concomitantly, the ability of platelets to adhere to vWF under flow conditions is drastically reduced, whereas the ability of the receptor to bind vWF under static conditions is preserved. These observations may in part explain the in vivo data presented by Sim et al.40 Decreased GPIb-IX-V palmitoylation reduces its raft localization and hence the ability of platelets to adhere to sites of vascular injury.

The article by Sim et al provides a platform for further study and characterization of the cellular effects of protein acylation. The results illustrate the intriguing observation that APT1 translocates to the membrane/cytoskeleton on activation, thereby placing it in proximity to its substrates. Does this then represent a possible mechanism for depalmitoylation of proteins and downregulation of platelet activation? Answers could be found by examining the raft localization of proteins in concert with palmitate turnover after cerulenin treatment. In addition, this article suggests the tantalizing possibility of being able to pharmacologically compare physiological responses to PAT inhibition to those derived with thioesterase inhibition.


*    Acknowledgments
 
Disclosures

None.


*    References
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Related Article:

Platelets Possess and Require an Active Protein Palmitoylation Pathway for Agonist-Mediated Activation and In Vivo Thrombus Formation
Derek S. Sim, James R. Dilks, and Robert Flaumenhaft
Arterioscler. Thromb. Vasc. Biol. 2007 27: 1478-1485. [Abstract] [Full Text] [PDF]




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