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• W1964140147 abstract "Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynyl-palmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo. Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic ω-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, ω-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with ω-alkynyl-myristate or ω-alkynyl-stearate, via an alkali sensitive thioester bond. Third, ω-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of ω-alkynyl-palmitate and ω-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with ω-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using ω-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The ω-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo. 2-bromopalmitate enhanced green fluorescent protein caspase-cleaved C-terminal p21 activated kinase 2 green fluorescent protein homogenization buffer 2-hydroxy-myristic acid 3-hydroxyl-3-methylglutaryl-CoA synthase long-chain fatty acids NeutrAvdin™-HRP N-ethylmaleimide N-myristoyl transferase protein acyltransferase polyvinylidene fluoride For lipid synthesis, energy production via β-oxidation, or for protein fatty acylation to occur, long-chain fatty acids (LCFAs) must be activated by conversion to their CoA derivatives (LCFA-CoAs) by fatty acyl-CoA synthetase (FAS). Protein fatty acylation is one of many types of posttranslational modifications of proteins by lipids, which also includes isoprenoids, glycosylphosphatidylinositols, and cholesterol. Typically, lipids covalently attached to proteins serve as hydrophobic membrane anchors (1Casey P.J. Protein lipidation in cell signaling.Science. 1995; 268: 221-225Crossref PubMed Scopus (728) Google Scholar, 2Dunphy J.T. Linder M.E. Signalling functions of protein palmitoylation.Biochim. Biophys. Acta. 1998; 1436: 245-261Crossref PubMed Scopus (315) Google Scholar, 3Linder M.E. Deschenes R.J. Palmitoylation: policing protein stability and traffic.Nat. Rev. Mol. Cell Biol. 2007; 8: 74-84Crossref PubMed Scopus (742) Google Scholar, 4Mumby S.M. Reversible palmitoylation of signaling proteins.Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (238) Google Scholar, 5Resh M.D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins.Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar, 6Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (427) Google Scholar). Protein fatty acylation is mainly divided into two categories: N-myristoylation and S-acylation. The corresponding reactions are catalyzed by N-myristoyl transferases (NMT1 and NMT2) and two families of protein acyltransferases (PATs) referred to as zinc finger, Asp-His-His-Cys PATs and membrane bound O-acyl-transferases [reviewed in (5Resh M.D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins.Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar, 7Miura G.I. Treisman J.E. Lipid modification of secreted signaling proteins.Cell Cycle. 2006; 5: 1184-1188Crossref PubMed Scopus (53) Google Scholar, 8Tsutsumi R. Fukata Y. Fukata M. Discovery of protein-palmitoylating enzymes.Pflugers Arch. 2008; 456: 1199-1206Crossref PubMed Scopus (80) Google Scholar)]. In S-acylation, several LCFAs (e.g., C16:0, C16:1, C18:0, C18:1, and even C14:0) are found covalently attached to cysteine residues of proteins (9Smotrys J.E. Linder M.E. Palmitoylation of intracellular signaling proteins: regulation and function.Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (476) Google Scholar, 10Resh M.D. Palmitoylation of ligands, receptors, and intracellular signaling molecules.Sci. STKE. 2006; 2006: re14Crossref PubMed Scopus (340) Google Scholar). Palmitate is the most abundant fatty acid and, consequently, is preferentially attached onto proteins. As such, S-acylation is commonly referred to as palmitoylation. In N-myristoylation, the saturated 14 carbon fatty acid is added to an N-terminal glycine residue in proteins (11Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). A conservative G→A mutation is sufficient to block N-myristoylation, has been used extensively to abrogate myristoylation, assess its impact on protein function (11Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). There are two types of myristoylation based on the timing of the reaction, cotranslational and posttranslational. Cotranslational myristoylation occurs on glycine residues exposed by the action of a methionyl-aminopeptidase on nascent polypeptides, whereas posttranslational myristoylation occurs at cryptic internal glycine residues exposed following cleavage by caspases during apoptosis (11Farazi T.A. Waksman G. Gordon J.I. The biology and enzymology of protein N-myristoylation.J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 12Zha J. Weiler S. Oh K.J. Wei M.C. Korsmeyer S.J. Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis.Science. 2000; 290: 1761-1765Crossref PubMed Scopus (472) Google Scholar, 13Utsumi T. Sakurai N. Nakano K. Ishisaka R. C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria.FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar, 14Sakurai N. Utsumi T. Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin.J. Biol. Chem. 2006; 281: 14288-14295Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 15Vilas G.L. Corvi M.M. Plummer G.J. Seime A.M. Lambkin G.R. Berthiaume L.G. Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events.Proc. Natl. Acad. Sci. USA. 2006; 103: 6542-6547Crossref PubMed Scopus (79) Google Scholar, 16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar, 17Bhatnagar R.S. Gordon J.I. Understanding covalent modifications of proteins by lipids: where cell biology and biophysics mingle.Trends Cell Biol. 1997; 7: 14-20Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 18Bhatnagar R.S. Futterer K. Waksman G. Gordon J.I. The structure of myristoyl-CoA:protein N-myristoyltransferase.Biochim. Biophys. Acta. 1999; 1441: 162-172Crossref PubMed Scopus (56) Google Scholar). The long exposure time required to detect the incorporation of [3H]fatty acids into protein (1–3 months or more) has long impeded the progress of investigators working on protein fatty acylation. The use of [125I]iodofatty acids has reduced this significantly but is typically associated with handling of large quantities (mCi) of the hazardous isotope 125I (19Berthiaume L. Peseckis S.M. Resh M.D. Synthesis and use of iodo-fatty acid analogs.Methods Enzymol. 1995; 250: 454-466Crossref PubMed Scopus (39) Google Scholar). Recently, we and others have used bio-orthogonal (analogs with functional handles that can be used by natural enzymes) ω-azido-fatty acid analogs to readily detect the acylation status of various fatty acylated proteins (16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar, 20Hang H.C. Geutjes E.J. Grotenbreg G. Pollington A.M. Bijlmakers M.J. Ploegh H.L. Chemical probes for the rapid detection of Fatty-acylated proteins in Mammalian cells.J. Am. Chem. Soc. 2007; 129: 2744-2745Crossref PubMed Scopus (166) Google Scholar, 21Kostiuk M.A. Corvi M.M. Keller B.O. Plummer G. Prescher J.A. Hangauer M.J. Bertozzi C.R. Rajaiah G. Falck J.R. Berthiaume L.G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.FASEB J. 2008; 22: 721-732Crossref PubMed Scopus (111) Google Scholar). Compared to using tritiated fatty acids in cell labeling reactions, the incorporation of the alkyl-azide analogs of fatty acids, ω-azido-dodecanoate (as an isosteric myristate analog), and ω-azido-tetradecanoate (as an isosteric palmitate analog) into proteins and their detection with a biotinylated-triarylphophine via the Staudinger reaction provided up to a million-fold increase in detection sensitivity (16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar). Using the palmitoyl-CoA analog ω-azido-tetradecanoyl-CoA as label, we identified 21 palmitoylated proteins in rat liver mitochondria, including 3-hydroxyl-3-methylglutaryl-CoA synthase (HMGCS), the rate-limiting enzyme in ketogenesis (21Kostiuk M.A. Corvi M.M. Keller B.O. Plummer G. Prescher J.A. Hangauer M.J. Bertozzi C.R. Rajaiah G. Falck J.R. Berthiaume L.G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.FASEB J. 2008; 22: 721-732Crossref PubMed Scopus (111) Google Scholar). Palmitoylated mitochondrial proteins are surprisingly numerous and the characterization of the role of their acylation is still pending in the vast majority of cases (21Kostiuk M.A. Corvi M.M. Keller B.O. Plummer G. Prescher J.A. Hangauer M.J. Bertozzi C.R. Rajaiah G. Falck J.R. Berthiaume L.G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.FASEB J. 2008; 22: 721-732Crossref PubMed Scopus (111) Google Scholar, 22Berthiaume L. Deichaite I. Peseckis S. Resh M.D. Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.J. Biol. Chem. 1994; 269: 6498-6505Abstract Full Text PDF PubMed Google Scholar, 23Corvi M.M. Soltys C.L. Berthiaume L.G. Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation.J. Biol. Chem. 2001; 276: 45704-45712Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Stucki J.W. Lehmann L.H. Siegel E. Acylation of proteins by myristic acid in isolated mitochondria.J. Biol. Chem. 1989; 264: 6376-6380Abstract Full Text PDF PubMed Google Scholar). In two characterized cases, the acylation of methylmalonyl semialdehyde dehydrogenase and carbamoyl phosphate synthetase 1 was shown to occur on the active site cysteine residues, thereby inhibiting these catabolic enzymes (22Berthiaume L. Deichaite I. Peseckis S. Resh M.D. Regulation of enzymatic activity by active site fatty acylation. A new role for long chain fatty acid acylation of proteins.J. Biol. Chem. 1994; 269: 6498-6505Abstract Full Text PDF PubMed Google Scholar, 23Corvi M.M. Soltys C.L. Berthiaume L.G. Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation.J. Biol. Chem. 2001; 276: 45704-45712Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition, Gross et al. (25Yang J. Gibson B. Snider J. Jenkins C.M. Han X. Gross R.W. Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity: a novel mechanism potentially underlying fatty acid induced insulin resistance.Biochemistry. 2005; 44: 11903-11912Crossref PubMed Scopus (28) Google Scholar) showed that the glycolytic metabolic enzyme GAPDH is also a palmitoylated protein. Altogether, the large number of these palmitoylated metabolic enzymes suggests an imminent and underappreciated role for protein palmitoylation in the regulation of metabolism. Another relatively recent breakthrough in the identification of palmitoylated proteins is the use of the acyl-biotin exchange reaction. The acyl-biotin exchange reaction is an indirect labeling method for the detection and purification of palmitoylated proteins from complex protein extracts. It substitutes biotinyl moieties for protein palmitoyl-modifications via a sequence of three chemical treatment steps. Now biotin-tagged proteins can be affinity-purified by streptavidin-agarose and protein identifications made by proteomic MS (26Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (360) Google Scholar, 27Roth A.F. Wan J. Bailey A.O. Sun B. Kuchar J.A. Green W.N. Phinney B.S. Yates III, J.R. Davis N.G. Global analysis of protein palmitoylation in yeast.Cell. 2006; 125: 1003-1013Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 28Wan J. Roth A.F. Bailey A.O. Davis N.G. Palmitoylated proteins: purification and identification.Nat. Protoc. 2007; 2: 1573-1584Crossref PubMed Scopus (301) Google Scholar). There are many different types of fatty acids attached to proteins. Therefore, the need for different chemical reporters to assess protein fatty acylation is essential. The alkynyl moiety has been used as a tag to generate bio-orthogonal analogs that could readily be reacted with azide-tagged probes using the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition, a type of reaction also known as “click chemistry” (29Beatty K.E. Xie F. Wang Q. Tirrell D.A. Selective dye-labeling of newly synthesized proteins in bacterial cells.J. Am. Chem. Soc. 2005; 127: 14150-14151Crossref PubMed Scopus (216) Google Scholar, 30Heal W.P. Wickramasinghe S.R. Leatherbarrow R.J. Tate E.W. N-Myristoyl transferase-mediated protein labelling in vivo.Org. Biomol. Chem. 2008; 6: 2308-2315Crossref PubMed Scopus (114) Google Scholar, 31Rostovtsev V.V. Green L.G. Fokin V.V. Sharpless K.B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes.Angew. Chem. Int. Ed. Engl. 2002; 41: 2596-2599Crossref PubMed Scopus (9845) Google Scholar, 32Speers A.E. Cravatt B.F. Profiling enzyme activities in vivo using click chemistry methods.Chem. Biol. 2004; 11: 535-546Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 33Wang Q. Chan T.R. Hilgraf R. Fokin V.V. Sharpless K.B. Finn M.G. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition.J. Am. Chem. Soc. 2003; 125: 3192-3193Crossref PubMed Scopus (1472) Google Scholar). To complement the ω-azido-fatty acid series of chemical reporters (16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar, 20Hang H.C. Geutjes E.J. Grotenbreg G. Pollington A.M. Bijlmakers M.J. Ploegh H.L. Chemical probes for the rapid detection of Fatty-acylated proteins in Mammalian cells.J. Am. Chem. Soc. 2007; 129: 2744-2745Crossref PubMed Scopus (166) Google Scholar, 21Kostiuk M.A. Corvi M.M. Keller B.O. Plummer G. Prescher J.A. Hangauer M.J. Bertozzi C.R. Rajaiah G. Falck J.R. Berthiaume L.G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.FASEB J. 2008; 22: 721-732Crossref PubMed Scopus (111) Google Scholar), we sought to investigate the potential of ω-alkynyl-fatty acids and click chemistry as a means to detect S-acylation and N-myristoylation. In addition to increasing the reaction rate, the copper (I) catalyst also sensitizes alkynes toward dipolar reagents such as azides in a specific manner (33Wang Q. Chan T.R. Hilgraf R. Fokin V.V. Sharpless K.B. Finn M.G. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition.J. Am. Chem. Soc. 2003; 125: 3192-3193Crossref PubMed Scopus (1472) Google Scholar). Therefore, in the absence of copper (I), the ω-alkynyl-fatty acids remain practically inert in biological systems. Because various LCFAs are known to be incorporated into cysteine residues of palmitoylated proteins, the introduction of a triple C-C bond between carbons 15 and 16 of palmitate to produce the reactive ω-alkynyl-palmitate should not interfere with the physiological properties of the fatty acid analog (34Hannoush R.N. Arenas-Ramirez N. Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.ACS Chem. Biol. 2009; 4: 581-587Crossref PubMed Scopus (103) Google Scholar). The use of ω-alkynyl-fatty acids to detect acylation of proteins has also recently been used by others (34Hannoush R.N. Arenas-Ramirez N. Imaging the lipidome: omega-alkynyl fatty acids for detection and cellular visualization of lipid-modified proteins.ACS Chem. Biol. 2009; 4: 581-587Crossref PubMed Scopus (103) Google Scholar, 35Charron G. Wilson J. Hang H.C. Chemical tools for understanding protein lipidation in eukaryotes.Curr. Opin. Chem. Biol. 2009; 13: 382-391Crossref PubMed Scopus (56) Google Scholar) and applied to perform proteomics studies on palmitoylated proteins in cells (26Martin B.R. Cravatt B.F. Large-scale profiling of protein palmitoylation in mammalian cells.Nat. Methods. 2009; 6: 135-138Crossref PubMed Scopus (360) Google Scholar). To assess the selectivity of incorporation of the ω-alkynyl-fatty acid analogs at a given site into proteins, we first took advantage of the fact that acylation of mitochondrial proteins occurred spontaneously and only required fatty acyl-CoA and purified enzymes. Second, another subset of well-characterized palmitoylated proteins is the Ras small GTPase family. All human Ras proto-oncogenes undergo extensive posttranslational modifications, including isoprenylation and carboxymethylation. In addition, H- and N-Ras (but not K-Ras) are also palmitoylated (6Resh M.D. Trafficking and signaling by fatty-acylated and prenylated proteins.Nat. Chem. Biol. 2006; 2: 584-590Crossref PubMed Scopus (427) Google Scholar, 36Buss J.E. Sefton B.M. Direct identification of palmitic acid as the lipid attached to p21ras.Mol. Cell. Biol. 1986; 6: 116-122Crossref PubMed Scopus (90) Google Scholar, 37Hancock J.F. Paterson H. Marshall C.J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane.Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (841) Google Scholar, 38Magee A.I. Gutierrez L. McKay I.A. Marshall C.J. Hall A. Dynamic fatty acylation of p21N-ras.EMBO J. 1987; 6: 3353-3357Crossref PubMed Scopus (219) Google Scholar) (supplementary Table I). To further validate the use of click chemistry for labeling of fatty acylated proteins in cultured cells and in vivo in mice, we also characterized the incorporation of the isosteric palmitic acid analog hexadec-15-ynoic acid (referred to as ω-alkynyl-palmitate) into a variety of H- and N-Ras constructs. To illustrate the versatility of this technique, we also show that an isosteric analog of myristic acid tetradec-13-ynoic acid (referred to as ω-alkynyl-myristate) can be incorporated co- and posttranslationally into a variety of proteins. In addition, to add further proof-of-concept and validation to this methodology, we show new results that confirm that the sites acylated by ω-alkynyl-palmitate are the same as palmitate by MS and provide new results that illustrate the versatility of the new method in vitro, in cells, and, promisingly, in vivo. We also show that labeling cells with ω-alkynyl-palmitate analog does not affect the subcellular fractionation pattern of N-Ras, which is predominantly found in the membrane fractions. All green fluorescent protein (GFP) antibodies were provided by Eusera (www.eusera.com, Edmonton, AB, Canada) or Abcam (Cambridge, UK). The following antibodies were used in this study: rabbit anti-GFP serum (Eusera EU-1 or Abcam Ab290; 1:50,000 dilution), goat anti-caspase-cleaved C-terminal p21 activated kinase (ctPAK2) (C19) (sc-1519, Santa Cruz Biotechnology; 1:1,000 dilution), affinity purified goat anti-GFP for immunoprecipitation (1 µl EU-4 from Eusera or Ab5450 from Abcam per sample), mouse anti-Fas for induction of apoptosis (clone CH11, Millipore; 150 ng/ml), rabbit anti-ctPAK2 serum for immunoprecipitation [10 µl per sample, prepared in the Berthiaume laboratory and described previously (16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar)], NeutrAvdin™ HRP (NA-HRP) conjugates (Pierce Biotechnology; 1:50,000), and rabbit anti-N-Ras (sc-519, Santa Cruz Biotechnology; 1:500). Crude rat hybridoma supernatants containing anti-pan Ras 259 or rat anti-H and K-Ras 238 (20 µl per sample) originally from ATCC were kind gifts from Dr. Jim Stone (University of Alberta, Canada). Monoclonal mouse anti-Ras clone RAS10 was from Millipore (used at 1:2,000). Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were of the highest purity available. The pEGFP-N1 vector was purchased from Invitrogen (Carlsbad, CA). The WT-ctPAK2-N15-EGFP and G2A-ctPAK2-N15-EGFP constructs were from previous work (15Vilas G.L. Corvi M.M. Plummer G.J. Seime A.M. Lambkin G.R. Berthiaume L.G. Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events.Proc. Natl. Acad. Sci. USA. 2006; 103: 6542-6547Crossref PubMed Scopus (79) Google Scholar) based on the pEGFP-N1 vector. Wild-type, full-length human Ras constructs, EGFP-H-Ras, EGFP-N-Ras, and EGFP-K-Ras, were gifts of Dr. Patrick Casey (Duke University Medical Center, Durham, NC). These constructs were based on the pEGFP-C1 vector (Clontech) in which the enhanced GFP was fused to the N-terminus of H-Ras, N-Ras, or K-Ras4B. COS-7 and Jurkat T cells were purchased from the ATCC (Manassas, VA). We obtained HeLa cells from Dr. T. Simmen (University of Alberta). All reagents for cell culture were purchased from Invitrogen. COS-7 and HeLa cells were maintained in DMEM while Jurkat cells were cultured in RPMI medium at 37°C and 5% CO2 in a humidified incubator. All the maintenance media were supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. COS-7 cells were transiently transfected with the indicated constructs using FuGene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) per the manufacturer's instructions in DMEM supplemented with 10% FBS in the absence of antibiotics. Transfection reagent-DNA complex was left in the medium for 16 h. The ω-alkynyl- and ω-azido-fatty acids synthesized as described in the supplementary material file (soon available from www.eusera.com) were added to cells as described in Martin et al. (16Martin D.D. Vilas G.L. Prescher J.A. Rajaiah G. Falck J.R. Bertozzi C.R. Berthiaume L.G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.FASEB J. 2008; 22: 797-806Crossref PubMed Scopus (83) Google Scholar) with the following modifications. Cells were deprived of fatty acids by incubating in their respective media supplemented with either 5% dextran-coated charcoal-treated FBS or 1% fatty acid-free BSA (Sigma Aldrich) for 1 h prior to labeling. The ω-azido-fatty acids, ω-alkynyl-fatty acids, or fatty acids were dissolved in DMSO to generate 20–100 mM stock solutions. To facilitate cellular uptake of ω-azido-fatty acids or fatty acids, prior to labeling, these were saponified by incubation with a 20% molar excess of potassium hydroxide at 65°C for 15 min. To do so, a 20× solution was made by dissolving the saponified fatty acids in prewarmed, serum-free culture media containing 20% fatty acid-free BSA at 37°C, followed by an additional 15 min incubation at 37°C. After deprivation of fatty acids, cells were washed with warm PBS and incubated in fresh media without supplement. One-twentieth volume of the 20× fatty acid-BSA conjugate in serum-free media was added to the cells, typically 200 µl to 3.8 ml media, so that the final concentration of BSA was 1% and the fatty acids were at the indicated concentrations. Saponification of the ω-alkynyl-fatty acids was also performed but was not required to increase incorporation into proteins. In control experiments, mock DMSO or unlabeled fatty acids were subjected to the saponification and conjugation to BSA as described above. Cells were labeled for times indicated at 37°C in a 5% CO2 humidified incubator. The inhibitors of N-myristoylation and S-acylation, 2-hydroxy-myristate (HMA) and 2-bromopalmitate (2-BP), respectively, were also saponified and conjugated to BSA prior to addition to cells. Cells (∼1 × 106 to 1 × 107) were washed with cold PBS, harvested, and lysed with cold EDTA-free RIPA buffer [0.1% SDS, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium-deoxycholate, 2 mM MgCl2, EDTA-free complete protease inhibitor (Roche)] by rocking for 15 min at 4°C. Cell lysates were centrifuged at 16,000 g for 10 min at 4°C and the postnuclear supernatants were collected. GFP fusion proteins were immunoprecipitated from approximately 1 mg of protein lysates with affinity purified goat anti-GFP cross-linked to Sepharose beads (www.eusera.com) by rocking for 2 h or overnight at 4°C. The beads were extensively washed with 0.1% SDS-RIPA, resuspended in 50 mM HEPES, pH 7.4, with 1% SDS, and heated for 15 min at 80°C. The supernatants containing the fusion proteins were collected. For the immunoprecipitation of endogenous Ras, rat anti-pan Ras 259 or rat anti-H and K-Ras 238 antibodies immobilized to protein G sepharose beads (GE Healthcare) were used as described above and immunoprecipitated from 4–5 mg of protein lysate. Endogenous PAK2 was immunoprecipitated from 300 µg of Jurkat protein lysate using rabbit anti-ctPAK2 serum. Transfected COS-7 cells (∼1 × 106 to 1 × 107) were deprived of fatty acids by incubating in serum free DMEM supplemented with 0.1% fatty acid-free BSA. [9,10(n)-3H] Myristic acid and [9,10(n)-3H] palmitic acid (500 μCi) were saponified by 20% molar excess of potassium hydroxide at 65°C for 15 min. Saponified fatty acids were dissolved and incubated in 0.1% fatty acid-free BSA in DMEM for 15 min at 37°C, followed by 5 min at 65°C, then returned to 37°C before adding to the cells. Cells were radiolabeled for 4 h at 37°C then lysed with 0.1% SDS-RIPA buffer. Radiolabeled proteins were immunoprecipitated as described above with goat anti-GFP Sepharose beads rocking" @default.
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• W1964140147 date "2010-06-01" @default.
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• W1964140147 title "Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry" @default.
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