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• W1998749044 abstract "Phosphatidylinositol (PtdIns) 4-kinase catalyzes the synthesis of PtdIns-4-P, the precursor of an array of lipid second messengers generated by additional phosphorylation by PtdIns-4-P 5-kinase and PtdIns 3-kinase. PtdIns 4-kinase activity is conserved from yeast to higher eukaryotes. Multiple isoforms of mammalian PtdIns 4-kinase have been purified, and the activities have been detected in almost all subcellular locations. We previously reported the cloning and characterization of the first mammalian PtdIns 4-kinase named PI4Kα (Wong, K., and Cantley, L. C. (1994) J. Biol. Chem. 269, 28878–28884). Alternatively spliced forms of PI4Kα have also been identified from several sources including bovine brain (Gehrmann, T., Vereb, G., Schmidt, M., Klix, D., Meyer, H. E., Varsanyi, M., and Heilmeyer, L. M., Jr. (1996) Biochim. Biophys. Acta1311, 53–63). Recently we isolated a distinct human PtdIns 4-kinase gene, named PI4Kβ, that encodes an enzyme that is wortmannin sensitive (Meyers, R., and Cantley, L. C. (1997) J. Biol. Chem. 272, 4384–4390). Here we report the locations of these enzymes and provide evidence for other yet unidentified isoforms present in specific organelles. PI4Kα is mostly membrane-bound and located at the endoplasmic reticulum; whereas PI4Kβ is in the cytosol and also present in the Golgi region. Neither of these isoforms accounts for the major type II PtdIns 4-kinase activity detected in the lysosomes and plasma membrane fraction. Phosphatidylinositol (PtdIns) 4-kinase catalyzes the synthesis of PtdIns-4-P, the precursor of an array of lipid second messengers generated by additional phosphorylation by PtdIns-4-P 5-kinase and PtdIns 3-kinase. PtdIns 4-kinase activity is conserved from yeast to higher eukaryotes. Multiple isoforms of mammalian PtdIns 4-kinase have been purified, and the activities have been detected in almost all subcellular locations. We previously reported the cloning and characterization of the first mammalian PtdIns 4-kinase named PI4Kα (Wong, K., and Cantley, L. C. (1994) J. Biol. Chem. 269, 28878–28884). Alternatively spliced forms of PI4Kα have also been identified from several sources including bovine brain (Gehrmann, T., Vereb, G., Schmidt, M., Klix, D., Meyer, H. E., Varsanyi, M., and Heilmeyer, L. M., Jr. (1996) Biochim. Biophys. Acta1311, 53–63). Recently we isolated a distinct human PtdIns 4-kinase gene, named PI4Kβ, that encodes an enzyme that is wortmannin sensitive (Meyers, R., and Cantley, L. C. (1997) J. Biol. Chem. 272, 4384–4390). Here we report the locations of these enzymes and provide evidence for other yet unidentified isoforms present in specific organelles. PI4Kα is mostly membrane-bound and located at the endoplasmic reticulum; whereas PI4Kβ is in the cytosol and also present in the Golgi region. Neither of these isoforms accounts for the major type II PtdIns 4-kinase activity detected in the lysosomes and plasma membrane fraction. The metabolism of phosphoinositides is a key event in transmitting mitogenic and developmental signals in response to a variety of hormones and growth factors. Two signal transduction pathways, each utilizing distinct phosphoinositide derivatives, have been characterized. In one pathway, phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; GST, glutathione S-transferase; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; HPLC, high performance liquid chromatography; HA, hemagglutinin; BiP, binding protein; TGN, trans-Golgi network; IRS, insulin receptor substrate. 1The abbreviations used are: PtdIns, phosphatidylinositol; GST, glutathione S-transferase; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; HPLC, high performance liquid chromatography; HA, hemagglutinin; BiP, binding protein; TGN, trans-Golgi network; IRS, insulin receptor substrate.-4,5-P2 is hydrolyzed by receptor-activated phospholipase C enzymes to generate the signaling molecules inositol 1,4,5-trisphosphate and diacylglycerol (4Berridge M.J. Annu. Rev. Biochem. 1987; 56: 159-193Crossref PubMed Scopus (2450) Google Scholar). PtdIns-4-P and PtdIns-4,5-P2 have also been shown to mediate actin rearrangement by directly regulating actin-binding proteins (5Janmey P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Abstract Full Text PDF PubMed Google Scholar, 6Janmey P.A. Lamb J. Allen P.G. Matsudaira P.T. J. Biol. Chem. 1992; 267: 11818-11823Abstract Full Text PDF PubMed Google Scholar). In a distinct pathway, phosphoinositides are phosphorylated at the C-3 position to generated a family of lipid messengers (7Carpenter C.L. Duckworth B.C. Auger K.R. Cohen B. Schaffhausen B.S. Cantley L.C. J. Biol. Chem. 1990; 265: 19704-19711Abstract Full Text PDF PubMed Google Scholar). These lipids, PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3 are not substrates for phospholipase C but are implicated in mitogenesis (8Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2186) Google Scholar), intracellular trafficking (9Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar) as well as actin rearrangement (10Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 11Hartwig J.H. Kung S. Kovacsovics T. Janmey P.A. Cantley L.C. Stossel T.P. Toker A. J. Biol. Chem. 1996; 271: 32986-32993Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Feeding into both PtdIns pathways is the precursor PtdIns-4-P, synthesized by PtdIns 4-kinases. The subcellular distribution of mammalian PtdIns 4-kinases has been studied. The majority of PtdIns 4-kinase activity in human cells is membrane-bound (12Carpenter C.L. Cantley L.C. Biochemistry. 1990; 29: 11147-11156Crossref PubMed Scopus (296) Google Scholar). PtdIns 4-kinase activity is detected in most membrane structures, including plasma membrane (13Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar), nuclear envelope (14Smith C.D. Wells W.W. J. Biol. Chem. 1983; 258: 9368-9373Abstract Full Text PDF PubMed Google Scholar), lysosome, Golgi apparatus (15Jergil B. Sundler R. J. Biol. Chem. 1983; 258: 7968-7973Abstract Full Text PDF PubMed Google Scholar), and endoplasmic reticulum (16Cockcroft S. Taylor J.A. Judah J.D. Biochim. Biophys. Acta. 1985; 845: 163-170Crossref PubMed Scopus (60) Google Scholar). PtdIns 4-kinase activity is also detected in coated vesicles, glucose transporter-containing vesicles (17Del Vecchio R.L. Pilch P.F. J. Biol. Chem. 1991; 266: 13278-13283Abstract Full Text PDF PubMed Google Scholar), and several specialized organelles, such as chromaffin granules (18Husebye E.S. Flatmark T. Biochim. Biophys. Acta. 1988; 968: 261-265Crossref PubMed Scopus (30) Google Scholar) and secretory vesicles from mast cells (19Kurosawa M. Parker C.W. J. Immunol. 1986; 136: 22-616Google Scholar). In addition, cytosolic PtdIns 4-kinases have been described (20Husebye E.S. Flatmark T. Biochim. Biophys. Acta. 1989; 1010: 250-257Crossref PubMed Scopus (16) Google Scholar). In view of such a wide subcellular distribution, it has long been speculated that different isoforms, targeted to different intracellular compartments, perform different physiological functions. While there is some evidence suggesting that distinct isozymes of PtdIns 4-kinase may be targeted to distinct organelles and be independently regulated, previous studies have been limited by lack of cDNA clones and isoform specific antibodies. In Saccharomyces cerevisiae, only two PtdIns 4-kinase isoforms are found in the entire genome. PIK1, the first PtdIns 4-kinase to be cloned, encodes a nuclear-associated PtdIns 4-kinase of 125 kDa that is indispensable for cell growth (21Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar, 22Garcia B.J. Marini F. Stevenson I. Frei C. Hall M.N. EMBO J. 1994; 13: 2352-2361Crossref PubMed Scopus (102) Google Scholar). Mutants arrest in G2 due to defects in cytokinesis. A second yeast gene, STT4, encodes a 200-kDa PtdIns 4-kinase that appears to be cytosolic and is dispensable for growth. Genetic studies of STT4 suggest its involvement in the protein kinase C pathway (23Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1172Abstract Full Text PDF PubMed Google Scholar, 24Yoshida S. Ohya Y. Nakano A. Anraku Y. Mol. Gen. Genet. 1994; 242: 631-640Crossref PubMed Scopus (77) Google Scholar). In contrast, the yeast PtdIns 3-kinase, Vps34, is required for vesicle trafficking from the Golgi apparatus to the vacuole (9Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar). Interestingly FAB1, a yeast gene that is homologous to the mammalian PtdIns-4-P 5-kinase, is required for normal vacuole function and morphology (25Yamamoto A. DeWald D.B. Boronenkov I.V. Anderson R.A. Emr S.D. Koshland D. Mol. Biol. Cell. 1995; 6: 525-539Crossref PubMed Scopus (234) Google Scholar). Because PtdIns 4-kinase generates PtdIns-4-P, the precursor for subsequent phosphorylation by downstream PtdIns kinases, PtdIns 4-kinases must play either a direct or an indirect role in vesicular trafficking (26Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Abstract Full Text PDF PubMed Scopus (248) Google Scholar). Previously, we reported the cloning and characterization of the first mammalian PtdIns 4-kinase, named PI4Kα (1Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). This protein is highly homologous to the yeast STT4 enzyme. Northern blot analysis of the poly(A)+ mRNAs from human tissues and cell lines revealed multiple alternatively spliced transcripts. A 230-kDa PtdIns 4-kinase made from an alternatively spliced form of the rat PI4Kα has recently been described (27Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). A bovine gene encoding a 170–200-kDa PtdIns 4-kinase that shares more than 95% identity with the human PI4Kα in the overlapping region has also been reported (2Gehrmann T. Vereb G. Schmidt M. Klix D. Meyer H.E. Varsanyi M. Heilmeyer Jr., L.M. Biochim. Biophys. Acta. 1996; 1311: 53-63Crossref PubMed Scopus (31) Google Scholar). It represents probably another splice variant of PI4Kα. More recently, we isolated another human cDNA that encodes a 110-kDa PtdIns 4-kinase (named PI4Kβ) that is more homologous to the yeast PIK1 gene. PI4Kβ is wortmannin-sensitive and may be the same enzyme recently reported to be involved in the hormone sensitive pools of inositol phospholipids (28Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (309) Google Scholar). To determine the relative roles of these enzymes in producing phosphoinositides for membrane trafficking, hormone sensitive PtdIns turnover, growth factor-dependent PtdIns-3,4,5-P3 production, and cytoskeletal rearrangement, it is first important to determine the subcellular locations of these enzymes. PtdIns, [γ-32-P] ATP, and silica gel plates were purchased from Avanti (Alabaster, AL), DuPont NEN, and E. Merck (Germany), respectively. Mouse monoclonal antibody 12CA5, reactive to the influenza virus hemagglutinin, was purchased from BabCo. Rabbit polyclonal anti-PI4Kα antibody 3334 was raised to a peptide corresponding to amino acids 501–512, KPYPKGDERKKA, coupled to keyhole limpet hemacyanin (1Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). Peptide antibodies were affinity-purified prior to immunoblotting and immunofluorescent staining experiments. Anti-PI4Kβ antibody was raised against a GST-fusion protein that was generated by polymerase chain reaction using oligonucleotide primers that generate amino acids 410–538 of PI4Kβ (3Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Anti-BiP and anti-γ-adaptin were purchased from StressGen Biotechnologies Corp. and Sigma, respectively. The 2.6-kilobase pair PI4Kα was tagged at the amino terminus with a 9-amino acid epitope (YPYDVPDYA) derived from influenza virus hemagglutinin by insertion into Bluescript encoding the epitope sequence. The tagged cDNA was subcloned into a mammalian expression vector pRC/CMV (Invitrogen). The construct was transiently transfected into CHO or HeLa cells using LipofectAMINE (Life Technologies, Inc.) according to the manufacture's procedures. The tagged cDNA was also subcloned into a baculoviral expression vector. Recombinant viruses were generated with the linear AcMNPV transfection module (Invitrogen) and were plaque-purified prior to use. The GST-PI4Kβ construct was generated by polymerase chain reaction as described in detail elsewhere (3Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and was expressed in Escherichia coliby standard procedures. The fusion protein is predicted to be 120 kDa since it lacks the amino-terminal 82 amino acids of PI4Kβ. Adherent parental CHO-IRS or CHO-IRS cells transfected with PI4Kα were harvested with phosphate-buffered saline containing 1 mmEDTA and 1 mm EGTA. Cells were pelleted and then resuspended in homogenizing buffer (10 mm Tris-HCl, pH 7.4, 20 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin). After swelling for 15 min on ice, the cells were Dounce-homogenized (25–35 strokes). Nuclei and unbroken cells were spun out at 1000 ×g for 10 min at 4 °C. The postnuclear supernatant was centrifuged at 100,000 × g for 1 h in a SW55Ti rotor (Beckman Instruments). The membrane pellet was solubilized with 1% Triton X-100 in homogenizing buffer. The cytosolic and particulate fractions were adjusted to equal volume. Fractionation procedures were performed according to Storrie and Madden (29Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar). Briefly, CHO-IRS cells transfected with PI4Kα were disrupted by low pressure nitrogen cavitation in 0.25 m sucrose, pH 7.4, containing various proteinase inhibitors. After centrifugation at 1300 × g for 10 min, the postnuclear supernatant was collected and overlaid on a hybrid percoll/metrizamide discontinuous density gradient (5 ml of 6% Percoll, 2 ml of 17% metrizamide, 2 ml of 35% metrizamide). The step gradient was centrifuged at 20,000 rpm for 30 min at 4 °C in a SW44Ti rotor (Beckman Instruments). Interfaces were sequentially removed from the top of the gradient as follows: first the top of the gradient, followed by the postnuclear supernatant in 0.25 m sucrose, the sucrose/Percoll interface, the 6% Percoll/17% metrizamide interface where lysosomes sediment, and finally the 17%/35% metrizamide interface where mitochondria sediment. Alkaline phosphodiesterase, cytochrome c oxidase, β-hexosaminidase, and α-mannosidase II were used as the marker enzymes for plasma membrane, mitochondria, lysosomes, and Golgi apparatus, respectively. Lactate dehydrogenase was used as a cytosol marker. Each of the enzyme assays was performed as described previously (29Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar). HeLa cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min. Fixed cells were permeabilized and nonspecific reactive sites were blocked for 30 min at room temperature in phosphate-buffered saline containing 0.1% Triton X-100, 5% normal goat and donkey serum. Cells were then incubated with the appropriate primary PtdIns 4-kinase isoform-specific antibody for 1 h at room temperature. Primary antibodies were detected by species-specific secondary antibodies, namely CyTM3-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated donkey anti-mouse IgG. ER was identified by staining BiP, a resident ER protein. Golgi was visualized using anti-γ-adaptin. The immunostained cells were observed by either conventional light or confocal microscopy. PtdIns kinase assays were performed as described previously (30Auger K.R. Cantley L.C. Cancer Cells. 1991; 3: 263-270PubMed Google Scholar). Briefly, the reaction mixture contained 0.3% Triton X-100, 50 μm ATP, 20 mm HEPES, pH 7.5, 10 mm MgCl2, 0.2 mg/ml sonicated lipids, 20 μCi of [γ-32P]ATP (3000 Ci/mmol; DuPont NEN) per sample. Assays were performed at 37 °C for 20 min and then stopped with 25 μl of 5 n hydrochloric acid. The lipid was extracted with 160 μl of 1:1 (v/v) chloroform:methanol. The organic layer was collected and analyzed by both thin layer chromatography and HPLC as described in detail elsewhere (31Auger K.R. Serunian L.A. Cantley L.C. Irvine R.F. Methods in Inositide Research. Raven Press, New York1990: 155-162Google Scholar). Since the 97-kDa form of PI4Kα and the PI4Kβ enzyme have similar mobilities on SDS-gels, it is critical to verify that the antibodies raised against the highly divergent regions of these two enzymes indeed do not cross-react. We assessed the isozyme specificity of the PtdIns 4-kinase antibodies by immunoblotting the recombinant PI4Kα and PI4Kβ that were expressed in Sf9 insect cells and in E. coli, respectively. Fig.1 shows that PI4Kα antibody detected a single band of 97-kDa protein only in Sf9 cells expressing PI4Kα (left panel, lane 3). No immunoreactivity was observed in either Sf9 cells expressing wild-type viral proteins (left panel, lane 4) or in E. coli expressing PI4Kβ (left panel, lane 2). Conversely, when the blot was reprobed with anti-PI4Kβ antibody, it reacted with the 120-kDa GST-PI4Kβ only in cells subjected to the induction of expression (right panel, lane 2) and not in the nontransformed E. coli (right panel, lane 1). The immunoreactivity was specific to PI4Kβ and not to the GST motif, since the GST-cleared antibodies still detected the 120-kDa PI4Kβ fusion protein (3Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). These results demonstrate that the antibodies are highly specific, and therefore suitable for immunocytochemical localization of PtdIns 4-kinase isozymes. CHO cells were selected to investigate the locations of PtdIns 4-kinases because these cells have been well characterized in subcellular fractionation studies and because they are convenient for transient gene expression. Since the PI4Kα and PI4Kβ genes are almost ubiquitously expressed in human tissues (1Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar, 3Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and since the antibodies raised against these enzymes react with the respective proteins in other mammalian tissues (not shown), we first investigated the ability of these antibodies to blot proteins in CHO cells. The anti-PI4Kα antibodies did not detect a 97-kDa PI4Kα isoform in the parental CHO cells (Fig. 2 A, left panel), but strongly reacted with an approximately 180-kDa protein (p180) of the size expected for the high molecular weight alternative splice of PI4Kα (2Gehrmann T. Vereb G. Schmidt M. Klix D. Meyer H.E. Varsanyi M. Heilmeyer Jr., L.M. Biochim. Biophys. Acta. 1996; 1311: 53-63Crossref PubMed Scopus (31) Google Scholar). This result is consistent with the ubiquitous expression of the 7.5-kilobase pair message for PI4Kα (1Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar, 27Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The 97-kDa form of PI4Kα was detected when this cDNA was introduced. The PI4Kβ antibody reacted with an approximately 110-kDa band (Fig.2 A, right panel), consistent with the size of this protein previously detected in Jurkat cells (3Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The postnuclear supernatants from the PI4Kα transfected cells were separated into soluble and particulate fractions by centrifugation at 100,000 ×g. The 97-kDa PI4Kα was predominantly associated with the particulate fraction (Fig. 2 B, left panel), as was p180. p110 PI4Kβ was found predominantly in the soluble fraction (Fig. 2 B, right panel). To further investigate the intracellular localization of PtdIns 4-kinase isoforms, cells overexpressing PI4Kα were fractionated using the hybrid Percoll/metrizamide discontinuous density gradient as described previously (29Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar). This gradient allows the isolation of lysosomes, mitochondria, and partial separation of plasma membrane from cytosol and organelles such as ER and Golgi. Interfaces sequentially collected from the top of the step gradient were adjusted to the same volume. Equal portions of individual fractions were then used for organelle marker enzyme assays, and for Western blot analysis with the anti-PtdIns 4-kinase antibodies. Monitoring the β-hexosaminidase activities, we found 85% of the lysosomes sedimented at the interface of Percoll/17% metrizamide as expected (Table I). The cytochrome c oxidase assays indicated that 69% of the mitochondria sedimented to the 17%/35% metrizamide interface, and 22% remained in the Percoll/17% metrizamide interface. Western blot revealed no significant amounts of anti-PI4Kα or anti-PI4Kβ reactive protein present in these two fractions. Instead, p97 PI4Kα, PI4Kβ, and the anti-PI4Kα reactive p180 concentrated at the top of the gradient and in the 0.25 m sucrose fraction (Fig.3), where over 95% of Golgi apparatus and 40% of plasma membrane cofractionated (Table I). About 40% of the plasma membrane marker activity was also present at the sucrose/Percoll interface, but PI4Kα and β were not detected in this fraction, suggesting that they are not associated with this fraction of the plasma membrane.Table ISummary of organelle marker enzyme assays and the distribution of PtdIns 4-kinase isozymesTop fractionSucroseSucrose/PercollPercoll/17% metrizamide17%/35% metrizamide%Alkaline phosphodiesterase I (plasma membrane)182239183α-Mannosidase II (golgi apparatus)4454110β-Hexosaminidase (lysosome)0312850Cytochrome c oxidase (mitochondria)1262269Total PtdIns 4-kinase activity2130341504C5G inhibitable activity15212611097-kDa PI4Kα++−−−p180-kDa form++−−−PI4Kβ++−−−Fractions of subcellular organelles obtained from the hybrid Percoll/metrizamide gradient (see “Experimental Procedures”) were assayed for organelle-specific enzyme activities. The percentage of total activity of each organelle marker is presented. Fractions from the same experiment were also assayed for lipid kinase activity. The PtdIns-4-P products generated in individual organelles were separated by TLC, quantified by a PhosphoImager (BioRad), and expressed as the percentage of total activity. The data shown represent the averages of three separate experiments. Open table in a new tab Fractions of subcellular organelles obtained from the hybrid Percoll/metrizamide gradient (see “Experimental Procedures”) were assayed for organelle-specific enzyme activities. The percentage of total activity of each organelle marker is presented. Fractions from the same experiment were also assayed for lipid kinase activity. The PtdIns-4-P products generated in individual organelles were separated by TLC, quantified by a PhosphoImager (BioRad), and expressed as the percentage of total activity. The data shown represent the averages of three separate experiments. The various subcellular fractions were also assayed for total PtdIns 4-kinase activity. Although half of the PtdIns 4-kinase activity was detected in the upper two factions where cytosol, Golgi apparatus, and endoplasmic reticulum partition, and where both PI4Kα and PI4Kβ fractionate (Fig. 3), a substantial amount of activity was detected in the region where plasma membranes sediment (34%) and where lysosomes sediment (15%) (Table I). The assay was carried out under conditions in which PtdIns 3-kinase is inactive (0.3% Triton X-100). HPLC analysis of the deacylated products generated under these conditions verified the absence of PtdIns-3-P (Fig. 4). The PtdIns 4-kinase activity was not significantly inhibited by 1 μmwortmannin, indicating that the wortmannin-inhibitable PI4Kβ is not a major component of total activity in these fractions (data not shown). Consistent with previous studies, an inhibitory antibody, 4C5G, raised against the 55-kDa type II PtdIns 4-kinase caused about 75% inhibition of the plasma membrane PtdIns 4-kinase (sucrose/Percoll fraction, TableI). This antibody also substantially inhibited PtdIns 4-kinase activity in other fractions. The 55-kDa type II PtdIns 4-kinase was previously shown to be the major PtdIns 4-kinase in red cell plasma membrane (32Graziani A. Ling L.E. Endemann G. Carpenter C.L. Cantley L.C. Biochem. J. 1992; 284: 39-45Crossref PubMed Scopus (34) Google Scholar) and in other cells. Thus, the results in Table I indicate that the membrane bound forms of PI4Kα and PI4Kβ are in compartments of the cell consistent with Golgi apparatus and/or endoplasmic reticulum, and that the major PtdIns 4-kinase activity in plasma membrane and lysosome is due to a type II PtdIns 4-kinase distinct from the PI4Kα isoforms and from PI4Kβ. To further discern the intracellular localization of PI4Kα and β, cytoimmunofluorescence studies were performed using both conventional and confocal microscopy. We chose HeLa cells for these studies since they grow better on coverslips and are hence more suitable for immunofluorescent staining experiments. HeLa cells transiently expressing HA-tagged PI4Kα were double stained with anti-PI4Kα (Fig. 5 A) and anti-HA epitope antibodies (Fig. 5 B). The two antibodies yielded identical patterns, suggesting that the staining obtained using anti-PI4Kα antisera was specific. The anti-HA antibody did not stain non-transfected cells (not shown). The anti-PI4Kα antibodies revealed punctate staining near the nucleus and in the cytoplasm, a pattern similar to that expected for ER. To determine whether PI4Kα is present in the ER, we double stained an ER resident protein Bip (Fig. 5 C) and PI4Kα (Fig. 5 D) in parental HeLa cells. Overlap of PI4Kα with the ER marker was clearly observed after merging the two images by confocal microscopy (Fig. 5 E). However, the colocalization was not complete; some punctate staining of PI4Kα in the peripheral regions was not observed to superimpose with BiP staining. As in CHO cells, the major endogenous protein in HeLa cells that reacts with the anti-PI4Kα antibody is the p180 protein (not shown), so this protein appears also to be located in the ER. Immunofluorescent studies with anti-PI4Kβ antibody revealed a pattern characteristic of the Golgi apparatus. Specifically, we noted a crescent of staining on one side of the nucleus (presumably the TGN), and some punctate staining within the cytoplasm which may represent the buds from extended TGN tubules (Fig. 5 G). The Golgi localization was confirmed by double staining γ-adaptin, which has previously been shown to be localized in the TGN and the late endosomes (Fig. 5 F) (33Ahle S. Mann A. Eichelsbacher U. Ungewickell E. EMBO J. 1988; 7: 919-929Crossref PubMed Scopus (251) Google Scholar, 34Robinson M.S. Kreis T.E. Cell. 1992; 69: 129-138Abstract Full Text PDF PubMed Scopus (286) Google Scholar). In this case, complete colocalization was observed (Fig. 5 H). Staining could be eliminated by preincubation of the primary antibody with the immunizing GST-PI4Kβ and not by GST alone (not shown), indicating that the observed signal is generated by PI4Kβ-specific antibodies. Our observation that much of the PI4Kβ protein is in the supernatant of a 100,000 ×g spin (Fig. 2 B) is consistent with several possibilities. First, the lysis and homogenization procedures dislodge some of this protein from the particulate fraction. Second, the cytosolic fraction is partially lost during cell fixation, or last, this fraction is not as easily visualized because of dilution. We compared the subcellular distribution of known mammalian PtdIns 4-kinases. The results demonstrate the differential localization of different isoforms and provide support for the hypothesis that individual members of this family of PtdIns kinases are targeted to distinct subcellular compartments and hence are performing different cellular functions. PI4Kα is detected predominantly in the particulate fraction, and PI4Kβ is present in the cytosol and to a lesser extent in the particulate fraction. Fractionation of CHO-IRS cells revealed that neither the α nor β isoform is located in the lysosomes or mitochondria, and probably not in the plasma membrane. Immunocytofluorescence studies demonstrate that PI4Kα is present in the endoplasmic reticulum, and PI4Kβ is localized to the Golgi region. While this work was in progress, Nakagawa et al. (27Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) reported the cloning and characterization of an alternative splice of rat PI4Kα gene that generates a protein predicted to be 230 kDa. Gehrmann et al. (2Gehrmann T. Vereb G. Schmidt M. Klix D. Meyer H.E. Varsanyi M. Heilmeyer Jr., L.M. Biochim. Biophys. Acta. 1996; 1311: 53-63Crossref PubMed Scopus (31) Google Scholar) also isolated a partial bovine cDNA encoding a 170–200-kDa PtdIns 4-kinase, that is almost certainly another splice variant of PI4Kα. The carboxyl-terminal half of both high molecular weight proteins contains the domains necessary for lipid catalysis, and share greater than 95% identity with the human p97 PI4Kα. We also noted an alternative splice of PI4Kα from human tissues that is predicted to encode a larger protein (1Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). Nakagawaet al. (27Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) found that the 97-kDa PI4Kα and the higher molecular weight form colocalize, and the later is predominantly associated with the particulate fraction. These results are consistent with our finding that the p180 and p97 cofractionate and colocalize. It is unlikely that they are integral membrane proteins, since neither splice variant contains a predicted transmembrane domain. However, it is clear from our studies that the membrane association does not require the extended amino-terminal sequence of the higher molecular weight form of PI4Kα. Nakagawa et al. (27Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) reported that Flag-tagged versions of both forms of PI4Kα localize to the Golgi membrane in COS cells. In our study, both the exogenously expressed p97 PI4Kα and the endogenous p180 localize to the ER rather than the Golgi of HeLa cells. The discrepancy cannot be explained by the cell type, since we also obtained ER staining when PI4Kα was expressed in COS cells (not shown). The possibility that the HA-tag affected localization is unlikely, since the endogenous p180 exhibited the same location as the HA-tagged p97. By separating intracellular structures on a step gradient and assaying each fraction for PtdIns kinase activity, we have shown that the lysosomal and plasma membrane-associated PtdIns 4-kinase activities are unlikely to be the result of PI4Kβ or any splice variants of PI4Kα. A plausible candidate is the previously described 55-kDa type II PtdIns 4-kinase. Historically, PtdIns 4-kinases are classified into two categories, types II and type III, on the basis of their molecular sizes, and the differences in their sensitivity to adenosine and nonionic detergent (13Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar). The type II PtdIns 4-kinase is dramatically activated by nonionic detergent but is potently inhibited by both adenosine and the 4C5G monoclonal antibody (32Graziani A. Ling L.E. Endemann G. Carpenter C.L. Cantley L.C. Biochem. J. 1992; 284: 39-45Crossref PubMed Scopus (34) Google Scholar). Type II PtdIns 4-kinase has been purified from erythrocyte membrane (32Graziani A. Ling L.E. Endemann G. Carpenter C.L. Cantley L.C. Biochem. J. 1992; 284: 39-45Crossref PubMed Scopus (34) Google Scholar) and also copurified with the epidermal growth factor receptor (35Cochet C. Gill G.N. Meisenhelder J. Cooper J.A. Hunter T. J. Biol. Chem. 1984; 259: 2553-2558Abstract Full Text PDF PubMed Google Scholar), as well as with the lymphocyte CD4 antigen (36Pertile P. Cantley L.C. Biochim. Biophys. Acta. 1995; 1248: 129-134Crossref PubMed Scopus (17) Google Scholar). Additional findings have also demonstrated that it is recruited to and activated by epidermal growth factor receptor upon ligand stimulation (37Kauffmann-Zeh A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 2685: 1188-1190Crossref Scopus (160) Google Scholar). We have extensively investigated the possibility that the type II 55-kDa enzyme is a proteolytic product of the 97-kDa PI4Kα since they share similar enzymatic properties. However, we are unable to detect proteins of 55 kDa resulting from the processing of the 97-kDa PI4Kα. Thus, the 55-kDa type II PtdIns 4-kinase appears to be encoded by a distinct but related gene. The finding of the differential ER and Golgi localization of PI4Kα and PI4Kβ suggests a direct role of PI4Kα and β in ER and Golgi function, as opposed to the ligand-stimulated phosphoinositide turnover at the plasma membrane. The finding that PI4Kβ is wortmannin-sensitive (IC50, 140 nm) and is located in the Golgi apparatus should caution future studies employing wortmannin inhibition as the standard of implicating the role of PtdIns 3-kinase in targeting proteins from Golgi apparatus to lysosome. Finally, our results do not exclude other sites of PtdIns 4-kinase function. For example, the colocalization of ER marker was not complete for PI4Kα. We also noticed a significant amount of PI4Kα in the particulate fraction that could not be extracted with detergent, implicating cytoskeletal association. Future studies with dominant negative forms or gene knockouts will elucidate the roles of these enzymes in mammalian cell function. We thank Fernando Agarraberes for his assistance in the cell fractionation procedures. We also thank Brian Duckworth and Anthony Couvillon for help in HPLC analysis, Dr. Lucia Rameh for advice in immunofluorescence studies, Dr. Richard Mitchell for insightful discussions, and Dr. Kermit Carraway III for critical review of the manuscript." @default.
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• W1998749044 title "Subcellular Locations of Phosphatidylinositol 4-Kinase Isoforms" @default.
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