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• W2014375034 abstract "PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport. PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyveWT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyveK1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P2 in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyveWT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyveWT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport. Multivesicular bodies (MVBs) 1The abbreviations used are: MVBmultivesicular bodyPtdInsphosphatidylinositolChchaperoninPphosphateP2bisphosphateGSTglutathione S-transferaseGSHglutathioneTGNtrans-Golgi networkEGFPenhanced green fluorescent proteinMPRmannose 6-phosphate receptorHSPhigh speed pelletLSPlow speed pelletIRAPinsulin-regulated aminopeptidasentnucleotide(s)CMVcytomegalovirusHAhemagglutinin. (referred to also as prelysosomal compartment or late endosomes) are morphologically defined organelles that are a crossing point of several membrane trafficking pathways (reviewed in Refs. 1Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (936) Google Scholar, 2Piper R.C. Luzio J.P. Traffic. 2001; 2: 612-621Crossref PubMed Scopus (168) Google Scholar, 3Mukherjee S. Maxfield F.R. Traffic. 2000; 1: 203-211Crossref PubMed Scopus (196) Google Scholar, 4Mellman I. Warren G. Cell. 2000; 100: 99-112Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 5Gruenberg J. Rev. Mol. Cell. Biol. 2001; 2: 721-730Crossref PubMed Scopus (584) Google Scholar, 6Miaczynska M. Zerial M. Exp. Cell Res. 2002; 272: 8-14Crossref PubMed Scopus (142) Google Scholar, 7Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1023) Google Scholar, 8Pfeffer S. Cell. 2003; 112: 507-517Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Cell surface endocytosed materials destined for degradation enter MVBs en route to lysosomes. Next, newly synthesized acid hydrolases that function in lysosomes are diverted from the secretory pathway through binding to one of the two mannose 6-phosphate-receptor (MPR) types, and reach MVBs by the biosynthetic pathway. Finally, several transport routes that deliver cargo to the trans-Golgi network emanate from MVBs. To dispatch cargo in the correct direction, MVBs ought to perform numerous sorting functions, the molecular mechanisms of which are just beginning to be unraveled. PIKfyve enzyme, a large protein of 2052 amino acids, is one of the multiple molecules thought important in MVB morphogenesis and function (9Ikonomov O.C. Sbrissa A. Foti M. Carpentier J.-L. Shisheva A. Mol. Biol. Cell. 2003; 14: 4581-4591Crossref PubMed Scopus (93) Google Scholar). Encoded by a single-copy evolutionarily conserved gene on locus 2q34 in humans, mammalian PIKfyve is responsible for intracellular PtdIns 5-P and PtdIns 3,5-P2 biosynthesis (reviewed in Ref. 10Shisheva A. Cell Biol. Internat. 2001; 25: 1201-1206Crossref PubMed Scopus (70) Google Scholar). PIKfyve could also act as a protein kinase, but the physiologically relevant substrates are currently unknown (11Sbrissa A. Ikonomov O.C. Shisheva A. Biochemistry. 2000; 39: 15980-15989Crossref PubMed Scopus (51) Google Scholar). The enzyme is primarily cytosolic with ∼30% associated with membranes (12Shisheva A. Rusin B. Ikonomov O.C. DeMarco C. Sbrissa D. J. Biol. Chem. 2001; 276: 11859-11869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Immunofluorescence microscopy data co-localize a subset of membrane-bound PIKfyve with late-endosome markers consistent with a plausible role of PIKfyve in the function of this compartment. The first indication for such a role came from cell studies utilizing PIKfyve point mutants deficient in the lipid kinase activity (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14Ikonomov O.C. Sbrissa D. Mlak M. Kanzaki M. Pessin J. Shisheva A. J. Biol. Chem. 2002; 277: 9206-9211Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Expression of these mutants in different cell types was found to induce a dramatic dominant-negative effect in the form of a progressive accumulation of dilated cytoplasmic vacuoles of endocytic origin indicating the role of PIKfyve in maintaining endosome membrane homeostasis. Recent ultrastructural studies identify the swollen compartment as MVBs, which, in addition to a significant gain of limiting membranes, display a lower number of internal vesicles (9Ikonomov O.C. Sbrissa A. Foti M. Carpentier J.-L. Shisheva A. Mol. Biol. Cell. 2003; 14: 4581-4591Crossref PubMed Scopus (93) Google Scholar). Ectopic expression of active PIKfyve at higher levels or cytoplasmic microinjection of the PIKfyve lipid product, PtdIns 3,5-P2, restores the normal cell morphology (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 14Ikonomov O.C. Sbrissa D. Mlak M. Kanzaki M. Pessin J. Shisheva A. J. Biol. Chem. 2002; 277: 9206-9211Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Together, these results specify the important role of PIKfyve PtdIns 3,5-P2-generating activity and yet-to-be identified PtdIns 3,5-P2 downstream effectors in MVB biogenesis and function. multivesicular body phosphatidylinositol chaperonin phosphate bisphosphate glutathione S-transferase glutathione trans-Golgi network enhanced green fluorescent protein mannose 6-phosphate receptor high speed pellet low speed pellet insulin-regulated aminopeptidase nucleotide(s) cytomegalovirus hemagglutinin. Besides the C-terminally positioned catalytic domain, the PIKfyve molecule harbors three other evolutionarily conserved modules: a FYVE finger that interacts specifically with PtdIns 3-P, a DEP domain, found in other proteins important in membrane association, and a chaperonin domain, homologous to the TCP-1 complex that associates with and facilitates the folding of newly synthesized actin and tubulin (10Shisheva A. Cell Biol. Internat. 2001; 25: 1201-1206Crossref PubMed Scopus (70) Google Scholar). While the FYVE finger domain was found to function as a major determinant for the PtdIns 3-P-dependent PIKfyve localization to late endosome membranes (15Sbrissa D. Ikonomov O.C. Shisheva A. J. Biol. Chem. 2002; 277: 6073-6079Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), the significance of the other two conserved domains in PIKfyve function is still unknown. With the premise that the latter two domains serve to interact with yet-to-be identified proteins relevant to the role of PIKfyve in maintaining MVB morphology and function, we have initiated yeast two-hybrid screens using the individual chaperonin and DEP domains as baits. Here we report the identification of p40 transport factor as a binding protein for the PIKfyve chaperonin domain. p40 has been discovered previously in a search for proteins interacting with Rab9, a GTPase localized on late endosomes and facilitating late endosome-to-Golgi transport of MPR (16Diaz E. Schimmöller F. Pfeffer S.R. J. Cell Biol. 1997; 138: 283-290Crossref PubMed Scopus (118) Google Scholar). Through selective binding to the GTP-loaded Rab9, p40 promotes the in vitro transport of MPR from late endosomes to TGN (16Diaz E. Schimmöller F. Pfeffer S.R. J. Cell Biol. 1997; 138: 283-290Crossref PubMed Scopus (118) Google Scholar). Because anti-p40 antibodies, but not the p40-depleted cytosols, inhibit this transport step, Pfeffer and collaborators (16Diaz E. Schimmöller F. Pfeffer S.R. J. Cell Biol. 1997; 138: 283-290Crossref PubMed Scopus (118) Google Scholar) suggest the membrane-associated p40 is the active species that, together with the active Rab9, functions in the return of MPR to TGN. Here we demonstrate that PIKfyve chaperonin domain associates with p40 and that p40 membrane association is strictly dependent on PIKfyve enzymatic activity. Because p40 was found to be an in vitro substrate for the PIKfyve protein kinase activity, we speculate that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchors p40 to discrete membranes facilitating late endosome-to-TGN transport. Cell Cultures—Human embryonic kidney (HEK) 293 or COS-7 cells were maintained in Dulbecco's modified Eagle's medium, containing 10% fetal bovine serum, and antibiotics as described previously (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The generation and maintenance of stably transfected doxycycline-inducible HEK293 cell lines expressing PIKfyveWT (clone 9), the dominant-negative kinase-deficient PIKfyveK1831E mutant (clone 5), or the control cell (TetOn) line were described elsewhere (17Ikonomov O.C. Sbrissa D. Mlak K. Shisheva A. Endocrinology. 2002; 143: 4742-4754Crossref PubMed Scopus (66) Google Scholar, 18Sbrissa D. Ikonomov O.C. Deeb R. Shisheva A. J. Biol. Chem. 2002; 277: 47276-47284Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Antibodies—Anti-PIKfyve antibodies (R7069) were characterized earlier (19Shisheva A. Sbrissa D. Ikonomov O. Mol. Cell. Biol. 1999; 19: 623-634Crossref PubMed Scopus (102) Google Scholar, 20Sbrissa D. Ikonomov O.C. Shisheva A. J. Biol. Chem. 1999; 274: 21589-21597Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Monoclonal anti-GFP and anti-phosphoserine-specific (1C8) antibodies were from Clontech and Calbiochem, respectively. The polyclonal antibodies were from the following sources: anti-HA and anti-GST, gifts by Mike Czech; anti-p40, a gift by Susan Pfeffer; and anti-IRAP, a gift by Paul Pilch. Plasmids for Yeast Two-hybrid System—Three baits encompassing the evolutionarily conserved domains of the PIKfyve protein were inserted in-frame with the LexA into pNLexNLS for yeast two-hybrid screening. The first one constituted the FYVE finger plus the DEP domain of PIKfyve (residues 99-473) and was generated by subcloning the EcoRI fragment of the pGEX-1-PIKfyveL (99-473) vector into the EcoRI digest of pNLexNLS. The second bait corresponded to the DEP domain consensus sequence in PIKfyve (residues 384-445) and was generated by PCR (sense primer 5′-CCCGGAATTCGATCACCGTTAC and antisense primer 5′-CCCGGGATCCTTACAACGCATATTC) designed with the appropriate restriction sites for subcloning into the EcoRI/BamHI sites of pNLexNLS. The third construct encompassed the chaperonin-like (Ch) domain consensus sequence in PIKfyve (residues 616-868) and was generated by two consecutive subclonings into pGEX-3X and pNLexNSL vectors. Specifically, first, the 2-kbp EcoRV fragment (nucleotides 1983-3919) released from pBluescript II SK(+)PIKfyveWT cDNA was blunt-ligated into the SmaI site of pGEX-3X. After confirmation of the correct orientation, the BamHI fragment (0.76 kbp) of the above vector was released and subcloned into the BamHI-digested pNLexNLS. The correct organization and sequence of all constructs were confirmed by restriction endonuclease digestion and DNA sequencing. Other Constructs—Generation of pCMV5-HA-PIKfyveSWT and pCMV5-HA-PIKfyveSΔ560-12231 was described elsewhere (20Sbrissa D. Ikonomov O.C. Shisheva A. J. Biol. Chem. 1999; 274: 21589-21597Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Recombinant adenoviruses, expressing HA-PIKfyveSWT were produced as described elsewhere (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Full-length human p40 was generated in BglII/HindIII restriction sites of pEGFP-C2 (Clontech) using pQE31-p40 cDNA (a kind gift by Dr. Susan Pfeffer) and the following strategy. First, the N-terminal DNA fragment (from the ATG initiation codon up to the HindIII site) was obtained by PCR using BglII-flanked sense primer (5-CGCAGATCTCCATGAAGCCAAC), HindIII-flanked antisense primer (5′-GAGAAGCTTCTGTTTGG) and pQE31-p40 cDNA as a template. The PCR product (∼180 bp) was digested with BglII/HindIII and then ligated together with the HindIII fragment of pQE31-p40 (∼1 kbp) into BglII/HindIII-digested pEGFP-C2. The PCR-amplified sequence and the correct orientation of the construct were verified by sequencing and restriction endonuclease mapping. Full-length p40 fused in-frame with GST was generated by first amplifying the coding sequence of p40 by PCR using primers with added EcoRI or XhoI restriction sites: sense 5′-CGGCGAATTCATGAAGCAAC-3′ and antisense 5′-CAGCCTCGAGTTAGTCCACTAC-3′, with the restriction sites underlined. The PCR product was digested with HindIII, and the resulting two fragments of 180 bp (nt 150-329 of p40 sequence GI:5032014) and 968 bp (nt 329-1297) were subsequently cut with EcoRI and XhoI, respectively, and ligated into the EcoRI/XhoI digest of pGEX5X-1 (Amersham Biosciences). Two C-terminal GST fusion peptides, residues 133-372, and 264-372, were also generated in pGEX5X-1 vector. For the first C-terminal peptide, the EcoRI/XhoI fragment (encompassing nt 546-1297) derived from clone YA#19 (isolated in the two-hybrid screen here, see below) was inserted into the corresponding digest of pGEX5X-1. For the second one, the PstI/SalI fragment (encompassing nt 941-1297) from pEGFP-p40 was subcloned initially into the corresponding digest of pBluescript II SK+ and then released by SmaI/XhoI digest for subcloning into SmaI/XhoI sites of pGEX5X-1. An N-terminal GST fusion peptide of p40 (residues 1-264) was generated by first subcloning the BglII/PstI fragment of pEGFP-p40 (nt 150-941) into pCMV5. The EcoRI/SalI fragment of the pCMV5-p40 construct was then ligated into EcoRI/SalI digest of pGEX4T-2 (Amersham Biosciences). The organization of the constructs was confirmed by restriction endonuclease digest and sequencing. Yeast Two-hybrid Screening—Interaction mating version of two-hybrid screen was performed following already published procedures (21Kolonin M.G. Zhong J. Finley Jr., R.L. Methods Enzymol. 2000; 328: 26-46Crossref PubMed Google Scholar). Briefly, the bait cDNA in pNLex (NLS) vector was transformed in RFY 309 (MATa) strain. The suitability of the bait was determined by interaction mating of the transformed MATa with a MATα strain (RFY231) pretransformed with human HeLa cell cDNA library (22Gyuris J. Golemis E. Chetkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1324) Google Scholar) or only with the empty library vector pJG4-5. The positives for interaction, as a result of the activation of the LEU2 reporter gene, showed galactose-dependent growth on plates without leucine. Due to its low background, i.e. low activation of the LEU2 reporter by itself, the PIKfyve-chaperonin (PIKfyve-Ch) domain bait was selected for further screening. To avoid the characterization of repetitive cDNA isolates, candidate clones were amplified by PCR using primers based on the sequence adjacent to the multiple cloning site of pJG4-5. The PCR products were digested with HaeIII, and the restriction patterns were compared after agarose gel separation. The candidates with different patterns were further studied by cross-mating assay with PIKfyve-Ch bait and several unrelated baits. The clones confirmed as true positives, i.e. selectively interacting with the PIKfyve-Ch bait and being dependent on galactose but not glucose for growth without leucine, were further characterized by sequencing. GST Protein Production and Purification—All GST fusion proteins were produced in transformed XA-90 Escherichia coli strain. Bacteria were grown for 1.5 h and then stimulated with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for an additional 6 h. The cells were lysed with 1 mg/ml lysozyme in a buffer containing 50 mm Hepes, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride. Cell lysates were treated with DNase (0.1 mg/ml) in the presence of 10 mm MgCl2. Purification of the GST fusion proteins was achieved on GSH-agarose beads (Sigma) as described previously (15Sbrissa D. Ikonomov O.C. Shisheva A. J. Biol. Chem. 2002; 277: 6073-6079Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In some experiments, the GST proteins were eluted from the beads and dialyzed against 50 mm Hepes, pH 7.4. The concentration and quality of the purified proteins, bound to or eluted from the beads, were determined electrophoretically by the intensity of the Coomassie Blue-stained protein bands versus bovine serum albumin standard (Pierce). Transient Co-transfection and Subcellular Fractionation—HEK293 cell stable lines inducibly expressing PIKfyveWT of PIKfyveK1831E, seeded at 750,000 cells/100-mm plates were co-transfected with pEGFP-p40 cDNA using LipofectAMINE as a transfection reagent. 24-40 h post-transfection, cells were rinsed in homogenization “HES++ buffer” (20 mm Hepes, pH 7.5, 1 mm EDTA, supplemented with 1× protease inhibitor mixture (1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml pepstatin, and 1 mm benzamidine) and 1× phosphatase inhibitor mixture (25 mm β-glycerophosphate, 10 mm sodium pyrophosphate, 50 mm NaF, and 2 mm NaVO3) at room temperature and then scraped in the same buffer at 4 °C. Cells were then homogenized in a motor-driven Teflon/glass homogenizer with 15 strokes at 1700 rpm. Homogenates were subjected to subcellular fractionation following published protocols established for 3T3-L1 adipocytes (12Shisheva A. Rusin B. Ikonomov O.C. DeMarco C. Sbrissa D. J. Biol. Chem. 2001; 276: 11859-11869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) with modifications. Briefly, homogenates were first centrifuged at 9,000 × g for 20 min at 4 °C (Sorval Instrument Division, SS-34 rotor) to obtain the low speed pellet (LSP). The supernatant was then centrifuged in a TLA 100.3 rotor (Beckman Instruments Inc.) at 4 °C for 24 min at 200,000 × g to separate the high speed pellet (HSP) from the fraction of the soluble proteins (referred to herein as cytosol). Pellets were resuspended in “HES++ buffer” to a protein concentration of ∼2 mg/ml (bicinchoninic protein assay kit, Pierce, Rockford, IL). Fractions were analyzed by immunoblotting with the indicated antibodies. Cytosolic fractions were subjected to immunoprecipitation with anti-HA or anti-PIKfyve antibodies as described below. In some experiments cultured cells were fractionated into total membrane and cytosol. In this case, cells were homogenized as above and then fractionated by two sequential centrifugations at 800 × g for 3 min to eliminate the nuclear pellet and then at 200,000 × g for 20 min in a TLA 100.3 rotor, to obtain the total membrane and cytosolic fractions. Total membrane pellets were resuspended as above, and the fractions were analyzed by immunoblotting or immunoprecipitation. Equilibrium Centrifugation in Self-formed Iodixanol Gradient—A total membrane fraction prepared from HEK293 cell stable lines inducibly expressing PIKfyveWT and resuspended in “HES++ buffer” were mixed with iodixanol (OptiPrep; Sigma) in a polyallomer Quick-Seal centrifuge tube to 30% iodixanol and 128 mm sucrose concentration as described previously (12Shisheva A. Rusin B. Ikonomov O.C. DeMarco C. Sbrissa D. J. Biol. Chem. 2001; 276: 11859-11869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A self-generating gradient was performed by centrifugation to equilibrium for 4 h as specified previously (12Shisheva A. Rusin B. Ikonomov O.C. DeMarco C. Sbrissa D. J. Biol. Chem. 2001; 276: 11859-11869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Fractions (∼ 0.37 ml) were collected from the bottom of the tube and were analyzed for protein concentration and by immunoblotting with antibodies indicated in the legend to Fig. 6. GST Pull-down Assay—GST-p40, GST alone or the fusion peptides, GST-p40-(133-372), GST-p40-(264-372), and GST-p40-(1-264), each at 5 μg/tube, were immobilized and purified on GSH-agarose beads. Cytosols derived from COS-7 cells infected or non-infected with adenoviruses encoding PIKfyveWT, prepared as previously described (13Ikonomov O.C. Sbrissa D. Shisheva A. J. Biol. Chem. 2001; 276: 26141-26147Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), were added and incubated with the beads for 18 h at 4 °C. Beads were then washed three times in a cytosol-washing buffer (20 mm Hepes, pH 7.4, 2 mm EDTA, 5 mm MgCl2, 0.1% Triton X-100, 150 mm NaCl, and 1× protease inhibitor mixture). Washed beads were then boiled in sample buffer, and the proteins were analyzed by SDS-PAGE and immunoblotting. Immunoblotting and Immunoprecipitation—Immunoblotting with the indicated polyclonal or monoclonal antibodies was performed subsequent to protein separation by SDS-PAGE and electrotransfer onto nitrocellulose membranes as previously described (19Shisheva A. Sbrissa D. Ikonomov O. Mol. Cell. Biol. 1999; 19: 623-634Crossref PubMed Scopus (102) Google Scholar). A chemiluminescence kit (Pierce) was used to detect the horseradish-peroxidase-bound secondary antibodies. Protein levels on the blots were quantified with a laser densitometer (Molecular Dynamics) by area integration scanning. Several exposures of each blot were quantified to assure that the chemiluminescence exposures were within the linear range of the film. PIKfyve proteins were immunoprecipitated from radioimmune precipitation assay buffer lysates of COS-7 cells transiently transfected with indicated PIKfyve constructs or from lysates of HEK293 cells infected with adenovirus encoding HA-PIKfyveWT using anti-HA or anti-PIKfyve antibodies as described previously (11Sbrissa A. Ikonomov O.C. Shisheva A. Biochemistry. 2000; 39: 15980-15989Crossref PubMed Scopus (51) Google Scholar). In some experiments PIKfyve proteins were immunoprecipitated from cytosols of a HEK293 cell line stably transfected with HA-PIKfyveWT. Control immunoprecipitates with preimmune serum or anti-HA immunoprecipitates from control non-transfected or non-infected cells were run in parallel. Immunoprecipitation was carried out for 16 h at 4 °C with protein A-Sepharose CL-4B added in the final 1.5 h of incubation. Immunoprecipitates were washed with radioimmune precipitation assay buffer or cytosol-washing buffer plus 1× protease inhibitor mixture and then solubilized in sample buffer. Proteins were resolved by SDS-PAGE and detected by immunoblotting as described above. p40 in Vitro Phosphorylation—The ability of PIKfyve to phosphorylate p40 was tested in vitro using PIKfyve immunopurified from HEK293 cells infected with adenovirus encoding PIKfyveWT following a published protocol (11Sbrissa A. Ikonomov O.C. Shisheva A. Biochemistry. 2000; 39: 15980-15989Crossref PubMed Scopus (51) Google Scholar). Briefly, anti-PIKfyve or preimmune precipitates, immobilized on protein A-Sepharose were washed three times in radioimmune precipitation assay buffer supplemented with 1× protease inhibitor mixture and then three times with 50 mm HEPES, pH 7.4. Washed beads were preincubated with the purified GST-p40 fusion protein or GST alone, each at 800 μg/ml for 10 min at 25 °C. The reaction was initiated by addition of the ATP/ion mix composed of [γ-32P]ATP (5 μCi) 25 μm ATP, 5 mm MnCl2, and 25 mm MgCl2 in 50 mm HEPES, pH 7.4, and continued for 15 min at 25 °C. The supernatants were removed. The beads were washed three times with ice-cold phosphate-buffered saline, supplemented with 50 mm NaF and 10 mm Na4P2O7, then boiled in sample buffer and analyzed by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and analyzed by autoradiography and immunoblotting with the indicated antibodies. Liposome Binding Assay—A possible specific interaction of p40 with PtdIns 5-P and PtdIns 3,5-P2 was examined exactly as described previously (15Sbrissa D. Ikonomov O.C. Shisheva A. J. Biol. Chem. 2002; 277: 6073-6079Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) using GST-p40 (5 μg) immobilized on GSH-agarose beads and PtdIns 5-P-enriched or PtdIns 3,5-P2-enriched liposomes. GST protein (5 μg) was used as a control. Identification of p40 as a PIKfyve-binding Protein by Yeast Two-hybrid Interaction—To identify proteins that interact with PIKfyve, we constructed the chaperonin domain (residues 616-868) and DEP domain (residues 384-445) of PIKfyve in pNLexNLS vector for screening a HeLa cell cDNA library. Initial tests, however, revealed that the DEP domain construct produced a high background. A similar high background was seen with a DEP domain bait expanded with some upstream sequence encompassing the N-terminally positioned FYVE finger (residues 99-473). Therefore, both baits containing the DEP domain were not used for further screening. Screening of 106 cDNA library transformants by the mating version of the yeast two-hybrid system (21Kolonin M.G. Zhong J. Finley Jr., R.L. Methods Enzymol. 2000; 328: 26-46Crossref PubMed Google Scholar) with the chaperonin-like domain as a bait resulted in 27 clones as potential interactors. Four of these clones encoded the p40 transport factor. The remaining clones encoded three other proteins, which are under characterization and will be described elsewhere. Pull-down and Immunoprecipitation Experiments Confirm PIKfyve-p40 Interaction—All the p40 clones isolated from our screening lacked the initial N-terminal part of the full-length molecule. The latter was generated by PCR amplification of the N-terminal amino acid segment using the full-length human p40 (16Diaz E. Schimmöller F. Pfeffer S.R. J. Cell Biol. 1997; 138: 283-290Crossref PubMed Scopus (118) Google Scholar) as a template as described under “Experimental Procedures.” To confirm the interaction of p40 with PIKfyve, a GST-conjugated peptide corresponding to full-length human p40 was used in pull-down experiments. Western blot analysis, illustrated in Fig. 1A, reveals that a substantial fraction of the immunoreactive PIKfyveWT binds to GST-p40 following incubation of bacterially produced purified GST-p40 with cytosols derived from COS cells infected with adenovirus encoding HA-PIKfyveWT. Control pull-down experiments with GST protein immobilized on GSH beads or GSH beads alone showed undetectable immunoreactive bands with electrophoretic properties of HA-PIKfyve (Fig. 1A). The interaction of PIKfyveWT with p40 was further verified by co-immunoprecipitation experiments in a HEK293 stable cell line inducibly expressing HA-PIKfyveWT and transiently co-transfected with pEGFP-p40 cDNA (Fig. 1" @default.
• W2014375034 created "2016-06-24" @default.
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• W2014375034 date "2003-12-01" @default.
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• W2014375034 title "Active PIKfyve Associates with and Promotes the Membrane Attachment of the Late Endosome-to-trans-Golgi Network Transport Factor Rab9 Effector p40" @default.
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