Absence Of Palmitoylation Research Articles

Phosphorylation and Palmitoylation Dynamically Regulate Sodium Hydrogen Exchanger Isoform 1 (NHE1) Activity: Implications in Health and Disease

The SLC9 family is made up of nine isoforms of sodium hydrogen exchangers (NHE) which regulate pH by exchanging an intracellular proton for an extracellular sodium ion. Isoform 1 (NHE1) is ubiquitously expressed and plays a vital role in multiple cellular processes in addition to regulating pH such as cellular migration and proliferation, control of cell volume, and stress fiber formation. Additionally, NHE1 is a scaffolding protein that organizes protein complexes to regulate various signaling pathways within the cell. Therefore, it is important to understand the factors contributing to NHE1 regulation as maintenance of these critical cellular functions is vital ensure a healthy cell. Regulation of NHE1 occurs onthe large intracellular C‐terminus where binding partners and posttranslational modifications (PTMs) influence the exchanger in both short‐ and long‐term manners. Phosphorylation and palmitoylation are both dynamic reversible posttranslational modifications (PTMs) that regulate proteins to influence critical cellular processes. Multiple studies have shown phosphorylation and palmitoylation can work together in a barcode to regulate proteins and alter cellular functions. Previous work in our lab has shown NHE1 is regulated by palmitoylation and that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as NHE1 associated cellular functions such as stress fiber formation and cell migration. NHE1 is regulated by phosphorylation of multiple sites through various kinase pathways. To better understand the relationship between palmitoylation and phosphorylation on NHE1 we used various stimuli known to regulate multiple kinase pathways that impact NHE1 phosphorylation and measured the impact on NHE1 palmitoylation. Using lysophosphatidic acid (LPA) and phorbol 12‐myristate (PMA) to increase NHE1 phosphorylation leads to increased palmitoylation of NHE1, while activation of the NHE1 phosphorylation with insulin leads to decreased NHE1 palmitoylation. Furthermore, we have shown that using these stimuli NHE1 activity increases with NHE1 palmitoylation and this can be attenuated by irreversibly inhibiting palmitoylation using 2‐bromopalmitate (2BP). Using this information, we have begun to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving the sodium hydrogen exchanger isoform 1 (NHE1). Future studies will target specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences. ND EPSCoRDoctoral Dissertation Assistantship UND0023386

Sodium Hydrogen Exchanger Isoform 1 (NHE1) Palmitoylation and Phosphorylation Barcoding: Implications on Regulation and Function

The sodium hydrogen exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by exchanging an intracellular proton for an extracellular sodium ion. Multiple cellular processes are associated with NHE1 activity including coordinated cell migration, cellular proliferation, and control of cell volume. NHE1 also functions as a membrane anchoring and scaffolding protein resulting in the organization of protein complexes, formation of stress fibers, and regulation of signaling pathways within the cell. The large C‐terminus (amino acids 503–815) of NHE1 extends intracellularly and functions as the major regulatory domain of the exchanger where binding partners and posttranslational modifications (PTM) influence the exchanger in both a short‐ and long‐term manner. Elucidating mechanisms of NHE1 regulation by posttranslational modifications has been focused on contributions from the many phosphorylation sites on functional cellular outcomes. Previously, we have shown NHE1 is also regulated by palmitoylation, a reversible lipid modification in which a 16‐carbon fatty acid is covalently linked to a cysteine residue via a thioester bond and have demonstrated that inhibition of palmitoylation in cells expressing NHE1 decreases NHE1 activity, as well as stress fiber formation and cell migration, two cellular functions that are regulated through NHE1 phosphorylation. Multiple studies demonstrating a relationship between phosphorylation and palmitoylation on protein regulation have been reported with our lab recently demonstrating a reciprocal relationship between phosphorylation and palmitoylation in regulation of the dopamine transporter. The goal of this study was to begin to understand how the presence or absence of palmitoylation and phosphorylation in a coordinated manner contributes to a barcode that dictates a regulatory outcome and cellular response involving NHE1. We utilized various stimuli including insulin, reduced serum, and lysophosphatidic acid (LPA), all of which are known to impact NHE1 phosphorylation and regulation through multiple kinase pathways. We show that increasing phosphorylation with phorbol 12‐myristate (PMA) and LPA results in increased palmitoylation of NHE1, while treatment with insulin which also increases NHE1 phosphorylation, results in decreased NHE1 palmitoylation. Using this information, we have begun studies to inhibit specific kinases involved in these pathways to further explore the mechanism through which palmitoylation is altered. Together, this data supports the idea of a barcode hypothesis where phosphorylation and palmitoylation and other PTMs work in concert to regulate protein function including NHE1. Future studies will identify the specific palmitoylation sites and their role in the barcode hypothesis pertaining to NHE1.Support or Funding InformationThis work was supported by the University of North Dakota School of Medicine and Health Sciences and P20 GM103442 (to U.N.D.) from the INBRE program of the National Institute of General Medical Sciences and the ND EPSCoR Doctoral Dissertation AssistantshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Consequences of dynamic changes in palmitoylation of Nav1.5 voltage-gated Na+ channels

Inhibition of cardiac Nav1.5 voltage-gated Na+ channels (VGSCs) contributes to toxicity by local anaesthetics (LAs). Inhibition is state-dependent; channels become more sensitive to block when inactivated. Nav1.5 undergoes palmitoylation, the addition of palmitoyl groups to intracellular cysteines, affecting Nav1.5 inactivation. Mutations associated with inherited arrhythmias disrupt Nav1.5 palmitoylation causing inactivation at more hyperpolarised potentials1. This may enhance their block by LAs increasing the vulnerability of affected individuals to toxicity. We are investigating whether palmitoylation influences state-dependent inhibition by the LA, lidocaine. However, it is unclear whether palmitoylation is a dynamic process or occurs through altered Nav1.5 expression. We therefore initially examined the consequences of acute inhibition of palmitoylation by 2-bromopalmitate (2-BP) on Nav1.5 function. Whole-cell voltage-clamp was used to record from HEK293 cells transiently expressing Nav1.52. Currents were evoked by depolarising from -80 mV and 2-BP was bath-applied. Current-voltage relationships and the voltage-dependence of activation and inactivation were established as described previously 2. Data are expressed as mean ± SEM. Comparisons were made using the paired Student's t-test. Acute application of 2-BP (1 μM) caused a reduction of 44 ± 4.5% (n = 12) in the peak Nav1.5 current amplitude elicited by depolarising from -80 to 0 mV, reducing peak current density from 487 ± 46 pA/pF to 205 ± 28 pA/pF (n = 12, p < 0.0001). The voltage-dependence of activation was shifted in the depolarising direction. The half-maximal voltage (V½) of activation changed from -30 ± 0.7 mV to -24 ± 1.7 mV (n = 12, p < 0.01). The slope of the activation curve was also significantly affected in the presence of 2-BP (8.8 ± 0.6 vs 14 ± 0.7, n = 12, p < 0.0001). The voltage-dependence of inactivation was shifted in a hyperpolarising direction, with a significant change in the V½ of inactivation from 75 ± 1.8 mV in control to -90 ± 2.3 mV in the presence of 2-BP (1 μM) (n = 12, p < 0.0001). Our results demonstrate that acute inhibition of palmitoylation shifts the voltage-dependence of inactivation in the hyperpolarising direction. A greater likelihood of inactivation caused by the absence of palmitoylation may enhance sensitivity to inhibition of Nav1.5 by LAs. We are currently investigating the effect of the palmitoylation status of Nav1.5 on block by lidocaine. 1.Pei Z, Xiao Y, Meng J et al. Nat Commun. 2016; 7: 12035.2.Baptista-Hon DT, Robertson FM, Owen SJ et al. Br J Anaesth. 2014; 113: i39–i48.

Evaluating the Inhibition of Palmitoylation and NHE1 on Cell Proliferation and Migration

The Na+‐ H+ Exchanger Isoform I (NHE1) is a key regulator of cell proliferation and migration in a range of cell types from both healthy and diseased tissues. Activation of NHE1 is an initial step in the development of the transformed phenotypes of cancer cells and therefore a key driver for tumor development and cancer progression. NHE1 also contributes to two of the hallmarks of cancer, the activation of invasion and metastasis and sustained proliferative growth. We have identified for the first time, that NHE1 undergoes reversible palmitoylation, the addition of a palmitic acid residue to the cytoplasmic regulatory domain of NHE1. We hypothesize that this process regulates NHE1 activation and will play a role in the control of cell proliferation and migration. This suggests that targeting the potential palmitoylation sites on NHE1 may provide a therapeutic target for cancer treatment. Our work characterizes the role of the palmitoylation of NHE1 in cell growth and migration. Using the palmitoylation inhibitor, 2‐bromopalmitate (2BP), we evaluated cell growth and migration in the presence and absence of palmitoylation. In proliferation assays, the rate of cell growth increased approximately 5‐fold when the serum present in the growth media was increased from 0.5% to 10% and approximately 2‐fold when 50 μM lysophosphatidic acid was added to the growth media. Under both conditions, the addition of 15 μM 2BP or 10 μM cariporide dramatically reduced or eliminated the increased proliferation. Cell migration was evaluated using Electric Cell‐substrate Impedance Sensing (ECIS). Once again, the rate of cell migration was reduced by 50% or more in the presence of 15 μM 2BP or 10 μM cariporide. These data demonstrated that inhibiting the palmitoylation of NHE1 decreases both cell proliferation and the rate of cell migration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 1216: c-Met protein expression is regulated by palmitoylation in prostate cancer cells

Abstract Prostate cancer (PCa) is the most common noncutaneous neoplasia diagnosed in men and represents roughly 10% of cancer-associated mortalities. The lethal phenotype of PCa is primarily characterized by progression of tumor cells to androgen-independence and metastasis. Organ-confined PCa is often curable with surgery and/or radiation therapy, however, as the disease becomes metastatic, the likelihood of survival becomes minimal. Key influences in the progression to metastasis are growth factor receptors including the receptor tyrosine kinase c-Met. Numerous bodies of evidence strongly link aberrant c-Met signaling with causation and/or progression of prostate cancer. Overexpression of c-Met can lead to activation in a ligand-independent manner and has been identified as a common mechanism of resistance to RTK-targeted therapy. In addition, multiple reports have identified the enzyme fatty acid synthase (FASN) as being commonly overexpressed in prostate cancer, and that this aberrant expression is an early event that becomes more pronounced with aggressive androgen-independent and metastatic disease. FASN is the sole enzyme responsible for de novo synthesis of the 16-carbon saturated fatty acid palmitate. In cancer, de novo lipids are more selectively partitioned into lipid rafts as phospholipids as well as utilized for post-translational acyl-modifications of signaling proteins. Previous findings have led us to identify a novel mechanism by which FASN activity regulates c-Met expression. Our work has determined that inhibition or shRNA knockdown of FASN results in a post-translational downregulation of already synthesized c-Met protein. This downregulation is prevented by the addition of exogenous palmitate. Here we provide evidence that inhibition of palmitoylation, using the palmitate analog 2-bromopalmitate, lowers total c-Met levels. This protein loss occurs post-transcriptionally and is associated with accumulation of c-Met in Golgi compartments. Using inhibitors to a number of internalization pathways, as well as surface biotinylation studies, our findings suggest the inhibition of palmitoylation reduces the stability of newly synthesized c-Met as opposed to inducing internalization and degradation. Moreover, both an acyl-biotin exchange technique and a click-chemistry based palmitate-labeling protocol have provided evidence suggesting c-Met itself is palmitoylated. From these data, we hypothesize that c-Met requires palmitoylation along a biosynthetic route to promote its stability and trafficking to the cell surface. In the absence of palmitoylation, c-Met accumulates within the Golgi and is disposed of through a yet to be determined degradation pathway. Our findings potentially reveal a novel mechanism for restricting the expression of c-Met as a means of preventing prostate cancer invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1216. doi:1538-7445.AM2012-1216

Palmitoylation of Pulmonary Surfactant Protein SP-C Is Critical for Its Functional Cooperation with SP-B to Sustain Compression/Expansion Dynamics in Cholesterol-Containing Surfactant Films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

The infection of mammalian and insect cells with SFV bearing nsP1 palmitoylation mutations

Semliki Forest virus (SFV), an alphavirus, replicates in vertebrate host and mosquito vector cells. The virus-specific part of the replicase complex constitutes nonstructural proteins 1–4 (nsP1–nsP4) and is bound to cytoplasmic membranes by an amphipathic helix inside of nsP1 and through the palmitoylation of cysteine residues in nsP1. In mammalian cells, defects in these viral functions result in a nonviable phenotype or the emergence of second-site compensatory mutations that have a positive impact on SFV infection. In most cases, these second-site compensatory mutations were found to compensate for the defect caused by the absence of palmitoylation in mosquito cells (C6/36). In C6/36 cells, however, all palmitoylation-defective viruses had severely reduced synthesis of subgenomic RNA; at the same time, several of them had very efficient formation of defective interfering genomes. Analysis of C6/36 cells that individually expressed either wild type (wt) or palmitoylation-deficient nsP1 forms revealed that similar to mammalian cells, the wt nsP1 localized predominantly to the plasma membrane, whereas its mutant forms localized to the cytoplasm. In contrast to transfected mammalian cells, all forms of nsP1 induced the formation of filopodia-like structures on some, but not all, transfected mosquito cells. These findings indicate that the plasma membrane and associated host factors may have different roles in alphavirus replicase complex formation in mammalian and mosquito cells. In general, the lack of nsP1 palmitoylation had a less severe effect on the function of the replication complex in mammalian cells when compared with that in mosquito cells.

Palmitoylation of CD36/FAT regulates the rate of its post-transcriptional processing in the endoplasmic reticulum

CD36/FAT is a transmembrane glycoprotein that functions in the cellular uptake of long-chain fatty acids and also as a scavenger receptor. As such it plays an important role in lipid homeostasis and, pathophysiologically, in the progression of type 2 diabetes and atherosclerosis. CD36 expression is tightly regulated at the levels of both transcription and translation. Here we show that its expression and location are also regulated post-translationally, by palmitoylation. Although palmitoylation of CD36 was not required for receptor maturation and cell surface expression, inhibition of palmitoylation either pharmacologically with cerulenin or by mutation of the relevant cysteines delayed processing at the ER and trafficking through the secretory pathway. The absence of palmitoylation also reduced the half life of the CD36 protein. Additionally, the CD36 palmitoylation mutant did not incorporate efficiently into lipid rafts, a site known to be required for its function of fatty acid uptake, and this reduced the efficiency of uptake of oxidized low density lipoprotein. These findings provide an added level of sophistication where translocation of CD36 to the plasma membrane may be physiologically regulated by palmitoylation.

Palmitoylation stabilizes unliganded rod opsin

S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane

Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms.

Palmitoylation is required for efficient Fas cell death signaling

Localization of the death receptor Fas to specialized membrane microdomains is crucial to Fas-mediated cell death signaling. Here, we report that the post-translational modification of Fas by palmitoylation at the membrane proximal cysteine residue in the cytoplasmic region is the targeting signal for Fas localization to lipid rafts, as demonstrated in both cell-free and living cell systems. Palmitoylation is required for the redistribution of Fas to actin cytoskeleton-linked rafts upon Fas stimulation and for the raft-dependent, ezrin-mediated cytoskeleton association, which is necessary for the efficient Fas receptor internalization, death-inducing signaling complex assembly and subsequent caspase cascade leading to cell death.

N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways

We have analysed the mechanism by which palmitoylation permits the progression of nitric oxide synthase 2 (NOS2) along the ER-Golgi-TGN pathway. Introduction of an additional myristoylation site at the N-terminus of NOS2 resulted in a chimera that displayed an enhanced association with the particulate fraction and with the plasma membrane but did not display increased enzymatic activity. In the absence of palmitoylation, introduction of a surrogate myristoylation site resulted in a mutant NOS2 with only 25% activity compared with the wild-type enzyme. Hence, the novel surrogate myristoyl moiety not only failed to increase NOS2 activity when introduced in a wild-type sequence environment, but was also unable to rescue the inactive phenotype of the Cys3Ser mutant. Introduction of an additional palmitoylatable Cys at position 2 of the wild-type sequence resulted in a chimera that associated to a larger degree with membranes and displayed decreased activity. Our data indicate that palmitoylation of inducible NOS at position 3 exquisitely determines its transit along the secretory pathway following a route that cannot be mimicked by a surrogate myristoylation or by a palmitate at position 2. In addition, the exit of NOS2 from the TGN and the accumulation in the cellular plasma membrane per se did not correlate with increased .NO synthesis.

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Palmitoylation of the three isoforms of human endothelin-converting enzyme-1

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation.

Palmitoylation of the three isoforms of human endothelin-converting enzyme-1

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation.

Palmitoylation of the V2 vasopressin receptor.

Palmitoylation of the V2 vasopressin receptor (V2R) and its functional role were investigated in transfected cells. Palmitoylation was assessed by incubating transfected cells with [3H]palmitic acid and immunoprecipitating the receptor with an antibody raised against a portion of the third intracellular loop of V2R. Wild-type and nonglycosylated V2R yielded tritium signals at 45-55 and 40 kDa, respectively, demonstrating that the V2R is palmitoylated and that receptor palmitoylation is independent of glycosylation. Substitution of CC341/342 for serines eliminated receptor palmitoylation, whereas replacement of a single amino acid, C341S or C342S, restored partial palmitoylation. Saturation binding assays revealed decreased cell surface expression of the nonpalmitoylated receptor compared with the wild-type; this effect was more pronounced when a truncated form of V2R (G345ter) was studied. The presence of either cysteine residue (C341S or C342S) elevated receptor expression to normal levels, most likely due to the partial restoration of palmitoylation. Ligand binding affinity, hormone-induced stimulation of adenylyl cyclase activity, receptor internalization, and desensitization were not affected by the absence of palmitoylation. No increase but rather a slight decrease in the extent of receptor palmitoylation was detected after exposure to vasopressin. It was concluded that the V2R is palmitoylated in both cysteines, each cysteine is palmitoylated independently from the other, and palmitoylation enhances cell surface expression of the V2R.

Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.

Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.