Marilyn D. Resh

Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins

Covalent attachment of myristate and/or palmitate occurs on a wide variety of viral and cellular proteins. This review will highlight the latest advances in our understanding of the enzymology of N-myristoylation and palmitoylation as well as the functional consequences of fatty acylation of key signaling proteins. The role of myristate and palmitate in promoting membrane binding as well as specific membrane targeting will be reviewed, with emphasis on the Src family of tyrosine protein kinases and alpha subunits of heterotrimeric G proteins. The use of myristoyl switches and regulated depalmitoylation as mechanisms for achieving reversible membrane binding and regulated signaling will also be explored.

Inhibition of Protein Palmitoylation, Raft Localization, and T Cell Signaling by 2-Bromopalmitate and Polyunsaturated Fatty Acids

The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

Palmitoylation of Ligands, Receptors, and Intracellular Signaling Molecules

Palmitate, a 16-carbon saturated fatty acid, is attached to more than 100 proteins. Modification of proteins by palmitate has pleiotropic effects on protein function. Palmitoylation can influence membrane binding and membrane targeting of the modified proteins. In particular, many palmitoylated proteins concentrate in lipid rafts, and enrichment in rafts is required for efficient signal transduction. This Review focuses on the multiple effects of palmitoylation on the localization and function of ligands, receptors, and intracellular signaling proteins. Palmitoylation regulates the trafficking and function of transmembrane proteins such as ion channels, neurotransmitter receptors, heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors, and integrins. In addition, immune receptor signaling relies on protein palmitoylation at many levels, including palmitoylated co-receptors, Src family kinases, and adaptor or scaffolding proteins. The localization and signaling capacities of Ras and G proteins are modulated by dynamic protein palmitoylation. Cycles of palmitoylation and depalmitoylation allow H-Ras and G protein α subunits to reversibly bind to and signal from different intracellular cell membranes. Moreover, secreted ligands such as Hedgehog, Wingless, and Spitz use palmitoylation to regulate the extent of long- or short-range signaling. Finally, palmitoylation can alter signaling protein function by direct effects on enzymatic activity and substrate specificity. The identification of the palmitoyl acyltransferases has provided new insights into the biochemistry of this posttranslational process and permitted new substrates to be identified. Many proteins contain covalently attached fatty acids. Two types of fatty acids can be linked to proteins: myristate, a 14-carbon fatty acid, or palmitate, a 16-carbon fatty acid. Some proteins contain both fatty acids. This Review focuses on the various ways that modification of proteins by palmitate regulates protein structure and function. Attachment of palmitate to intracellular signaling proteins can help target these proteins to specific membranes where their action is needed for signal transduction. Palmitoylation is often a dynamic reaction: Removal of the bound palmitate results in dissociation of the depalmitoylated protein from the membrane. Palmitate can also be attached to transmembrane proteins, which helps the modified proteins to traffic through the cell, to become enriched in specialized membrane domains (termed rafts), and to signal effectively. Surprisingly, even secreted proteins can be palmitoylated. In this case, palmitoylation modulates the ability of these proteins to signal close to and far away from the producing cell. Moreover, palmitoylation can regulate enzyme activity as well as protein-protein interactions. Thus, modification of ligands, receptors, and intracellular signaling proteins by palmitate has multiple effects on their localization and signaling functions.

Localization of Human Immunodeficiency Virus Type 1 Gag and Env at the Plasma Membrane by Confocal Imaging

ABSTRACT Budding of lentiviruses occurs at the plasma membrane, but the preceding steps involved in particle assembly are poorly understood. Since the Gag polyprotein mediates virion assembly and budding, studies on the localization of Gag within the cell should provide insight into the mechanism of particle assembly. Here, we utilize biochemical fractionation techniques as well as high-resolution confocal imaging of live cells to demonstrate that Gag is localized at the plasma membrane in a striking punctate pattern. Mutation of the N-terminal myristoylation site results in the formation of large cytosolic complexes, whereas mutation of the N-terminal basic residue cluster in the matrix domain redirects the Gag protein to a region partially overlapping the Golgi apparatus. In addition, we show that Gag and Env colocalize at the plasma membrane and that mistargeting of a mutant Gag to the Golgi apparatus alters the pattern of surface expression of Env.

Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains

ABSTRACT The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges.” Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.

Regulation of cellular signalling by fatty acid acylation and prenylation of signal transduction proteins

Covalent modification by fatty acylation and prenylation occurs on a wide variety of cellular signalling proteins. The enzymes that catalyze attachment of these lipophilic moieties to proteins have recently been identified and characterized. Each lipophilic group confers unique properties to the modified protein, resulting in alterations in protein/protein interactions, membrane binding and targeting, and intracellular signalling. The biochemistry and cell biology of protein myristoylation, farnesylation and geranylgeranylation is reviewed here, with emphasis on the Src family of tyrosine kinases, Ras proteins and G protein coupled signalling systems.

Membrane Targeting of Lipid Modified Signal Transduction Proteins

Covalent attachment of lipophilic moieties to proteins influences interaction with membranes and membrane microdomains, as well as signal transduction. The most common forms of fatty acylation include modification of the N-terminal glycine of proteins by N-myristoylation and/or attachment of palmitate to internal cysteine residues. Protein prenylation involves attachment of farnesyl or geranylgeranyl moieties via thio-ether linkage to cysteine residues at or near the C-terminus. Attachment of each of these lipophilic groups is catalyzed by a distinct enzyme or set of enzymes: N-myristoyl transferase for N-myristoylation, palmitoyl acyl transferases for palmitoylation, and farnesyl or geranylgeranyl transferases for prenylation. The distinct nature of the lipid modification determines the strength of membrane interaction of the modified protein as well as the specificity of membrane targeting. Clusters of basic residues can also synergize with the lipophilic group to promote membrane binding and targeting. The final destination of the modified protein is influenced by multiple factors, including the localization of the modifying enzymes, protein/protein interactions, and the lipid composition of the acceptor membrane. In particular, much interest has been focused on the ability of fatty acylated proteins to preferentially partition into membrane rafts, subdomains of the plasma membrane that are enriched in cholesterol and glycosphingolipids. Lipid raft localization is necessary for efficient signal transduction in a wide variety of systems, including signaling by T and B cell receptors, Ras, and growth factor receptors. However, certain membrane subdomains, such as caveolae, can serve as reservoirs for inactive signaling proteins. Heterogeneity in the types of membrane subdomains, as well as in the types of lipophilic groups that are attached to proteins, provide an additional level of complexity in the regulation of signaling by membrane bound proteins.

Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes.

Differentiation of oligodendrocyte progenitor cells requires activation of the Src family kinase Fyn. The signals that are upstream and downstream of Fyn in oligodendrocytes remain essentially unknown. Here we show that extracellular matrix engagement regulates the morphology of oligodendrocytes and activates Fyn. Infection of primary oligodendrocyte cultures with recombinant adenovirus revealed that expression of Fyn or its downstream target p190RhoGAP induced process extension. This phenotypic change was not observed when kinase-inactive Fyn or GAP-defective p190 mutants were expressed. Because Rho family proteins are regulated by p190, we monitored the effects of introducing dominant-negative (DN) or constitutively activated (CA) versions of Rho, Rac1, or Cdc42 into primary oligodendrocyte cultures. Expression of DN Rho, CA Rac1, or CA Cdc42 induced outgrowth of oligodendrocyte processes, whereas introduction of CA Rho, DN Rac1, or DN cdc42 inhibited oligodendrocyte differentiation, indicating that Rho and Cdc42-Rac1 exert opposing effects on oligodendrocyte differentiation. Direct measurement of Rho family activity revealed that RhoA was downregulated, and Cdc42 and Rac1 were upregulated during differentiation of primary oligodendrocytes. Moreover, inhibition of integrin engagement or of Fyn activation blocked activation of Rac1 and cdc42 as well as myelin basic protein expression. Taken together, these results suggest a linear signal transduction pathway of integrin-Fyn-Rho family GTPases that controls morphologic differentiation of oligodendrocytes.

Hhat Is a Palmitoylacyltransferase with Specificity for N-Palmitoylation of Sonic Hedgehog

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.

Cholesterol Depletion from the Plasma Membrane Triggers Ligand-independent Activation of the Epidermal Growth Factor Receptor

We recently demonstrated that depletion of plasma membrane cholesterol with methyl-β-cyclodextrin (MβCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617–34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MβCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MβCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MβCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MβCD-induced tyrosine phosphorylation of EGFR, indicating that MβCD activation of EGFR is ligand-independent. Moreover, MβCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MβCD-induced phosphorylation of EGFR, SHC, phospholipase C-γ, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MβCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MβCD, in addition to its ability to disrupt membrane rafts.