Post-translational Palmitoylation Research Articles

GTP-binding-protein-coupled receptor kinases--two mechanistic models.

Six vertebrate protein kinases (G-protein-coupled receptor kinases; GRKs) that regulate the function of G-protein-coupled receptors (GPCRs) were recently cloned; several distinct properties set them apart from conventional second-messenger regulated protein kinases. It appears that GRKs bind GPCR* through two separate sites: a high-affinity site, which involves intracellular loops of the activated receptor, and the lower-affinity site, encompassing the phosphorylation region. The high-affinity interaction may involve complementary structural elements of GRKs and GPCRs* rather than precise amino acid alignment, thus allowing broad and overlapping specificities of these kinases, in spite of differences in the sequences of GPCRs. In addition, GRK structures are modified by several posttranslational modifications, including phosphorylation, autophosphorylation, prenylation, carboxymethylation, and palmitoylation, probably affecting properties of these enzymes. While GRKs phosphorylate and inactivate receptor molecules which are engaged in G-protein activation, controversy surrounds whether GRKs might be activated and phosphorylate unstimulated GPCRs, leading to a desensitization of a larger population of the receptors. In this review, mechanistic aspects of GPCR* phosphorylation related to the distinct properties, regulation and modes of action of GRKs are described.

Fatty Acylation of Synaptotagmin in PC12 Cells and Synaptosomes

Synaptotagmin I is localized to synaptic vesicles where it functions in the calcium-triggered release of neurotransmitters. Here we demonstrate that synaptotagmin I covalently incorporated [3H]palmitate after metabolic labeling of PC-12 cells and rat brain synaptosomes. Labeling was localized to a tryptic fragment that contains a cluster of cysteine residues adjacent to the molecule's single transmembrane anchor. Neutral hydroxylamine released the [3H]palmitate from this fragment and increased its electrophoretic mobility, demonstrating that acylation occurs at the membrane-proximal cysteine cluster. In addition, hydroxylamine-induced mobility shifts were also apparent for synaptotagmins II and III, suggesting that posttranslational palmitoylation via thioester bonds may be a general modification of all synaptotagmins.

Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation

Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.

Biosynthesis and Palmitoylation of Endothelial Nitric Oxide Synthase: Mutagenesis of Palmitoylation Sites, Cysteines-15 and/or -26, Argues against Depalmitoylation-Induced Translocation of the Enzyme

The presence of myristoylated endothelial nitric oxide synthase (eNOS) in cytosolic fractions of bovine aortic endothelial cells (BAEC) suggests that N-myristoylation of eNOS is not sufficient for membrane localization. Therefore, we examined if posttranslational palmitoylation was another molecular signal for the membrane attachment of eNOS. Metabolic labeling showed incorporation of [3H]palmitic acid into the membrane-bound, but not the cytosolic, form of eNOS. Fatty acid analysis demonstrated the labeled fatty acid incorporated into eNOS was palmitate, linked via a hydroxylamine-labile thioester bond. Biosynthesis and turnover studies show that the turnover of palmitate is much faster than the protein itself. However, the rates of palmitoylation and depalmitoylation were not affected by bradykinin or ionomycin treatment of BAEC. To examine the contribution of palmitoylation to the membrane association of eNOS, we mutated cysteine-15 and -26. Both mutations markedly diminished palmitoylation of eNOS, but did not significantly alter its membrane association. Additionally, [3H]palmitic acid was not incorporated into nonmyristoylated mutant eNOS (G2A eNOS), suggesting that myristoylation is necessary for subsequent palmitoylation of the enzyme. Taken together, our data imply that palmitoylation does not play a major role in membrane association of eNOS and other amino acid sequences, in conjunction with N-myristoylation, are necessary and sufficient for membrane association of the enzyme.

Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin.

The alpha subunit of the heterotrimeric Gs protein that couples the beta-adrenergic receptor to adenylyl cyclase undergoes post-translational palmitoylation. We examined the dynamics of this modification of alpha s by metabolic labeling of COS and S49 lymphoma cells under different conditions. The endogenous alpha s proteins were immunoprecipitated with a peptide-specific antibody, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by fluorography and densitometry. A pulse-chase study of COS cells incubated with [3H]palmitate or [35S]methionine showed that for alpha s the palmitate turnover (t1/2 approximately 50 min) was significantly faster than the protein degradation. Treatment of cells with 10 microM isoproterenol, a beta-adrenergic receptor agonist, in the presence of [3H]palmitate led to a rapid 4-10-fold increase in the palmitoylation of alpha s. This increase in palmitoylation was concentration-dependent (EC50 approximately 0.9 microM) and blocked by the antagonist propranolol. The mutant alpha s proteins in the unc and H21a S49 cell lines did not show an increase in [3H]palmitate incorporation with isoproterenol treatment. Cholera toxin treatment of COS cells increased the [3H]palmitate incorporation into the alpha s subunits. These data indicate that palmitoylation of the alpha s subunit is dynamic and regulated by activation of the alpha s subunit.

G protein—coupled receptor kinases

G protein‐coupled receptor kinases (GRKs) constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist-bound, or activated, G protein‐coupled receptors (GPCRs) as their primary substrates. GRK-mediated receptor phosphorylation rapidly initiates profound impairment of receptor signaling, or desensitization. This review focuses on the regulation of GRK activity by a variety of allosteric and other factors: agonist-stimulated GPCRs, fl∞ subunits of heterotrimeric GTP-binding proteins, phospholipid cofactors, the calcium-binding proteins calmodulin and recoverin, posttranslational isoprenylation and palmitoylation, autophosphorylation, and protein kinase C‐mediated GRK phosphorylation. Studies employing recombinant, purified proteins, cell culture, and transgenic animal models attest to the general importance of GRKs in regulating a vast array of GPCRs both in vitro and in vivo.

Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.

The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.

The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.

Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain.

Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.

Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein.

The 32-kDa herbicide-binding protein, a component of photosystem II, is synthesized as a membrane-associated 33.5-kDa precursor within the chloroplast. We show that membrane attachment of the precursor and processing to the 32-kDa form occur in the unstacked stromal lamellae. Once processed, the 32-kDa protein translocates, within the thylakoids, to the topologically distinct stacked granal lamellae. Posttranslational palmitoylation of the processed 32-kDa protein is also shown to occur. This modification takes place in a membrane-protected domain and is mainly confined to the protein assembled in the granal lamellae, where functional photosystem II centers are concentrated.