A Phosphatidylinositol 4-Kinase Pleckstrin Homology Domain That Binds Phosphatidylinositol 4-Monophosphate
1998; Elsevier BV; Volume: 273; Issue: 35 Linguagem: Inglês
10.1074/jbc.273.35.22761
ISSN1083-351X
AutoresJill M. Stevenson, Imara Y. Perera, Wendy F. Boss,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoPleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio. Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio. Since the first report of changes in phosphoinositide metabolism in response to light (1Morse M.J. Crain R.C. Satter R.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7075-7078Crossref PubMed Google Scholar), there have been many studies of the metabolism of inositol phospholipids in plants (2Drøbak B.K. Biochem. J. 1992; 288: 697-712Crossref PubMed Scopus (145) Google Scholar, 3Cote G.G. Crain R.C. Bioessays. 1994; 16: 39-46Crossref Scopus (46) Google Scholar, 4Perera I.Y. Boss W.F. Briggs W.R. Heath R.L. Tobin E.M. Regulation of Plant Growth and Development by Light. American Society of Plant Physiologists, Rockville, MD1996: 114-126Google Scholar). These studies reveal two distinguishing features of phosphoinositide metabolism in higher plants: 1) [3H]PI-4-P 1The abbreviations used are: PI-4-Pphosphatidylinositol 4-monophosphatePI-45-P2, phosphatidylinositol 4,5-bisphosphatePI4Kphosphatidylinositol 4-kinasePIphosphatidylinositolPH domainpleckstrin homology domainPLCphospholipase CPI-3-Pphosphatidylinositol 3-monophosphatePI-34-P2, phosphatidylinositol 3,4-bisphosphatePAphosphatidic acidPCRpolymerase chain reaction, RACE, rapid amplification of cDNA endskbkilobaseseEF-1αelongation factor-1αPAGEpolyacrylamide gel electrophoresisNBD12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). is 10–20-fold higher than [3H]PI-4,5-P2, and 2) changes in [3H]PI-4-P are detectable. Conversely, in the most responsive animal systems, the ratio of PI-4-P to PI-4,5-P2 is ∼1:1, and there is only a transient change in PI-4,5-P2, with little to no change in PI-4-P even though inositol 1,4,5-trisphosphate may increase 40-fold (5Cunningham E. Thomas G.M.H. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). One explanation that we are exploring for these differences is that the synthesis of PI-4,5-P2 is rate-limiting in plant cells because PI-4-P is sequestered by cytoskeletal or other proteins and not readily available for phosphorylation to PI-4,5-P2 or other metabolic pathways. phosphatidylinositol 4-monophosphate 5-P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-kinase phosphatidylinositol pleckstrin homology domain phospholipase C phosphatidylinositol 3-monophosphate 4-P2, phosphatidylinositol 3,4-bisphosphate phosphatidic acid polymerase chain reaction, RACE, rapid amplification of cDNA ends kilobases elongation factor-1α polyacrylamide gel electrophoresis 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). The first committed step in the biosynthesis of PI-4,5-P2is the phosphorylation of PI by PI 4-kinase to form PI-4-P. PI-4-P is phosphorylated by PI-4-P 5-kinase to form PI-4,5-P2. Biochemically, PI 4-kinase and PI-4-P 5-kinase activities have been studied in plant membranes (6Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Crossref Scopus (82) Google Scholar, 7Sandelius A.S. Sommarin M. Morré D.J. Boss W.F. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss, New York1990: 139-161Google Scholar, 8Gross W. Yang W. Boss W.F. Biochim. Biophys. Acta. 1992; 1134: 73-80Crossref PubMed Scopus (17) Google Scholar, 9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar) and cytoskeletal (10Tan Z. Boss W.F. Plant Physiol. ( Bethesda ). 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar, 11Xu P. Lloyd C.W. Staiger C.J. Drøbak B.K. Plant Cell. 1992; 4: 941-951Crossref PubMed Google Scholar) and soluble (12Okpodu C.M. Gross W. Burkhart W. Boss W.F. Plant Physiol. ( Bethesda ). 1995; 107: 491-500Crossref PubMed Scopus (18) Google Scholar) fractions. Recently, a putative PI-4-P 5-kinase was cloned fromArabidopsis (13Satterlee J.S. Sussman M.R. Plant Physiol. ( Bethesda ). 1997; 115: 864Google Scholar), but the genes encoding PI 4-kinases in plants have remained elusive. A clear distinction between two structurally different isoforms of the PI 4-kinase has emerged from the cloning and sequencing of PI 4-kinase from yeast (14Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (174) Google Scholar, 15Garcia-Bustos J.F. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (103) Google Scholar, 16Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1171Abstract Full Text PDF PubMed Google Scholar), human (17Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar, 18Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), rat (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and bovine (20Gehrmann T. Vereb G. Schmidt M. Klix D. Meyer H.E. Varsanyi M. Heilmeyer Jr., L.M.G. Biochim. Biophys. Acta. 1996; 1311: 53-63Crossref PubMed Scopus (31) Google Scholar, 21Balla T. Downing G.J. Jaffe H. Kim S. Zolyomi A. Catt K.J. J. Biol. Chem. 1997; 272: 18358-18366Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) tissues. The two isoforms differ in size, amino acid sequence homology, and putative location and function within the cell. The smaller type encodes a polypeptide of ∼110–125 kDa that is found in the cytosol and is associated with the Golgi apparatus (22Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The second type encodes a larger protein of ∼200–230 kDa that is membrane-associated (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 22Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). A distinctive feature of the group of higher molecular mass PI 4-kinases is that they contain putative PH domains. PH domains are poorly conserved protein modules of ∼100 amino acids in length (23Shaw G. Bioessays. 1996; 18: 35-46Crossref PubMed Scopus (256) Google Scholar, 24Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 25Ingley E. Hemmings B.A. J. Cell. Biochem. 1994; 56: 436-443Crossref PubMed Scopus (67) Google Scholar, 26Musacchio A. Gibson T. Rice P. Thompson J. Saraste M. Trends Biochem. Sci. 1993; 18: 343-348Abstract Full Text PDF PubMed Scopus (496) Google Scholar). These motifs exist in proteins that associate with membranes during signal transduction. PH domains bind a variety of ligands ranging from other signal transduction proteins such as G-protein βγ subunits (27Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar) to polyphosphorylated inositol lipids (28Lomasney J.W. Cheng H.-F. Wang L.-P. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 30Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (309) Google Scholar, 31Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (687) Google Scholar)in vitro. N-terminal regions of the PH domain of phospholipase C-δ1 (PLCδ1) (28Lomasney J.W. Cheng H.-F. Wang L.-P. Liu S.-M. Fesik S.W. King K. J. Biol. Chem. 1996; 271: 25316-25326Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), β-adrenergic receptor kinase (29Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), β-spectrin (30Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (309) Google Scholar), pleckstrin, Tsk (T-cell-specific kinase), and Ras GTPase-activating protein (31Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (687) Google Scholar) bind to inositol phospholipids. The affinity of PH domains for PI-4-P, PI-4,5-P2, PI-3-P, PI-3,4-P2, and PI 3,4,5-trisphosphate varies with the type of protein (32Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). This means that rapid cellular changes in the levels of the inositol phospholipids could affect the location and regulation of specific PH domain-containing proteins. Identifying PH domains and their ligand affinities should increase the understanding of how a protein is regulated. The PH domains of the PI 4-kinases have been described based only on primary sequence homology with other PH domains and have not been characterized biochemically. Here we show biochemical and molecular evidence for a large molecular mass PI 4-kinase in both carrot (DcPI4Kα) andArabidopsis (AtPI4Kα), and we show for the first time the affinity of a PI 4-kinase PH domain for specific phosphoinositides. The active enzyme was purified using antibodies raised against the expressed conserved domains. The molecular mass of the PI 4-kinase was estimated to be 205 kDa based on Western blot analysis of the purified protein. Western blots also indicated thatDcPI4Kα is associated with an F-actin fraction. More important, using a new technique, Fat Western blotting, we determined that the recombinant carrot PI 4-kinase PH domain binds specifically to phosphatidic acid (PA), PI-4-P, and PI-4,5-P2. The lipid binding data give new insights into potential mechanisms for regulating PI 4-kinase activity and its distribution. Total RNA was extracted from carrot cells grown in suspension culture (33Chen Q. Boss W.F. Plant Physiol. ( Bethesda ). 1990; 94: 1820-1829Crossref PubMed Scopus (39) Google Scholar) by hot borate/phenol/chloroform extraction (34Hall T.C. Ma Y. Buchbinder B.U. Pyne J.W. Sun S.M. Bliss F.A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3196-3200Crossref PubMed Scopus (164) Google Scholar, 35Perera I. Zielinski R. Plant Physiol. ( Bethesda ). 1992; 100: 812-819Crossref PubMed Scopus (19) Google Scholar). cDNA was synthesized from 5 μg of total RNA by Moloney murine leukemia virus reverse transcriptase (Promega) and primed with random hexamers (Boehringer Mannheim) as described (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Amplification of cDNA by the polymerase chain reaction was achieved with degenerate oligonucleotides deduced from conserved amino acid sequences of known PI 4-kinases. The conserved regions were used by Nakagawa et al. (19Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) to clone the rat PI 4-kinase. The sequences of these regions are (V/T)GDDCRQ and HIDFGF(M/V). Arabidopsis codon usage was followed to design the degenerate primers, and EcoRI sites were added to the 5′-ends of the primers to facilitate subcloning of PCR products into pBluescript (Stratagene). The sequences of the primers, using the International Union of Biochemistry code for degeneracy, were CGG AAT TCR YTG GWG AYG AYT GYC GTC AR for the sense primer and CGG AAT TCN ATR AAW CCR AAR TCD ATR TG for the antisense primer. The PCRs containing 10 μl of the cDNA synthesis mixture, 20–25 pmol of each primer, and 5 units of Taq polymerase (Promega) were amplified by 25 cycles of the following: 97 °C for 30 s, 45 °C for 1 min, and 72 °C for 1 min. The PCR products were resolved by agarose gel (1%, w/v) electrophoresis, gel-purified, digested with EcoRI, and subcloned into pBluescript. Sequence of the cDNA was determined by the dideoxy chain termination method (37Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5468Crossref PubMed Scopus (55549) Google Scholar) using the Sequenase kit (U. S. Biochemical Corp.). After determining the sequence of the PCR product, gene-specific primers were used to amplify cDNA 3′ and 5′ to the original PCR product by the rapid amplification of cDNA ends (RACE) method exactly as described previously (38Frohman M.A. Methods Enzymol. 1993; 218: 340-356Crossref PubMed Scopus (468) Google Scholar). The ArabidopsisPI 4-kinase was cloned by using the carrot 5′-RACE product as a probe to screen an Arabidopsis λYES cDNA library (a gift of Dr. Ralph Dewey, North Carolina State University). The probe was labeled with [α-32P]dCTP and random hexamers. The plated library (3 × 106 clones) was hybridized to the carrot cDNA at 55 °C for 16 h in hybridization buffer containing 6× saline/sodium phosphate/EDTA (SSPE; 1× SSPE = 10 mm NaH2PO4, 1 mm EDTA, and 149 mm NaCl) and 5× Denhardt's solution (100× Denhardt's solution = 2% (w/v) fatty acid-free bovine serum albumin, 2% (w/v) polyvinylpyrrolidone, 2% (w/v) Ficoll 400, 0.5% (w/v) SDS, and 100 μg of calf thymus DNA (Sigma)). The final wash was in 1× SSPE and 0.1% (w/v) SDS at 55 °C for 1 h. The nylon membrane was exposed to x-ray film for 24 h at −80 °C. Two hybridization-positive clones were carried through three successive screens. The larger clone was 2.5 kb and was used as a probe to screen an Arabidopsis λZap II cDNA library that had been selected for large cDNAs (39Kieber J.J. Rothenberg M. Roman G. Feldman K. Ecker J.R. Cell. 1993; 72: 427-441Abstract Full Text PDF PubMed Scopus (1525) Google Scholar). Again, two positive clones were carried through three sequential screening steps, and the largest clone (3.1 kb) was analyzed for its complete sequence by the Iowa State Sequencing Facility. Total RNA was fractionated by formaldehyde-containing agarose gel electrophoresis (40Zielinski R.E. Plant Physiol. ( Bethesda ). 1987; 84: 937-943Crossref PubMed Google Scholar). The RNA was transferred by capillary transfer and UV-cross-linked to nylon membrane and then hybridized with the Arabidopsis cDNA labeled by random priming with [α-32P]dCTP. Hybridization was at 42 °C in hybridization buffer containing 50% (v/v) formamide. The final wash of the blot was in 0.1× SSPE and 0.1% (w/v) SDS at 65 °C for 1 h. The nylon was then exposed to x-ray film for 3 days at −80 °C. A cDNA encoding the carrot PI 4-kinase PH domain was generated by PCR amplification with primers that flanked either end of the domain sequence. The sequences of the primers were CGG GAT CCC CCC TGG TTA GGC AAC ACA TT (sense) and GGA ATT CCA ACC TTG AAA ACG CAA GCT T (antisense). The primers contained BamHI (sense primer) and EcoRI (antisense primer) sites on their 5′-ends to facilitate directional subcloning into the bacterial expression vector pRSET-A (Invitrogen). The PCR product was gel-purified, digested with BamHI andEcoRI, and ligated into pRSET-A. BL21(DE3) pLys S cells were transformed with the recombinant plasmid, and expression was induced with the addition of isopropyl-β-d-thiogalactopyranoside (1 mm final concentration) to the cell culture. Bacterial cells expressing the His-tagged PH domain were lysed by sonication and solubilized in 6 m guanidine hydrochloride, and the recombinant polypeptide was purified by metal affinity chromatography using ProBond resin (Invitrogen). Because the PH domain was insoluble, purification was carried out under denaturing conditions with solutions containing 8 m urea. Column fractions were dialyzed sequentially to remove urea and to promote refolding. Tomato eEF-1α (a gift from Christine K. Shewmaker, Calgene Inc.) was expressed and purified using the same protocol. Total microsomes were prepared from carrot suspension culture cells 5 days after transfer or from whole Arabidopsis thaliana plants. Suspension culture cells were filtered by gravity and homogenized in an equal volume of buffer containing 10 mm KCl, 1 mm EDTA, 1 mm MgCl2, 50 mm Tris (pH 7.5), 95 mm LiCl, 2 mm EGTA, polyvinylpolypyrrolidone (0.1 g/g of cells), 8% (w/v) sucrose, 1 mm dithiothreitol, 2 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mg/100 ml leupeptin, and 2 mm benzamidine. Homogenization was with an equal volume of 0.2-mm glass beads in a Virtis homogenizer in 30-s pulses for 2 min at 4 °C. Arabidopsis plants were coarsely macerated and ground in a Virtis homogenizer with an equal volume of buffer containing 3 mm EDTA, 2 mmEGTA, 30 mm Tris (pH 7.4), 250 mm sucrose, 14 mm β-mercaptoethanol, 2 mm dithiothreitol, 2 μg/ml aprotinin, 0.1 mm phenylmethylsulfonyl fluoride, 1 mg/100 ml leupeptin, and 2 mm benzamidine. The homogenate was centrifuged at 2000 × g for 5 min. The resultant supernatant was centrifuged at 40,000 × g for 60 min to obtain a microsomal fraction. Microsomes were resuspended in 30 mm Tris (pH 7.4). F-actin-rich fractions were prepared from the microsomal fraction isolated from 5-day-old carrot suspension culture cells as described previously (10Tan Z. Boss W.F. Plant Physiol. ( Bethesda ). 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar). PCR was used to amplify the reading frame of the largest AtPI4Kα clone. The primers used were CGG GAT CCG TTC AGT CAC ATA TAT TAG AA (sense) and G GAA TTC TTA CTT CTC GAT GCC TTG (antisense). An internalBamHI site 45 nucleotides upstream of the region encoding the lipid kinase unique domain and the EcoRI site of the antisense primer allowed the PCR product to be digested with these two enzymes, purified, and ligated into pRSET-B. Expression and purification of the recombinant protein were exactly the same as for the recombinant PH domain described above, except that the protein was not dialyzed. Instead it was concentrated in a Centricon 10 (Amicon, Inc.), and resolved by SDS-PAGE so that ∼50 μg of recombinant protein were present in each lane of the gel. The gel was stained with 0.05% (w/v) Coomassie Brilliant Blue R-250 in water and washed copiously with water until the bands were visible. The bands containing the recombinant protein were excised from the gel and sent to Zeneca LifeScience Molecules for injection into two rabbits (662 and 663). The rabbits were given seven boosts of the recombinant protein over the course of 3 months. Sera from test bleeds and production bleeds were analyzed for cross-reactivity to the recombinant protein by immunoblotting (data not shown). Antiserum from test bleed 2 of rabbit 662 was purified for IgG on a protein A-Sepharose column (Sigma). Unspecific and His tag-generated antibodies were removed by incubating the purified IgG with an acetone precipitate of E. coli cells expressing His-tagged eEF-1α and removing the aggregates by centrifugation (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). A protein A-Sepharose-antibody affinity column was made by direct coupling with dimethyl pimelimidate (Sigma) as described previously (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Production bleed antisera from both rabbits (0.5 ml total) were pooled and added to 1 ml of protein A-Sepharose beads in 30 mm Tris (pH 7.4). After coupling the antibody to the beads, the column was washed with 20 bed volumes of 30 mm Tris (pH 8.0). The efficiency of coupling of antibody to protein A beads was analyzed by SDS-PAGE before and after the addition of dimethyl pimelimidate. Heavy chain IgG bands were present at 55 kDa before coupling, but not after. Arabidopsis microsomes (12.5 mg) were solubilized for 30 min at 4 oC in buffer used to solubilize cytoskeletal proteins (2% (v/v) Triton X-100, 100 mm Tris (pH 7.4), 10 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 150 μg/ml leupeptin, and 670 μg/ml DNase I) (42Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. Oxford University Press, New York1992Google Scholar). Solubilized microsomes were centrifuged at 40,000 × g for 30 min. The supernatant was incubated with antibody-coupled beads in a 10 × 33-mm column resuspended in 1 bed volume of 100 mm Tris (pH 7.4) at 4 °C with shaking for 2 h. The bed was allowed to settle, and the flow-through fraction was collected. The column was washed with 10 bed volumes of 100 mm Tris (pH 7.4). In addition, to prepare the column for elution and to ensure adequate washing, the column was washed with 10 mm phosphate buffer (pH 7.0) until the flow-through fraction had a spectrophotometric absorbance reading of <0.010 at 280 nm. Bound proteins were eluted with 100 mm glycine (pH 3.0). Fractions of 1 bed volume were collected, immediately neutralized with 0.05 volume of 1m phosphate (pH 8.0), and analyzed by SDS-PAGE and immunoblotting and for PI 4-kinase activity. Each fraction eluted from the immunoaffinity column was assayed in duplicate (60 μl/assay) to determine the PI 4-kinase activity. The reaction mixture contained final concentrations of 7.5 mm MgCl2, 1 mm sodium molybdate, 0.5 mg/ml PI, 0.1% (v/v) Triton X-100, 0.9 mmATP, 30 mm Tris (pH 7.2), and 20 μCi of [γ-32P]ATP (7000 Ci/mmol) in a total volume of 100 μl. Stock PI (5 mg/ml) was solubilized in 1% (v/v) Triton X-100. The reactions were incubated at 25 °C for 2 h with intermittent shaking. The reactions were stopped with 1.5 ml of ice-cold CHCl3/MeOH (1:2) and kept at 4 °C until the lipids were extracted. Lipids were extracted as described previously (9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar). Extracted lipids were vacuum-dried, solubilized in CHCl3/MeOH (2:1), and spotted onto Whatman LK5D silica gel plates that had been completely dried in a microwave oven for 5 min after presoaking in 1% (w/v) potassium oxalate for 80 s. The lipids were separated in either a CHCl3/MeOH/NH4OH/H2O (86:76:6:16) solvent system (9Cho M.H. Shears S.B. Boss W.F. Plant Physiol. ( Bethesda ). 1993; 103: 637-647Crossref PubMed Scopus (52) Google Scholar) or a borate/pyridine-based solvent (43Walsh J.P. Caldwell K.K. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9184-9187Crossref PubMed Scopus (106) Google Scholar) and quantitated with a Bioscan System 500 Imaging Scanner. The plates were subsequently exposed to a phosphor screen for 2 days and visualized by a Storm PhosphorImager (Molecular Dynamics, Inc.). Carrot microsomes were solubilized in 1% (v/v) Triton X-100 at 4 °C overnight. To preclear unspecific antibodies and antigens, solubilized microsomes were incubated for 1 h on ice with 0.2 volume of preimmune serum from rabbit 662. 0.33 volume of protein A-Sepharose was added and incubated for 1.5 h at 4 °C with shaking. The beads were pelleted, and the supernatant was saved and used for subsequent steps. Immunoprecipitation, as described previously (41Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), was carried out by adding to the supernatant either preimmune serum or partially purified antiserum from test bleed 2 of rabbit 662 (described above). The protein A-antibody-antigen complex was washed three times with 30 mm Tris (pH 7.2) and resuspended in SDS-PAGE sample buffer for subsequent analysis by electrophoresis and immunoblotting. Protein was separated by SDS-PAGE using 8% (w/v) polyacrylamide and transferred onto polyvinylidene difluoride membrane (44Bjerrum O.J. Schafer-Nielsen C. Dunn M.J. Analytical Electrophoresis. Verlag Chemie, Weinheim1986: 315Google Scholar). The membrane was incubated for 1 h with anti-AtPI4Kα antiserum (1:1000) at 25 °C. Cross-reactivity was detected by incubation with goat anti-rabbit IgG, F(ab′)2 conjugated to horseradish peroxidase (Pierce), and subsequent chemiluminescent detection by exposing the blot to X-Omat AR film (Eastman Kodak Co.) for the amount of time indicated. Phospholipids were from Sigma, except for PI-3-P and PI-3,4-P2, which were from Matreya, Inc., and NBD-labeled PA, NBD-labeled phosphatidylcholine, and rhodamine-labeled phosphatidylethanolamine, which were from Avanti Polar Lipids. Phospholipids were solubilized in chloroform as stock solutions of 1 mg/ml. A minimum of 10 μl containing 0.5, 1.0, or 5.0 μg of lipid were spotted onto nitrocellulose (NitroBind, MSI) at a time. The membrane and lipids were dried at 24 °C for 1 h. The nitrocellulose was incubated with 3% (w/v) fatty acid-free bovine serum albumin (isolated by cold ethanol precipitation; Sigma A-6003) in TBST (10 mm Tris (pH 8.0), 140 mm NaCl, and 0.1% (v/v) Tween 20) for 1 h and then placed in a solution containing the His-tagged fusion proteins (PH domain or eEF-1α) diluted in TBST (0.5 μg/ml) at 4 °C overnight with shaking. The nitrocellulose was washed with TBST three times for 10 min each and then incubated with T7 tag monoclonal antibody (Novagen) to the His-tagged region diluted 1:10,000 in TBST for 1 h at 24 °C. The nitrocellulose was washed three times for 10 min in TBST at 24 °C and then incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (Pierce) at a titer of 1:30,000 in TBST for 1 h at 24 °C. The nitrocellulose was washed again in TBST three times for 10 min and then incubated for 5 min in a 1:1 mixture of peroxidase substrate and luminol/enhancer (Pierce) for subsequent chemiluminescent detection. The nitrocellulose was exposed to X-Omat AR film for 0.5–5 min as indicated. Using degenerate primers based on highly conserved regions of yeast PIK1 and STT4 PI 4-kinase lipid kinase domains, we amplified a cDNA of 394 nucleotides from carrot RNA. Sequence analysis of this PCR product showed that the deduced amino acid sequence was 50% identical and 64% similar to yeast STT4 and only 35% identical and 54% similar toArabidopsis PI 3-kinase (AtVPS34). 5′- and 3′-RACE were used to amplify regions of sequence beyond this initial PCR product. The 3′-RACE product was 1.1 kb in size and spanned the rest of the catalytic domain and extended through the 3′-untranslated region to the poly(A) tail. The 5′-RACE product contained 1.4 kb and had sequence homology to the lipid kinase unique domain and PH domain of the other PI 4-kinases reported in GenBankTM. The 5′-RACE product from carrot was used as a probe to screen anArabidopsis λYES cDNA library. Two positive
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