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• W2029878872 abstract "Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIα (PI4KIIα) and IIβ (PI4KIIβ). Nearly all of PI4KIIα, and about half of PI4KIIβ, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIα converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIα to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIα in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIα and associates more weakly with lipid rafts. Phosphatidylinositol 4-kinases play essential roles in cell signaling and membrane trafficking. They are divided into type II and III families, which have distinct structural and enzymatic properties and are essentially unrelated in sequence. Mammalian cells express two type II isoforms, phosphatidylinositol 4-kinase IIα (PI4KIIα) and IIβ (PI4KIIβ). Nearly all of PI4KIIα, and about half of PI4KIIβ, associates integrally with membranes, requiring detergent for solubilization. This tight membrane association is because of palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domains of both type II isoforms. Deletion of this motif from PI4KIIα converts the kinase from an integral to a tightly bound peripheral membrane protein and abrogates its catalytic activity (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Sudhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). Here we identify the first two cysteines in the CCPCC motif as the principal sites of palmitoylation under basal conditions, and we demonstrate the importance of the central proline for enzymatic activity, although not for membrane binding. We further show that palmitoylation is critical for targeting PI4KIIα to the trans-Golgi network and for enhancement of its association with low buoyant density membrane fractions, commonly termed lipid rafts. Replacement of the four cysteines in CCPCC with a hydrophobic residue, phenylalanine, substantially restores catalytic activity of PI4KIIα in vitro and in cells without restoring integral membrane binding. Although this FFPFF mutant displays a perinuclear distribution, it does not strongly co-localize with wild-type PI4KIIα and associates more weakly with lipid rafts. Phosphatidylinositol 4-phosphate (PI4P) 3The abbreviations used are: PI4P, phosphatidylinositol 4-phosphate; 2-BP, 2-bromopalmitate; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; PI4K, phosphatidylinositol 4-kinase; PMSF, phenylmethylsulfonyl fluoride; TGN, trans-Golgi network; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; Ni2+-NTA, nickel nitrilotriacetic acid; PI4KII, phosphatidylinositol 4-kinase II; WT, wild type; DRM, detergent-resistant membranes. is a regulator of membrane trafficking as well as a substrate in the biosynthesis of other phosphoinositides. It is especially abundant in the Golgi apparatus where it partners with the Arf1 GTPase to recruit adaptor proteins essential in vesicular trafficking from the trans-Golgi network (TGN) (1Di Paolo G. De Camilli P. Nature. 2006; 443: 651-657Crossref PubMed Scopus (2080) Google Scholar, 2De Matteis M.A. D'Angelo G. Biochem. Soc. Symp. 2007; 74: 107-116Crossref PubMed Scopus (19) Google Scholar). The generation of PI4P is catalyzed by two distinct classes of phosphatidylinositol 4-kinase, type II and type III. Mammals have an α and β form of each type (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar, 4Heilmeyer Jr., L.M. Vereb Jr., G. Vereb G. Kakuk A. Szivak I. IUBMB Life. 2003; 55: 59-65Crossref PubMed Scopus (53) Google Scholar, 5Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). PI4KIIα and PI4KIIIβ contribute primarily to Golgi-localized PI4P, and they are responsible for about 50% of the PI4P in the Golgi. The pools of Golgi PI4P produced by each enzyme are distinct but interconnected by cross-talk (6Weixel K.M. Blumental-Perry A. Watkins S.C. Aridor M. Weisz O.A. J. Biol. Chem. 2005; 280: 10501-10508Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). PI4KIIα resides primarily in the TGN and in endosomes (7Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Biochem. J. 2003; 373: 57-63Crossref PubMed Scopus (53) Google Scholar, 9Chang F.S. Han G.S. Carman G.M. Blumer K.J. J. Cell Biol. 2005; 171: 133-142Crossref PubMed Scopus (36) Google Scholar). The critical roles of PI4KIIα in Golgi and endosome functions are evident by the dramatic effects of PI4KIIα depletion by RNA interference. These include impaired recruitment of clathrin adaptor protein AP-1 complexes and GGA to the TGN (10Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 11Wang J. Sun H.Q. Macia E. Kirchhausen T. Watson H. Bonifacino J.S. Yin H.L. Mol. Biol. Cell. 2007; 18: 2646-2655Crossref PubMed Scopus (127) Google Scholar), inhibition of constitutive secretion from the TGN (10Wang Y.J. Wang J. Sun H.Q. Martinez M. Sun Y.X. Macia E. Kirchhausen T. Albanesi J.P. Roth M.G. Yin H.L. Cell. 2003; 114: 299-310Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), inhibition of phagocytosis (12Pizarro-Cerda J. Payrastre B. Wang Y.J. Veiga E. Yin H.L. Cossart P. Cell. Microbiol. 2007; 9: 2381-2390Crossref PubMed Scopus (65) Google Scholar), and decreased trafficking of epidermal growth factor receptors through the late endosomal pathway (13Minogue S. Waugh M.G. De Matteis M.A. Stephens D.J. Berditchevski F. Hsuan J.J. J. Cell Sci. 2006; 119: 571-581Crossref PubMed Scopus (108) Google Scholar). In neuronal cells, PI4KIIα is also found on secretory granules and synaptic vesicles (14Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 15Salazar G. Craige B. Wainer B.H. Guo J. De Camilli P. Faundez V. Mol. Biol. Cell. 2005; 8: 3692-3704Crossref Google Scholar), and it has been implicated in exocytosis and endocytosis of regulated secretory vesicles (15Salazar G. Craige B. Wainer B.H. Guo J. De Camilli P. Faundez V. Mol. Biol. Cell. 2005; 8: 3692-3704Crossref Google Scholar, 16Wenk M.R. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8262-8269Crossref PubMed Scopus (259) Google Scholar). Despite the importance of PI4KIIα, very little is known about its enzymatic regulation or the basis of its localization in cells. It behaves like an integral membrane protein, requiring detergent for solubilization, despite its lack of a bona fide transmembrane domain (14Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 17Minogue S. Anderson J.S. Waugh M.G. dos Santos M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). We have previously reported that this behavior is probably because of palmitoylation in a cysteine-rich motif, residues 173CCPCC177 in rat PI4KIIα, as en bloc deletion of these five residues results in loss of palmitoylation and in conversion of the kinase from an integral to a peripheral membrane protein. In addition, the deletion mutant was catalytically inactive (14Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, we did not definitively prove that palmitoylation, rather than the deletion itself, was responsible for these altered properties. The loss of catalytic activity is especially problematic because the CCPCC motif resides within the catalytic domain, in close proximity to two residues that we had shown to be essential for kinase activity (18Barylko B. Wlodarski P. Binns D.D. Gerber S.H. Earnest S. Sudhof T.C. Grichine N. Albanesi J.P. J. Biol. Chem. 2002; 277: 44366-44375Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Moreover, the proline residue within the motif is also necessary for expression of activity (see below). Here we use multiple alternative approaches to show conclusively that palmitoylation is indeed required for critical properties of the kinase, including enzymatic activity, intracellular localization, integral membrane association, and partitioning into “rafts.” We identify two cysteine residues within the CCPCC motif that are preferentially palmitoylated under basal conditions. In addition, replacing the four cysteines with highly hydrophobic phenylalanine residues can partially restore endomembrane targeting and catalytic activity, although the phenylalanine mutant behaves as a peripheral protein. Materials-l-α-Phosphatidylinositol was from Avanti Polar Lipids, Inc. (Alabaster, AL). [γ-32P]ATP and [9,10-3H]palmitic acid were from PerkinElmer Life Sciences. Dimethyl pimelimidate was from Pierce. Primers were obtained from Sigma Genosys and IDT (Coralville, IA). Cloning and mutagenesis reagents were from Stratagene (La Jolla, CA). Lipofectamine 2000 and cDNA purification kits were from Invitrogen. Nickel nitrilotriacetic acid (Ni2+-NTA)-agarose was from Qiagen (Valencia, CA). Triton X-100 and reagents for electrophoresis and immunoblotting were from Bio-Rad. Other reagents, including 2-bromopalmitate, ATP, Brij 98, buffers, and protease inhibitors, were from Sigma. Polyclonal anti-PI4KIIα antibodies were prepared as described in Ref. 18Barylko B. Wlodarski P. Binns D.D. Gerber S.H. Earnest S. Sudhof T.C. Grichine N. Albanesi J.P. J. Biol. Chem. 2002; 277: 44366-44375Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar. Monoclonal anti-Myc antibody 9E10 was obtained from the National Cell Culture Center (Minneapolis, MN); recombinant protein G-Sepharose 4B conjugate was from Zymed Laboratories Inc., and sheep anti-TGN46 was from Serotec (Raleigh, NC). 125I-Labeled secondary antibodies were from Amersham Biosciences, and fluorescently conjugated secondary antibodies were from Molecular Probes (Eugene, OR). TLC plates were from Whatman. Purification of Recombinant PI4KIIα-Recombinant PI4KIIα containing a His6 tag at its amino terminus was expressed in Sf9 cells and in Escherichia coli. The procedure for Sf9 expression is described in Ref. 18Barylko B. Wlodarski P. Binns D.D. Gerber S.H. Earnest S. Sudhof T.C. Grichine N. Albanesi J.P. J. Biol. Chem. 2002; 277: 44366-44375Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar. For expression in E. coli, PI4KIIα was subcloned from the pCMV5myc1 vector into a pQE-80L vector (Qiagen) using the flanking BamHI (5′) and PstI (3′) restriction sites. Cells were resuspended in buffer A containing 1% Triton X-100, 20 mm Hepes (pH 8.0), 100 mm NaCl, 1 mm β-mercaptoethanol, a protease inhibitor mixture consisting of 10 μg/ml each of Nα-p-tosyl-l-lysine chloromethyl ester, Nα-p-tosyl-l-arginine methyl ester, Nα-p-tosyl-l-lysine chloromethyl ketone, leupeptin, pepstatin A, and 0.2 mm phenylmethylsulfonyl fluoride (PMSF). In the case of E. coli, lysozyme (0.05 mg/ml) was added to extract the kinase. Cell lysates were centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatants were mixed with Ni2+-NTA resin for 1 h at 4 °C. The resin was washed with buffer A supplemented with 30 mm imidazole (pH 8.0) and 300 mm NaCl but containing 0.1% Triton X-100. The kinase was then eluted with 20 mm Hepes (pH 8.0), 150 mm imidazole (pH 8.0), 100 mm NaCl, 0.1% Triton X-100, 1 mm β-mercaptoethanol, and 0.2 mm PMSF. When prepared from E. coli, the kinase eluted from Ni2+-NTA resin was further purified on an SP-Sepharose column using a 0.05-0.3 m NaCl linear gradient without detergent. Aliquots of the protein were frozen in liquid N2 and could be stored at -70 °C for several months without significant loss of activity. Generation of Point Mutants-Rat pCMV5-Myc-PI4KIIα cDNAs was used as a template for generation of constructs containing mutations in the 173CCPCC177 palmitoylation motif. Site-directed mutagenesis was performed using the Stratagene QuickChange site-directed mutagenesis kit according to the manufacturer’s protocol. Preparation of Cytosol and Membrane Fractions-Twenty hours after transfection, COS-7 cells expressing Myc-PI4KIIα were washed with ice-cold phosphate-buffered saline (PBS) and scraped from Petri dishes in a solution containing 0.25 m sucrose, 20 mm Tris-HCl (pH 7.5), 0.1 m NaCl, 1 mm EDTA, protease inhibitors as described above and phosphatase inhibitors (50 mm sodium fluoride, 50 mm glycerophosphate, 1 mm sodium orthovanadate, 10 μm microcystin). Cells were lysed by two freeze-thaw cycles and passage through a 27-gauge needle. The lysates were centrifuged at 1,000 × g for 5 min to remove cell debris and nuclei. The supernatant (post-nuclear supernatant) was then centrifuged at 200,000 × g for 15 min to separate cytosol from membranes. The resulting membrane pellets were homogenized in a solution containing 20 mm Tris-HCl (pH 7.5), 1 m NaCl, and 1 mm EDTA and then centrifuged at 200,000 × g for 15 min to obtain weakly binding peripheral membrane proteins in the supernatant. The resulting pellets were then homogenized in 0.1 m sodium carbonate (pH 11) and centrifuged as above to obtain tightly bound peripheral membrane proteins in the supernatant. Finally, the pellets of this centrifugation were homogenized in 1% Triton X-100 and centrifuged to obtain integral membrane proteins in the supernatant. All solutions contained the protease and phosphatase inhibitors described above. Subcellular Fractionation of Cells-Post-nuclear supernatants of COS-7 cells prepared as described above were fractionated on a discontinuous sucrose gradient according to Waugh et al. (8Waugh M.G. Minogue S. Anderson J.S. Balinger A. Blumenkrantz D. Calnan D.P. Cramer R. Hsuan J.J. Biochem. J. 2003; 373: 57-63Crossref PubMed Scopus (53) Google Scholar). Briefly, samples were layered on a gradient consisting of successive layers of 10, 15, 20, 25, 30, and 40% sucrose and centrifuged for 20 h at 180,000 × g in an SW40 rotor. One-ml fractions were collected from the bottom of the tubes, and equal aliquots were subjected to SDS-gel electrophoresis and immunoblotting. Preparation of Low Buoyant Density Membrane Fractions-Low buoyant density “membrane raft” fractions were prepared using Brij 98 as described by Claas et al. (19Claas S. Stipp C.S. Hemler M.E. J. Biol. Chem. 2001; 276: 7974-7984Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Cells were homogenized in a solution containing 1% Brij 98, 150 mm NaCl, 5 mm MgCl2, 25 mm MES (pH 6.5), 10 mm sodium pyrophosphate, 2 mm sodium fluoride, 0.1 mm sodium orthovanadate, and protease inhibitors. The lysate was adjusted to 40% sucrose (1.5 ml of lysate mixed with 1.5 ml of 80% w/v sucrose) and overlaid with 5.5 ml of 35% sucrose and 3.5 ml of 5% sucrose in the buffer described above but without Brij 98. Following centrifugation at 210,000 × g for 16 h at 4 °C in an SW40 rotor, 1-ml fractions were collected from the bottom of the tubes, and equal aliquots were subjected to SDS-gel electrophoresis and immunoblotting. Analysis of [3H]Palmitate Incorporation-Sixteen hours after transfection with plasmids encoding wild-type (WT) or mutant PI4KIIα, COS-7 cells were labeled with [3H]palmitate (0.3 mCi/ml) for 3 h as described by Linder et al. (20Linder M.E. Kleuss C. Mumby S.M. Methods Enzymol. 1995; 250: 314-330Crossref PubMed Scopus (36) Google Scholar). Cells were washed with ice-cold PBS and solubilized in RIPA buffer consisting of 0.15 m NaCl, 50 mm Tris-HCl (pH 8.0), 1% Nonidet P40, 0.5% deoxycholate, 0.05% SDS, 2 mm EDTA, 0.2 mm PMSF, and the protease and phosphatase inhibitors described above. The kinases were immunoprecipitated with anti-Myc antibody and, after five washes with RIPA buffer, each immunoprecipitate was divided into 2 aliquots and electrophoresed. One gel was stained with Coomassie Blue, and [3H]palmitate incorporation was detected by autoradiography. The radioactive band (containing PI4KIIα) was cut from the gel, solubilized with 30% H2O2 for 16 h at 60 °C, and subjected to liquid scintillation counting. The other aliquot was used for immunoblotting with anti-Myc antibodies and 125I-labeled secondary antibodies to estimate relative amounts of PI4KIIα. Blots were scanned by phosphorimaging (Fujifilm BAS 1500). Levels of [3H]palmitate incorporation were normalized to the amount of kinase in each sample. Immunoprecipitation-Total cell lysates or solubilized membranes were cleared of insoluble material by centrifugation at 15,000 × g for 15 min. Resulting supernatants were then precleared by incubation with recombinant protein G-Sepharose 4B conjugate for 30 min. Myc-tagged proteins were immunoprecipitated following 4 h of incubation with anti-Myc antibodies that were chemically cross-linked with dimethyl pimelimidate to protein G-Sepharose. Cross-linking prevents the elution from protein G-Sepharose of the IgG heavy chain, which has a similar molecular weight as PI4KIIα. The immunoprecipitates were washed extensively before analysis. Inhibition of Palmitoylation with 2-Bromopalmitate (2-BP)-COS-7 cells expressing WT Myc-PI4KIIα were treated with 2-BP according to a procedure described by Webb et al. (21Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Briefly, 12 h after transfection, COS-7 cells were incubated for an additional 12 h in DMEM containing 2.5% dialyzed fetal bovine serum (FBS), 0.25% defatted BSA, with or without 100 μm 2-BP (from a 0.1 m stock solution dissolved in ethanol). To measure [3H]palmitate incorporation into PI4KIIα,[3H]palmitate was added for the last 4 h of incubation with 2-BP or vehicle. Lipid Kinase Assays-PI4K activity was determined by measuring phosphorylation of phosphatidylinositol (PI) using [γ-32P]ATP (10 mCi/ml) as radioactive phosphate donor. Typically, assays were performed on 1% Triton X-100 extracts of membranes from cells expressing WT or mutant Myc-PI4KIIα. These extracts were incubated with PI/Triton X-100 micelles at a 1:10 molar ratio of lipid to detergent, and assays were initiated by addition of MgCl2 and ATP. Final concentrations of PI, ATP, and MgCl2 were 1, 0.5, and 15 mm, respectively. The PI/Triton X-100 micelles were prepared by sonicating PI dissolved (after evaporating chloroform under N2) in a solution containing 50 mm Tris-HCl (pH 7.5), 6 mm Triton X-100, and 0.5 mg/ml BSA. Following incubation, lipids were extracted according to Bligh and Dyer (22Bligh E.G. Dyer W.J. Can. J. Med. Sci. 1959; 37: 911-917Google Scholar) and separated by TLC in a solvent system consisting of n-propyl alcohol/H2O/NH4OH (65:20:15). Radioactive PI4P spots were detected by autoradiography, scraped, and subjected to scintillation counting to quantify radioactivity. The relative amounts of kinase in samples were estimated by quantitative immunoblotting using 125I-labeled secondary antibodies. When assaying purified kinases (as in Fig. 3B), PI4P production was measured directly by scintillation counting without resolving the lipids on TLC. In some experiments (Fig. 7, C and D), PI4K activity was measured in intact membranes in the absence of detergent, with or without PC/PI liposomes. To prepare liposomes, PC and PI (in chloroform) were mixed at the molar ratio of 9:1, dried under N2, and resuspended in 0.3 m sucrose solution to obtain final concentration of 18 and 2 mm PC and PI, respectively. After overnight incubation at 4 °C on a rotator, the suspensions were passed 20 times through a 0.1 μm filter using a Mini-Extruder (Avanti Polar Lipids). Activities of the expressed kinase were estimated by subtracting the activity of membrane extracts from mock-transfected cells. Expression levels were similar as determined by immunoblotting.FIGURE 7Activity of WT and FFPFF mutant of PI4KIIα. Kinase activities of WT PI4KIIα and the FFPFF mutant are shown. A, activities assayed using PI/Triton X-100 micelles as substrate. Membranes prepared from transfected COS-7 cells were extracted with Triton X-100, and the extracts were assayed as described in Fig. 2B. Data are from triplicate measurements of three experiments. B, phosphorylation of PI and PIP in COS-7 cells transfected with WT PI4KIIα or the SSPSS or FFPFF mutants. Cells were labeled with 32Pi for 4 h, and lipids were extracted and separated by TLC. Levels of radioactive PIP and PIP2 are expressed as relative to mock-transfected cells (control). Results represent the mean ± S.E. of single measurements from six experiments. Expression levels of WT and mutant kinases were nearly identical within each experiment, as determined by immunoblotting. C, phosphorylation of endogenous PI in membranes isolated from transfects of COS-7 cells. Membranes were incubated for 10 min with 0.5 mm [γ-32P] ATP, and then lipids were extracted and subjected to TLC to estimate incorporation of radioactive phosphate into PI4P. Activities of membranes from mock-transfected cells (∼2-3 pmol/mg/min) were subtracted. Relative expression levels were estimated by immunoblotting with 125I-labeled secondary antibodies. Results are the average of duplicate measurements from a representative experiment. D, activities assayed using PI/PC liposomes as substrate. Intact membranes from transfected COS-7 cells were preincubated with 0.5 mm [γ-32P] ATP for 30 min to maximally phosphorylate endogenous PI and then further incubated for the indicated time with PC/PI liposomes (18 mm PC, 2 mm PI). The basal activity in the absence of exogenous PI was subtracted. Results are the average of duplicate measurements of one experiment, which is representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Quantification of Phosphoinositides in Cells-Sixteen hours after transfection with plasmids encoding WT or mutant PI4KIIα, COS-7 cells were labeled for 4 h with 25 μCi/ml [32P]orthophosphate in phosphate-free DMEM, then scraped into methanol/concentrated HCl (10:1), and transferred into tubes, and lipids were extracted with chloroform. The organic phase was collected and washed twice with an equal volume of methanol and 1 n HCl (1:1). Aliquots were counted in a scintillation counter, and equal amounts of radioactivity were spotted onto TLC plates and developed as described above. Immunofluorescence Microscopy-COS-7 and HeLa cells grown on coverslips were transfected with WT or mutant MycPI4KIIα. Sixteen hours after transfection, cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min on ice, blocked with 1% BSA and 3% donkey serum in PBS, and stained with anti-Myc antibody followed by rhodamine-conjugated secondary antibody. Fluorescence was visualized using a Zeiss LSM510 confocal microscope. Other Procedures-COS-7 and HeLa cells were grown in DMEM containing 10% fetal bovine serum (FBS) and antibiotics. Cells were transiently transfected using Lipofectamine 2000 according to the manufacturer's instructions and were used 16-24 h after transfection. Protein concentrations were determined using a modified Lowry method (23Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar, 24Peterson G.L. Anal. Biochem. 1979; 100: 201-220Crossref PubMed Scopus (884) Google Scholar) with BSA as a standard. SDS-PAGE was carried out according to the method of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Immunoblot analysis was carried out by the method of Towbin et al. (26Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar) as described previously (27Wagner M.C. Barylko B. Albanesi J.P. J. Cell Biol. 1992; 119: 163-170Crossref PubMed Scopus (151) Google Scholar). Effect of Palmitoylation on the Enzymatic Activity of PI4KIIα-This study was motivated by our earlier observation that deletion of a cysteine-rich motif, 173CCPCC177, from rat PI4KIIα abolishes both its ability to incorporate [3H]palmitate in cells and its catalytic activity in vitro (14Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To ascertain that the loss of activity is because of the failure of the mutant to undergo palmitoylation, and not to the deletion itself, we inhibited palmitoylation using the nonmetabolizable palmitate analog 2-BP (21Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). Treatment of cells with 100 μm 2-BP for 12 h reduced [3H]palmitate incorporation into Myc-tagged PI4KIIα by 75% (Fig. 1A) and its kinase activity by 80% (Fig. 1B). Despite this reduction in palmitoylation, the kinase remained tightly associated with membranes, even in the presence of 1 m NaCl (Fig. 1C). However, unlike PI4KIIα from untreated cells, which exists almost entirely as an integral membrane protein, PI4KIIα from 2-BP-treated cells behaved predominantly as a peripheral membrane protein that was extractible by 0.1 m Na2CO3 (pH 11) in the absence of detergent. Similar results were obtained when PI4KIIα palmitoylation was prevented by mutating all four cysteines in the 173CCPCC177 motif to serines. However, in this case no palmitoylation was detectable (Fig. 2A), and the mutant expressed much lower catalytic activity (Fig. 2B). As expected, this SSPSS mutant did not bind integrally to membranes (Fig. 2C). Moreover, its association with membranes was considerably weaker than that of the wild-type kinase from 2-BP treated cells, with 25% of the mutant being cytosolic (extractible in 0.1 m NaCl) and another 40% solubilized by 1 m NaCl. This weaker membrane binding may be due to the greater intrinsic hydrophobicity of cysteine than serine (28Nagano N. Ota M. Nishikawa K. FEBS Lett. 1999; 458: 163-170Crossref Scopus (116) Google Scholar). To further demonstrate the importance of palmitoylation for catalysis, we compared the activities of recombinant PI4KIIα purified from Sf9 cells or E. coli. The bacterially expressed kinase is unpalmitoylated, as E. coli cells do not express protein acyltransferases, whereas PI4KIIα incorporates [3H]palmitate when expressed in Sf9 cells (Fig. 3A). The Sf9-derived kinase had an activity of ∼150 min-1, whereas the activity of bacterially expressed PI4KIIα never exceeded 10 min-1 (Fig. 3B). These results confirm that palmitoylation strongly enhances PI4KIIα activity. Moreover, because the unpalmitoylated enzymes are largely membrane-associated, albeit not integrally, the role of this modification in catalysis is not merely to tether PI4KIIα to substrate-containing membranes or micelles. Mutational Analysis of the Palmitoylation Motif of PI4KIIα-Site-directed mutagenesis was used to identify the acylated residue(s) within the 173CCPCC177 motif. Individual and multiple cysteines in the motif were mutated to serine, a structurally similar amino acid. Individual mutations of Cys-173, Cys-174, or Cys-176 resulted in 35-54% decrease in [3H]palmitate incorporation, which corresponds closely to the reduction in integral membrane binding of the mutants (Fig. 4A). In contrast, the C177S mutant was palmitoylated normally, although it showed a 20% reduction in integral association with membranes. Perhaps this cysteine contributes to membrane binding by virtue of its intrinsic hydrophobicity (28Nagano N. Ota M. Nishikawa K. FEBS Lett. 1999; 458: 163-170Crossref Scopus (116) Google Scholar) rather than by undergoing acylation. Kinase activity was more strongly inhibited by mutation of Cys-173 or Cys-" @default.
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• W2029878872 date "2009-04-01" @default.
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