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• W2101133471 abstract "Diacylglycerol kinase ζ is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase ζ has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase ζ interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase ζ-interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase ζ. Because diacylglycerol kinase ζ is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase ζ regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase ζ showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase ζ-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase ζ in the modulation of membrane trafficking, which may also help to define SNX27 function. Diacylglycerol kinase ζ is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase ζ has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase ζ interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase ζ-interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase ζ. Because diacylglycerol kinase ζ is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase ζ regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase ζ showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase ζ-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase ζ in the modulation of membrane trafficking, which may also help to define SNX27 function. Intracellular membrane traffic requires a complex molecular machinery with a plethora of small GTPases, adaptors, and coat components that must be assembled and disassembled in different steps to ensure correct vesicle formation. Membrane lipids are key components in this process; many proteins involved in vesicle formation have lipid-binding domains, and modulation of lipid-modifying enzymes profoundly alters secretion and/or endocytosis (1Gruenberg J. Lipids in endocytic membrane transport and sorting.Curr. Opin. Cell Biol. 2003; 15: 382-388Crossref PubMed Scopus (94) Google Scholar). Diacylglycerol (DAG) 1The abbreviations used are: DAG, diacylglycerol; Ab, antibody; Ank, ankyrin; BAR, Bin/amphiphysin/Rvs; CT, C-terminal; DGK, diacylglycerol kinase; EEA1, early endosomal antigen 1; ERC, endocytic recycling compartment; FL, full-length; X-α-gal, 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside; GFP, green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; HIV-1, human immunodeficiency virus, type 1; IF, immunofluorescence; IS, immunological synapse; PA, phosphatidic acid; PDZbm, PDZ-binding motif; PI, phosphatidylinositol; PI3K, phosphoinositide-3-OH kinase; PKC, protein kinase C; PX, Phox homology; PDZ, postsynaptic density protein, disc-large, and zonula occludens-1; PLD, phospholipase D; RA, Ras association; RNAi, RNA interference; SNX, sorting nexin; TCR, T cell receptor; TfR, transferrin receptor; Tf-Rhod, transferrin tetramethylrhodamine; WB, Western blot; SD, synthetic dropout. is a lipid with important functions in membrane trafficking. When generated in restricted membrane regions, the characteristic negative curvature of DAG promotes the membrane constriction essential for fission and the instability required for fusion (2Goni F.M. Alonso A. Structure and functional properties of diacylglycerols in membranes.Prog. Lipid Res. 1999; 38: 1-48Crossref PubMed Scopus (212) Google Scholar, 3Burger K.N. Greasing membrane fusion and fission machineries.Traffic. 2000; 1: 605-613Crossref PubMed Scopus (112) Google Scholar, 4Lentz B.R. Malinin V. Haque M.E. Evans K. Protein machines and lipid assemblies: current views of cell membrane fusion.Curr. Opin. Struct. Biol. 2000; 10: 607-s615Crossref PubMed Scopus (133) Google Scholar, 5Jun Y. Fratti R. Wickner W. Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion.J. Biol. Chem. 2004; 279: 53186-53195Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In addition to modifying membrane characteristics, DAG binds to and activates various proteins needed for vesicle formation, such as protein kinase D and ADP-ribosylation factor (Arf) GTPase-activating protein (6Baron C. Malhotra V. Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane.Science. 2002; 295: 325-328Crossref PubMed Scopus (341) Google Scholar, 7Wong T. Fairn G. Poon P. Shmulevitz M. McMaster C. Singer R. Johnston G. Membrane metabolism mediated by Sec14 family members influences Arf GTPase activating protein activity for transport from the trans-Golgi.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12777-12782Crossref PubMed Scopus (23) Google Scholar). In addition to DAG, phosphatidic acid (PA) also confers the negative curvature that facilitates vesicle fission or fusion (3Burger K.N. Greasing membrane fusion and fission machineries.Traffic. 2000; 1: 605-613Crossref PubMed Scopus (112) Google Scholar, 8Huttner W.B. Zimmerberg J. Implications of lipid microdomains for membrane curvature, budding and fission.Curr. Opin. Cell Biol. 2001; 13: 478-484Crossref PubMed Scopus (198) Google Scholar, 9Kooijman E. Chupin V. de Kruijff B. Burger K. Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid.Traffic. 2003; 4: 162-174Crossref PubMed Scopus (301) Google Scholar, 10Kooijman E.E. Chupin V. Fuller N.L. Kozlov M.M. de Kruijff B. Burger K.N. Rand P.R. Spontaneous curvature of phosphatidic acid and lysophosphatidic acid.Biochemistry. 2005; 44: 2097-2102Crossref PubMed Scopus (218) Google Scholar). PA can also bind to and activate enzymes that participate in membrane trafficking, such as coatomer, Arf, N-ethylmaleimide-sensitive factor, kinesin, phosphatidylinositol-4-phosphate 5-kinase, and Arf6 GTPase-activating protein (11Jenkins G. Fisette P. Anderson R. Type I phosphatidylinositol 4-phosphate 5-kinase isoforms are specifically stimulated by phosphatidic acid.J. Biol. Chem. 1994; 269: 11547-11554Abstract Full Text PDF PubMed Google Scholar, 12Jones D.R. Sanjuan M.A. Merida I. Type Iα phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid.FEBS Lett. 2000; 476: 160-165Crossref PubMed Scopus (66) Google Scholar, 13Manifava M. Thuring J.W. Lim Z.Y. Packman L. Holmes A.B. Ktistakis N.T. Differential binding of traffic-related proteins to phosphatidic acid- or phosphatidylinositol (4,5)-bisphosphate-coupled affinity reagents.J. Biol. Chem. 2001; 276: 8987-8994Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Jovanovic O. Brown F. Donaldson J. An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling.Mol. Biol. Cell. 2006; 17: 327-355Crossref PubMed Scopus (64) Google Scholar, 15Jackson T.R. Brown F.D. Nie Z. Miura K. Foroni L. Sun J. Hsu V.W. Donaldson J.G. Randazzo P.A. ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.J. Cell Biol. 2000; 151: 627-638Crossref PubMed Scopus (154) Google Scholar). The diacylglycerol kinase (DGK) family is an evolutionarily conserved family of lipid kinases that phosphorylate DAG to produce PA (16Luo B. Regier D.S. Prescott S.M. Topham M.K. Diacylglycerol kinases.Cell. Signal. 2004; 16: 983-989Crossref PubMed Scopus (0) Google Scholar). All members of the family have at least two N-terminal cysteine-rich domains (C1) and a conserved catalytic domain. These proteins have additional functional domains that allow their classification into five subgroups (I–V). Their structural diversity, distinct tissue expression, and specific intracellular localization confer on each DGK isoform the ability to regulate different DAG and PA pools and thus to participate in diverse signaling complexes (17Kanoh H. Yamada K. Sakane F. Diacylglycerol kinases: emerging downstream regulators in cell signaling systems.J. Biochem. (Tokyo). 2002; 131: 629-633Crossref PubMed Scopus (116) Google Scholar). DGKζ belongs to the type IV DGK family, characterized by a MARCKS (myristoylated alanine-rich protein kinase C (PKC) kinase substrate) homology domain as well as a C-terminal region with four ankyrin (Ank) repeats and an ETAV sequence (18Bunting M. Tang W. Zimmerman G.A. McIntyre T.M. Prescott S.M. Molecular cloning and characterization of a novel human diacylglycerol kinase ζ.J. Biol. Chem. 1996; 271: 10230-10236Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). These four amino acids can bind the class I PDZ (post-synaptic density protein, disc-large, and zonula occludens-1) domain, thus constituting a PDZ-binding motif (PDZbm) (19Sheng M. Sala C. PDZ domains and the organization of supramolecular complexes.Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1044) Google Scholar). DGKζ is expressed ubiquitously and is associated with cell cycle regulation, cytoskeletal reorganization, and modulation of the immune response among other functions (20Avila-Flores A. Santos T. Rincon E. Merida I. Modulation of the mammalian target of rapamycin pathway by diacylglycerol kinase-produced phosphatidic acid.J. Biol. Chem. 2005; 280: 10091-10099Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 21Olenchock B.A. Guo R. Silverman M.A. Wu J.N. Carpenter J.H. Koretzky G.A. Zhong X.P. Impaired degranulation but enhanced cytokine production after FcεRI stimulation of diacylglycerol kinase ζ-deficient mast cells.J. Exp. Med. 2006; 203: 1471-1480Crossref PubMed Scopus (49) Google Scholar, 22Topham M.K. Bunting M. Zimmerman G.A. McIntyre T.M. Blackshear P.J. Prescott S.M. Protein Kinase C regulates the nuclear localization of DGKζ.Nature. 1998; 394: 697-700Crossref PubMed Scopus (253) Google Scholar, 23Yakubchyk Y. Abramovici H. Maillet J.C. Daher E. Obagi C. Parks R.J. Topham M.K. Gee S.H. Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase ζ.Mol. Cell. Biol. 2005; 25: 7289-7302Crossref PubMed Scopus (54) Google Scholar, 24Zhong X. Hainey E.A. Olenchock B.A. Jordan M.S. Maltzman J.S. Nichols K.E. Shen H. Koretzky G.A. Enhanced T cell responses due to diacylglycerol kinase ζ deficiency.Nat. Immunol. 2003; 4: 882-890Crossref PubMed Scopus (185) Google Scholar). DGKζ is expressed abundantly in T lymphocytes; studies using GFP-coupled DGKζ chimeras in live T cells demonstrated receptor-dependent membrane translocation of this enzyme (25Santos T. Carrasco S. Jones D.R. Merida I. Eguinoa A. Dynamics of diacylglycerol kinase ζ translocation in living T-cells. Study of the structural domain requirements for translocation and activity.J. Biol. Chem. 2002; 277: 30300-30309Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This analysis indicated that the C-terminal region of the protein confers specificity for protein translocation, suggesting the importance of regulation based on protein-protein interactions. Accordingly recent studies showed DGKζ interaction with several proteins such as PKC, Rac, syntrophins, leptin receptor, and Src (23Yakubchyk Y. Abramovici H. Maillet J.C. Daher E. Obagi C. Parks R.J. Topham M.K. Gee S.H. Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase ζ.Mol. Cell. Biol. 2005; 25: 7289-7302Crossref PubMed Scopus (54) Google Scholar, 26Hogan A. Shepherd L. Chabot J. Quenneville S. Prescott S.M. Topham M.K. Gee S.H. Interaction of γ1-syntrophin with diacylglycerol kinase-ζ. Regulation of nuclear localization by PDZ interactions.J. Biol. Chem. 2001; 276: 26526-26533Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Liu Z. Chang G.Q. Leibowitz S.F. Diacylglycerol kinase ζ in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation.J. Biol. Chem. 2001; 276: 5900-5907Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Luo B. Prescott S. Topham M. Association of diacylglycerol kinase ζ with protein kinase C α: spatial regulation of diacylglycerol signaling.J. Cell Biol. 2003; 160: 929-937Crossref PubMed Scopus (91) Google Scholar, 29Davidson L. Pawson A.J. De Maturana R.L. Freestone S.H. Barran P. Millar R.P. Maudsley S. Gonadotropin-releasing hormone-induced activation of diacylglycerol kinase-ζ and its association with active c-src.J. Biol. Chem. 2004; 279: 11906-11916Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), indicating that DGKζ functions may be largely dependent on the formation of distinct protein complexes. Using a mass spectrometry-based analysis of DGKζ-interacting proteins, we identified sorting nexin 27 (SNX27), a member of the SNX family of proteins involved in membrane traffic and protein sorting (30Worby C.A. Dixon J.E. Sorting out the cellular functions of sorting nexins.Nat. Rev. Mol. Cell. Biol. 2002; 3: 919-931Crossref PubMed Scopus (332) Google Scholar, 31Carlton J. Bujny M. Rutherford A. Cullen P. Sorting nexins—unifying trends and perspectives.Traffic. 2005; 6: 75-82Crossref PubMed Scopus (159) Google Scholar). The association between these proteins was direct and was mediated by interaction of the SNX27 PDZ domain with the C terminus of DGKζ. SNX27 was expressed in hematopoietic cells and localized to the endocytic recycling system of T lymphocytes. Finally we traced transferrin receptor (TfR) recycling, which was accelerated in cells with diminished DGKζ levels. Our results identify SNX27 as a new DGKζ-binding protein and unveil a function for SNX27·DGKζ complex in the control of protein trafficking of T cells. Orthovanadate, PMSF, poly(dl-lysine) and Igepal CA-630 were from Sigma. Leupeptin and aprotinin were purchased from Roche Applied Science, wortmannin was from Calbiochem, and transferrin tetramethylrhodamine (Tf-Rhod) was from Molecular Probes (Leiden, The Netherlands). Rabbit polyclonal anti-DGKζ antibody (Ab) raised against a C-terminal peptide was a generous gift of Dr. Kaoru Goto (Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, Japan) (32Hozumi Y. Ito T. Nakano T. Nakagawa T. Aoyagi M. Kondo H. Goto K. Nuclear localization of diacylglycerol kinase ζ in neurons.Eur. J. Neurosci. 2003; 18: 1448-1457Crossref PubMed Scopus (82) Google Scholar). Rabbit polyclonal anti-DGKζ Ab raised against an N-terminal peptide was a generous gift of Dr. M. K. Topham (University of Utah, Salt Lake City, UT) (33Abramovici H. Hogan A. Obagi C. Topham M. Gee S. Diacylglycerol kinase-ζ localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.Mol. Biol. Cell. 2003; 14: 4499-4511Crossref PubMed Scopus (76) Google Scholar). To generate the polyclonal anti-SNX27, the N-terminal 265-residue fragment of SNX27, which contains PDZ and phox homology (PX) domains, was produced as a GST fusion protein in bacteria. The fragment was cleaved with thrombin and used to immunize rabbits with Freund's adjuvant (Invitrogen). To affinity purify antibodies to SNX27, serum from immunized rabbits was incubated with the antigen coupled to cyanogen bromide-activated Sepharose (Amersham Biosciences). Bound antibody was eluted with Immuno-Pure IgG elution buffer (Pierce), neutralized with PBS, pH 7.4, and then dialyzed against the same solution. We used the following mouse monoclonal antibodies: anti-hemagglutinin (anti-HA) (Babco, Richmond, CA), -Myc (Cell Signaling Technology, Danvers, MA), -GST (Santa Cruz Biotechnology, Santa Cruz, CA), -GFP (Roche Applied Science), -tubulin (Sigma), -early endosomal antigen 1 (EEA1), -GM130 and -SNX2 (BD Transduction Laboratories), and -CD63 (Oncogene Research, San Diego, CA). Mouse anti-human TfR and rabbit anti-Rab11 were from Zymed Laboratories Inc. The monoclonal Ab to human LAMP1 developed by J. Thomas August and James E. K. Hildreth was obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD, National Institutes of Health and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA). Cy3- and Cy5-conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA), and Alexa 488-conjugated Ab was from Molecular Probes. The pcDNA3MycDGKζ, GFP-DGKζ, and HA-DGKζCT constructs were described previously (25Santos T. Carrasco S. Jones D.R. Merida I. Eguinoa A. Dynamics of diacylglycerol kinase ζ translocation in living T-cells. Study of the structural domain requirements for translocation and activity.J. Biol. Chem. 2002; 277: 30300-30309Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For generation of GST fusion proteins, pcDNA3MycDGKζ was BglII-digested, blunted, and KpnI-digested, and the 3.9-kb fragment was subcloned in the pEBG eukaryotic GST vector digested with KpnI/ClaI (GST-DGKζFL). The construct encoding the C-terminal region of the protein (GST-DGKζCT, including the four ankyrin repeats and the PDZbm) was excised from pGEM-T with NotI and then subcloned in pEBG vector digested with NotI. The human Myc-tagged SNX27b full length (Myc-SNX27bFL) was described previously (34Joubert L. Hanson B. Barthet G. Sebben M. Claeysen S. Hong W. Marin P. Dumuis A. Bockaert J. New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.J. Cell Sci. 2004; 117: 5367-5379Crossref PubMed Scopus (125) Google Scholar). Myc-tagged deletion mutants (Myc-SNX27bΔRA/-SNX27bΔPX/-SNX27bRA) were generated from the full-length SNX27b using PCR and subcloned into pDMyc-neo vector, which is a modified version of the pCIneo vector (Stratagene) (35Seet L.F. Hong W. Endofin, an endosomal FYVE domain protein.J. Biol. Chem. 2001; 276: 42445-42454Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), with the same restriction enzyme sites as above. All constructs were confirmed by sequencing. The coding region corresponding to mouse SNX27a cloned into the pSPORT1 vector was obtained from Open Biosystems (Clone number 6431126, GenBank™ accession number BC053495); pSPORT1-SNX27a was digested with SalI/BamHI, and the 1.7-kb fragment was subcloned in pEGFP-C2 digested with SalI/BamHI. To generate GFP-SNX27aΔPDZ, the sequence 336TCCGAG341 in GFP-SNX27a was mutated to GTCGAC to generate a SalI site. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). The plasmid was cleaved with SalI, and the resulting 6.4-kbp fragment lacking the PDZ domain was then religated. For yeast two-hybrid interaction assays, four DGKζ constructs were subcloned in pGBKT7 vector fused to GAL4BD as bait, the SNX27 N-terminal region (containing the PDZ and PX domains) was subcloned into pGADT7 vector fused to GAL4AD, and pGBKT7DGKζFL was generated by digesting pcDNA3MycDGKζ with EcoRI, and the 3.4-kb fragment was subcloned into EcoRI-digested pGBKT7. pGBKT7DGKζΔAnk was generated from pGBKT7DGKζFL digested with SacI and religated. To generate pGBKT7DGKζΔPDZbm, GFP-DGKζΔPDZbm was digested with NcoI, blunted, and digested with EcoRI/AflII; the 2.9-kb fragment was subcloned in pGBKT7 digested with EcoRI/SmaI. The pGBKT7CT construct, including the four ankyrin repeats and PDZbm, was generated by PCR from GFP-DGKζFL with appropriate primers (AnkPDZ1, 5′-GAATTCGCACTGCCCCAAGGTGAAG-3′; AnkPDZ, 5′-GTCGACTACACAGCTGTCTCCTGGTCC-3′), including two restriction sites, EcoRI and SalI. The 430-bp PCR product was subcloned in the pGEM-T Easy vector and then excised with EcoRI/SalI for subcloning in EcoRI/SalI-digested pGBKT7. To generate pGADT7SNX27ΔRA, pCIneoSNX27 was digested with XhoI/XbaI, and the 730-bp fragment containing the N-terminal portion of the protein was subcloned in pCDNA3 digested with XhoI/XbaI (pCDNA3SNX27ΔRA). pCDNA3SNX27ΔRA was digested with XbaI, blunted, and EcoRI-digested; the 730-bp fragment was subcloned in pGADT7 digested with EcoRI/SmaI. The rat basophilic leukemia mast cell line was provided by Dr. S. Corbalán García (Departamento de Bioquímica y Biología Molecular, Universidad de Murcia, Murcia, Spain). The following cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA): K562 human chronic myelogenous leukemia, the CTLL2 mouse cytotoxic cell line, the EL4 mouse T lymphoma, Jurkat human acute T cell leukemia, and HEK293/HEK293T human embryonic cell lines. Thymocytes were obtained from BALB/c mice following standard protocols, and dendritic cells were from Dr. C. F. Ardavín (Centro Nacional de Biotecnología/CSIC, Madrid, Spain). Jurkat, HEK293, and HEK293T cell lines were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Sigma) and 2 mm glutamine (37 °C, 5% CO2). Jurkat cells in logarithmic growth phase were transfected (1.2 × 107 in 400 ml of complete medium) with 20 μg of plasmid DNA by electroporation with a Gene Pulser (Bio-Rad; 270 V, 975 microfarads); cells were immediately transferred to 10 ml of complete medium and assayed after 24 h. HEK293T and HEK293 cells were transfected using Jet-PEI reagent (PolyPlus Transfection, Illkirch, France) and Lipofectamine Plus (Invitrogen), respectively. For transfection, HEK293T cells were plated in 150-mm culture dishes. When cells reached 60% confluence (24 h), GST, GST-DGKζFL, and GST-DGKζCT transfection was carried out using Jet-PEI. After 24 h, cells were lysed in Nonidet P-40 buffer (150 mm NaCl, 10 mm NaF, 10 mm Na4P2O7, 50 mm Tris-HCl, pH 7.5, 1% Igepal CA-630, and 0.5 mm PMSF/protease inhibitor mixture), and lysates were centrifuged (20,800 × g, 10 min, 4 °C). Supernatants were incubated with glutathione-Sepharose 4B (Amersham Biosciences) (overnight, 4 °C) to batch purify GST recombinant proteins. Beads were then washed extensively with BC500 buffer (25 mm Tris-HCl, pH 7.8, 500 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 0.2% Igepal CA-630, 1% Triton X-100, and 0.1% sodium deoxycholate). Finally bound proteins were eluted with 5× Laemmli buffer (36Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). Aliquots of eluted proteins were analyzed by 7.5% SDS-PAGE and visualized by Coomassie Blue staining. Bands of interest were excised and analyzed by MS. Coomassie Blue-stained bands were excised manually from gels, deposited in 96-well plates, and processed automatically in an Investigator ProGest protein digestion station (Genomics Solutions, Cambridgeshire, UK) where samples were in-gel-reduced, alkylated with iodoacetamide, and trypsin-digested (37Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar). Resulting peptides were analyzed by MALDI-TOF MS; 0.3 μl of matrix solution (5 mg/ml 2,5-dihydrobenzoic acid in 33% (v/v) aqueous acetonitrile and 0.1% (v/v) trifluoroacetic acid) was added to an AnchorChip MALDI target (Bruker Daltonics GmbH, Bremen, Germany) and allowed to dry at room temperature. A 0.3-μl aliquot of each peptide mixture was then deposited onto matrix surfaces and dried at room temperature. MALDI mass spectra were acquired automatically on a Bruker Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics) by FlexControl 1.1 software and processed by Xtof 5.1.1 software to analyze raw data. Each spectrum was calibrated internally with two trypsin autolysis reference ions, specifically 842.510- and 2211.105-Da peptides, to reach a typical mass measurement accuracy of ±30 ppm in the 800–3000 m/z range. All known contaminants were excluded during the process. The parameters used to analyze data were a signal-to-noise threshold of 20 and resolution higher than 4000. For protein identification, tryptic peptide masses were transferred to the BioTools 2.0 interface (Bruker Daltonics) to search in the National Center for Biotechnology non-redundant (NCBInr) database using Mascot software (Matrix Science, London, UK). Search parameters were set as follows: carbamidomethyl cysteine as fixed modification by the treatment with iodoacetamide, oxidized methionines as variable modification, peptide mass tolerance of 80 ppm, and one missed cleavage site. In all protein identifications, the probability Mowse scores were greater than the minimum score fixed as significant (78 in all cases) with a p value less than 0.05. DGKζ constructs were subcloned in pGBKT7 vector fused to GAL4BD as bait, and SNX27ΔRA was subcloned into pGADT7 vector fused to GAL4AD. The interaction assay was developed according to the manufacturer's protocols (Clontech). The AH109 yeast strain was co-transformed by the LiAc method with pGADT7SNX27ΔRA with each of the DGKζ constructs or with control empty vector. To select co-transformed yeast, cells were plated on SD medium lacking leucine and tryptophan. Growing colonies were replated on high stringency SD medium lacking leucine, tryptophan, alanine, and histidine plus 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside (X-α-gal) to confirm interacting proteins. We performed subcellular fractionation of Jurkat cells as described previously (38Palacios S. Lalioti V. Martinez-Arca S. Chattopadhyay S. Sandoval I.V. Recycling of the insulin-sensitive glucose transporter GLUT4. Access of surface internalized GLUT4 molecules to the perinuclear storage compartment is mediated by the Phe5-Gln6-Gln7-Ile8 motif.J. Biol. Chem. 2001; 276: 3371-3383Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Briefly 3 × 107 Jurkat cells in logarithmic growth phase were washed twice with PBS at 4 °C and harvested by centrifugation. Cells were resuspended in homogenization buffer A (250 mm sucrose, 20 mm Hepes, pH 7.4, 1 mm EDTA, leupeptin, pepstatin, aprotinin, 50 mm NaF, 50 mm glycerophosphate, 1 mm orthovanadate, and 1 mm PMSF) and then disrupted using a 23-gauge needle. Whole cells and nuclei were removed by centrifugation (800 × g, 10 min, 4 °C). All subsequent manipulations were performed at 4 °C. The postnuclear supernatant was centrifuged (20,000 × g, 20 min, 4 °C), and the high density microsome fraction was pelleted from the resulting supernatant by centrifugation (45,000 × g, 30 min, 4 °C). Low density microsomes were collected from the resulting supernatant by further centrifugation (180,000 × g, 90 min, 4 °C). The supernatant from this last centrifugation contained the cytosolic fraction. The pellet resulting from the 20,000 × g centrifugation contained the crude plasma membrane; it was collected and resuspended in buffer A, overlaid on 1 ml of 35% sucrose prepared in buffer A, and then centrifuged (100,000 × g, 1 h). The purified plasma membrane was collected from the top of the interphase, mixed with buffer B (buffer A without sucrose), and concentrated by centrifugation (108,000 × g, 40 min, 4 °C). All pellets were resuspended in the same volume of buffer A using a 25-gauge needle. Samples were analyzed by SDS-PAGE, loading the same volume for each fraction (the cytosol sample represented 1/25 of the total cytosol). Jurkat or HEK293 cells, transiently transfected with selected plasmids, were lysed in Nonidet P-40 buffer and cleared by centrifugation. Protein lysates (400 μg) were incubated with the indicated antibodies (2 μl, 1 h, 4 °C) followed by G protein coupled to Sepharose (1 h, 4 °C). Immunoprecipit" @default.
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• W2101133471 title "Proteomics Identification of Sorting Nexin 27 as a Diacylglycerol Kinase ζ-associated Protein" @default.
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