The Protein Kinase C-dependent Phosphorylation of Serine 166 Is Controlled by the Phospholipid Species Bound to the Phosphatidylinositol Transfer Protein α
2000; Elsevier BV; Volume: 275; Issue: 28 Linguagem: Inglês
10.1074/jbc.m002203200
ISSN1083-351X
AutoresClaudia M. van Tiel, Jan Westerman, Marten Paasman, K.W.A. Wirtz, Gerry T. Snoek,
Tópico(s)Pancreatic function and diabetes
ResumoThe charge isomers of bovine brain PI-TPα (i.e. PI-TPαI containing a phosphatidylinositol (PI) molecule and PI-TPαII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V max values for PI-TPαI and II were 2.0 and 6.0 nmol/min, respectively; theK m values for both charge isomers were about equal,i.e. 0.7 μm. Phosphorylation of charge isomers of recombinant mouse PI-TPα confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32P-labeled PI-TPαs showed that serine was the major site of phosphorylation. Degradation of 32P-labeled PI-TPα by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one32P-labeled peptide (amino acids 104–190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPα. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPα to perinuclear Golgi structures concomitant with a 2–3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPα expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPα(S166A) and Myc-tagged PI-TPα(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPα is linked to the degradation of PI. The charge isomers of bovine brain PI-TPα (i.e. PI-TPαI containing a phosphatidylinositol (PI) molecule and PI-TPαII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V max values for PI-TPαI and II were 2.0 and 6.0 nmol/min, respectively; theK m values for both charge isomers were about equal,i.e. 0.7 μm. Phosphorylation of charge isomers of recombinant mouse PI-TPα confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32P-labeled PI-TPαs showed that serine was the major site of phosphorylation. Degradation of 32P-labeled PI-TPα by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one32P-labeled peptide (amino acids 104–190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPα. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPα to perinuclear Golgi structures concomitant with a 2–3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPα expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPα(S166A) and Myc-tagged PI-TPα(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPα is linked to the degradation of PI. phosphatidylinositol transfer protein mouse recombinant PI-TPα phosphatidylinositol phosphatidylcholine phosphatidylglycerol lysophosphatidylinositol phorbol 12-myristate 13-acetate protein kinase C platelet-derived growth factor polyacrylamide gel electrophoresis tosylphenylalanyl chloromethyl ketone-treated trypsin wild type Phosphatidylinositol transfer protein (PI-TP)1 is a ubiquitous protein that has been shown to play an essential role in secretion (vesicle flow) and in phospholipase C-dependent signaling as was established in reconstituted systems (1.Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 2.Ohashi M. de Vries K.J. Frank R. Snoek G.T. Bankaitis V. Wirtz K.W.A. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (169) Google Scholar, 3.Thomas G.M. Cunningham E. Fensome A. Ball A. Totty N.F. Truong O. Hsuan J.J. Cockcroft S. Cell. 1993; 74: 919-928Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 4.Cockcroft S. Ball A. Fensome A. Hara S. Jones D. Prosser S. Swigart P. Biochem. Soc. Trans. 1997; 25: 1125-1131Crossref PubMed Scopus (11) Google Scholar). In addition, PI-TP may have a role in delivering substrate to the phosphatidylinositol 3-kinase complex (5.Panaretou C. Domin J. Cockcroft S. Waterfield M.D. J. Biol. Chem. 1997; 272: 2477-2485Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 6.Kular G. Loubtchenkov M. Swigart P. Whatmore J. Ball A. Cockcroft S. Wetzker R. Biochem. J. 1997; 325: 299-301Crossref PubMed Scopus (49) Google Scholar). Two isoforms of PI-TP have been identified, PI-TPα and PI-TPβ (7.de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar, 8.de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell. Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar, 9.Tanaka S. Hosaka K. J. Biochem. (Tokyo). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar). Both isoforms transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membranes in vitro (10.Wirtz K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar). PI-TPβ expresses an additional activity for sphingomyelin (7.de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar, 11.Westerman J. de Vries K.J. Somerharju P. Timmermans-Hereijgers J.L. Snoek G.T. Wirtz K.W.A. J. Biol. Chem. 1995; 270: 14263-14266Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In studies with the reconstituted systems, PI-TPα and β behaved similarly. Recently, PI-TPα overexpressed in NIH3T3 cells was shown to enhance the constitutive levels of lysophosphatidylinositol (lysoPI) (12.Snoek G.T. Berrie C.P. Geijtenbeek T.B. van der Helm H.A. Cadee J.A. Iurisci C. Corda D. Wirtz K.W.A. J. Biol. Chem. 1999; 274: 35393-35399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Overexpression of PI-TPβ in these cells had no effect on lysoPI formation but stimulated the conversion of ceramide into sphingomyelin (13.Van Tiel C.M. Luberto C. Snoek G.T. Hannun Y.A. Wirtz K.W.A. Biochem. J. 2000; 346: 537-543Crossref PubMed Scopus (31) Google Scholar). In addition to these soluble PI-TPs, a membrane-bound form of PI-TP was detected containing a PI-TPα homology domain at the N terminus (amino acids 1–257) and six putative membrane-spanning domains. This retinal degeneration B (RdgB) protein was originally identified inDrosophila (14.Vihtelic T.S. Goebl M. Milligan S. O'Tousa J.E. Hyde D.R. J. Cell Biol. 1993; 122: 1013-1022Crossref PubMed Scopus (159) Google Scholar). Localization studies by indirect immunofluorescence and by microinjection of fluorescently labeled PI-TPs have shown that PI-TPα is mainly localized in the nucleus and in the cytosol and that PI-TPβ is mainly associated with the Golgi membranes (7.de Vries K.J. Heinrichs A.A. Cunningham E. Brunink F. Westerman J. Somerharju P.J. Cockcroft S. Wirtz K.W.A. Snoek G.T. Biochem. J. 1995; 310: 643-649Crossref PubMed Scopus (89) Google Scholar, 8.de Vries K.J. Westerman J. Bastiaens P.I. Jovin T.M. Wirtz K.W.A. Snoek G.T. Exp. Cell. Res. 1996; 227: 33-39Crossref PubMed Scopus (81) Google Scholar). Upon stimulation of Swiss mouse 3T3 fibroblasts by growth factors that activate the PI signaling pathway or by phorbol 12-myristate 13-acetate (PMA), PI-TPα became rapidly associated with the Golgi membranes. Under these conditions PI-TPα was found to be phosphorylated, suggesting that this modification may be a prerequisite for its association with the Golgi. (15.Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). PI-TPα was also a substrate for protein kinase C (PKC) in vitro in agreement with the presence of five putative phosphorylation sites: Thr-59, Thr-169, Thr-198, Thr-251, and Ser-166 (15.Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar, 16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). Two charge isomers of PI-TPα are present in tissues and cells of which one isomer carries a PI molecule (PI-TPαI) and the other a PC molecule (PI-TPαII) (17.van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar). The cellular concentration of PI-TPαI is about 8-fold higher as compared with that of PI-TPαII. Because the affinity of PI-TPα for PC is about 16-fold lower than the affinity for PI, the relative amounts of the two isomers reflect the accessible pools of PI and PC in the cell. To date, no comparable charge isomers of PI-TPβ have been detected. The physiological significance of the two charge isomers of PI-TPα is not yet known. It has been suggested that the yeast analogue of PI-TP, SEC14p, acts as a sensor of these phospholipid pools in the Golgi inasmuch as that the two charge isomers affect PI and PC metabolism differently (18.Skinner H.B. McGee T.P. McMaster C.R. Fry M.R. Bell R.M. Bankaitis V.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 112-116Crossref PubMed Scopus (136) Google Scholar). Given the ability of PI-TPα to be involved in both PI and PC metabolism (12.Snoek G.T. Berrie C.P. Geijtenbeek T.B. van der Helm H.A. Cadee J.A. Iurisci C. Corda D. Wirtz K.W.A. J. Biol. Chem. 1999; 274: 35393-35399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19.Monaco M.E. Alexander R.J. Snoek G.T. Moldover N.H. Wirtz K.W.A. Walden P.D. Biochem. J. 1998; 335: 175-179Crossref PubMed Scopus (20) Google Scholar), one may expect a regulatory mechanism in the cell to be able to discriminate between PI-TPαI and II. Because PI-TPα is a substrate for PKC, we have investigated whether the phospholipid bound to PI-TPα has an effect on the in vitro phosphorylation. It will be shown that PI-TPαII is more rapidly phosphorylated by PKC than PI-TPαI. Both charge isomers have one major phosphorylation site (Ser-166), replacement of which with Ala or Asp completely abolished the transfer activity. A model will be presented in which the phosphorylation of PI-TPα is linked to the agonist-induced production of lysoPI. Egg yolk PC, soybean PI, phosphatidic acid, phosphatidylserine, PMA, ATP, phosphoserine, phosphothreonine, and phosphotyrosine were obtained from Sigma. The pBluescript SK− vector and the Quickchange site-directed mutagenesis kit were purchased from Stratagene (La Jolla, CA). The oligonucleotides were synthesizsed by Eurogentec, Belgium. The pET-15b vector was obtained from Novagen (Madison, WI). The Escherichia colistrain BL21(DE3) was obtained from Dr. J. H. Veerkamp (Department of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands). Isopropyl-β-d-thiogalactopyranoside was purchased from Promega (Madison, WI). Ni2+-High Bond matrix was from Invitrogen (San Diego, CA). [γ-32P]ATP (3000 Ci/mmol) was obtained from Amersham Pharmacia Biotech. Cellulose TLC plates and TPCK-trypsin were purchased from Merck KGaA. The FuGENE6 Transfection Reagent and the anti-c-Myc monoclonal antibody were from Roche Molecular Biochemicals. PKC was purified from rat brain by a modified procedure previously described by Huang et al. (20.Huang K.P. Chan K.F. Singh T.J. Nakabayashi H. Huang F.L. J. Biol. Chem. 1986; 261: 12134-12140Abstract Full Text PDF PubMed Google Scholar). Rat brains (20–40 g of tissue) were homogenized, and the cytosolic fraction was subsequently purified on DEAE-Sepharose, Sephacryl 200 and phenyl-Sepharose columns. The purified enzyme has a specific activity of 200 nmol of phosphate/min/mg protein when assayed with histone IIIs as substrate. The purified enzyme is stable for several months when kept at −80 °C in 50% glycerol and 0.01% Triton X-100. PI-TPαI and II were purified from bovine brain cytosol as described by van Paridon et al. (17.van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar). The cDNA encoding mouse PI-TPα was expressed in E. coli, and the protein was purified as described by Geijtenbeek et al.(16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). The mouse recombinant PI-TPα (recPI-TPα) purified from E. colicontains one molecule of phosphatidylglycerol (PG) (16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). To exchange the PG molecule for a PI or PC molecule, recPI-TPα (28 nmol) was incubated with PI (385 nmol) present in unilamellar vesicles consisting of PI:PC (30:70 mol %) or with PC (900 nmol) present in vesicles consisting of PC:phosphatidic acid (70:30 mol %). Vesicles were prepared in 20 mm Tris buffer, pH 7.6, 50 mmNaCl by sonication under nitrogen for 10 min. Vesicles and protein were incubated for 15 min at 37 °C. The reaction was stopped by the addition of MgCl2 to a final concentration of 5 mm. Protein and vesicles were separated on a DEAE-cellulose column that was equilibrated in 20 mm Tris, pH 7.6, 50 mm NaCl, 5 mm MgCl2. The recPI-TPα eluted in the run-through and the negatively charged vesicles were retarded on the column (17.van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar). The protein content was determined using the Bradford assay (21.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217487) Google Scholar). The products of the exchange reaction were analyzed by isoelectric focussing (16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). The PI-TPα cDNA cloned into the pBluescript vector (pBlue-wtPI-TPα) (16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar) was used for site-directed mutagenesis using the Quickchange site-directed mutagenesis method according to the manufacturer's instruction (Stratagene). Ser-166 was replaced by Ala using the following mutagenic oligonucleotides: sense primer, 5′-CCAGCAAAATTTAAG GCTGTCAAAACAGGACGC-3′; antisense primer, 5′-GCGTCCTGTTTTGACAGC CTTAAATTTTGCTGG-3′. The bold nucleotides encode the mutated amino acid (Ser-166 to Ala-166), and the underlined nucleotide is a mutation that does not result in a change in amino acid composition, but it deletes a DraI restriction site. Incorporation of the mutagenic oligonucleotides into the construct (pBlue-PI-TPα(S166A) was checked by restriction enzyme analysis and by sequencing. Both the mutated and wtPI-TPα cDNAs were cloned into the pET-15b expression vector. Expression of these constructs yielded wtPI-TPα or PI-TPα(S166A) fused to an N-terminal peptide containing six histidine residues. A mutant PI-TPα in which Ser-166 was replaced with Asp-166 was obtained in a similar way using the above oligonucleotides except that the bold nucleotides were replaced for GAT (sense primer) and ATC (antisense primer). The E. coli strain BL21(DE3) was transformed by either the wtPI-TPα-pET15b or the mutant PI-TPα-pET-15b construct. A 10-ml culture of each transformant grown overnight in LB medium containing 50 μg/ml ampicillin was used to inoculate 1 liter of LB medium (also containing 50 μg/ml ampicillin). Bacteria were grown at 37 °C. At an A 600 of 0.8, the cultures were induced with 0.5 mmisopropyl-β-d-thiogalactopyranoside and grown for an additional 3 h. His6-tagged wtPI-TPα or His6-tagged mutant PI-TPα were purified from these cultures. All further manipulations were performed at 4 °C. Bacteria were harvested by centrifugation at 5,000 × g for 30 min. The pellet was resuspended in 50 ml of STE buffer (0.5m NaCl, 10 mm Tris/HCl, 0.1 mmEDTA, pH 7.5) and sonicated for 5 × 30 s at 80 W output with a macrotip of a Branson Sonifier B12. The homogenate was centrifuged at 17,500 × g. The supernatant was collected and extensively dialyzed against buffer A (18.3 mmNa2HPO4, 1.3 mmNaH2PO4, 500 mm NaCl, pH 7.8). The dialyzed supernatant was applied to a Ni2+-High Bond column (20 ml) and eluted with a linear gradient (200 ml) of 0–500 mm imidazol in buffer A (flow rate, 0.5 ml/min; fractions, 5 ml). The fractions were assayed for PI-TPα by measuring PI transfer activity and by Western blotting using an antibody against PI-TP. Purified His6-tagged wtPI-TPα and His6-tagged mutant PI-TPα were used for the in vitro phosphorylation experiments. PI and PC transfer activities were determined in a continuous fluorescence assay using 2-pyrenyl-decanoyl-PI or -PC as substrates. (11.Westerman J. de Vries K.J. Somerharju P. Timmermans-Hereijgers J.L. Snoek G.T. Wirtz K.W.A. J. Biol. Chem. 1995; 270: 14263-14266Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 17.van Paridon P.A. Visser A.J. Wirtz K.W.A. Biochim. Biophys. Acta. 1987; 898: 172-180Crossref PubMed Scopus (64) Google Scholar). Measurements were performed using a fluorimeter (Photon Technology International) equipped with a thermostated cuvette holder and a stirring device. PI-TPα (0.1–5 μg) was phosphorylated in a reaction volume of 60 μl containing 20 mm Tris/HCl, pH 7.5, 7.5 mm magnesium acetate, 10 μg/ml leupeptin, 10 μm ATP, and 1–2 μCi of [γ-32P]ATP. The Ca2+/phospholipid-independent phosphorylation was determined in the presence of 1 mm EGTA, and the Ca2+/phospholipid-dependent phosphorylation was determined in the presence of 1 mm Ca2+, 96 μg/ml phosphatidylserine, and 3.2 μg/ml diacylglycerol. The mixture was incubated for 10 min at 37 °C, and the reaction was terminated by the addition of 600 μl of cold acetone. Bovine serum albumin (1 μg) was added, and after 30 min on ice, the precipitated protein was spun down, dissolved in sample buffer (125 mm Tris/HCl, pH 6.8, 5% (w/v) SDS, 12.5% (v/v) 2-mercaptoethanol, and 10% (v/v) glycerol) and analyzed by SDS-PAGE (15% gel) followed by autoradiography. Swiss mouse 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% newborn calf serum and buffered with NaHCO3 (44 mm) in a 7.5% CO2 atmosphere. Near-confluent cell cultures in 75-cm2 flasks were labeled for 4.5 h with 1.5 mCi of carrier-free [32P]Pi in 6 ml of phosphate-free Ham's F-12 (DF) medium containing 5% newborn calf serum. PMA (100 ng/ml) was present during the last 15 min of the labeling. The membrane-free supernatant was prepared, and the immunoprecipitation procedure was carried out as described by Snoeket al. (15.Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar). After identification by autoradiography, the 32P-labeled PI-TPα bands were excised from the dried gel and eluted as described by Boyleet al. (22.Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Briefly, the gel slices were homogenized in 50 mm ammonium bicarbonate, pH 7.3–7.6, SDS (final concentration, 0.1%), and 2-mercaptoethanol (final concentration, 1%) were added, and the samples were boiled for 5 min. After incubation of the mixture at 37 °C for 2 h, the gel was spun down and the supernatant containing the 32P-labeled proteins was collected. A second elution with 0.1% SDS and 1% 2-mercaptoethanol was carried out on the gel pellet. Carrier protein (boiled RNase, 10 μg) and trichloroacetic acid (final concentration, 12%) was added to the combined supernatant fractions, and the samples were incubated on ice for 1 h. The trichloroacetic acid precipitate was washed with cold ethanol and dried. For phosphoamino acid analysis, the pellet was dissolved in 6 m HCl and hydrolyzed for 1 h at 110 °C. The HCl was removed by lyophilization, and the pellet was dissolved in pH 1.9 buffer (glacial acetic acid:formic acid (88%):H2O; 78:25:897 v/v/v). A mixture of phosphoserine, phosphothreonine, and phosphotyrosine (1 μg of each) was added. The32P-labeled phosphoamino acids were separated by two-dimensional electrophoresis on 20 × 20-cm cellulose TLC plates. The first dimension was in buffer pH 1.9, and the second dimension was in glacial acetic acid:pyridine:H2O (50:5:945 v/v/v), pH 3.5. After electrophoresis the plates were dried, the phosphoamino acids were visualized by staining with 0.2% (w/v) ninhydrin in acetone, and the 32P-labeled amino acids were identified by autoradiography. For phosphopeptide mapping the trichloroacetic acid pellet was dissolved in performic acid, and oxidation was performed for 1–2 h on ice. After lyophilization the sample was and incubated with TPCK-trypsin in 50 mm ammonium bicarbonate (200 μg/ml) at 37 °C for 5 h. The incubation was repeated by the addition of fresh trypsin, and the sample was lyophilized. The phosphopeptides were separated on cellulose TLC plates. In the first dimension electrophoresis was performed using the pH 1.9 buffer; in the second dimension TLC was performed in n-butanol:pyridine:glacial acetic acid:H2O (75:50:15:60 v/v/v/v). Radioactive phosphopeptides were identified by autoradiography. PI-TPα was phosphorylated as described above with the following changes. The ATP concentration was 1 mm with a trace of [γ-32P]ATP, and the incubation time was 2–4 h at 30 °C. The proteins were separated by SDS-PAGE, eluted, and precipitated with 10% trichloroacetic acid as described above. The pellet was digested with cyanogen bromide (2.5 mg/ml in 70% formic acid, 50 nmol cyanogen bromide/nmol protein) by incubation for 24 h in the dark at room temperature. After lyophilization, the sample was dissolved in 6 m guanidine HCl in 0.085% trifluoroacetic acid, and the peptides were separated on a reverse phase column C2/C18 (Amersham Pharmacia Biotech, SMART system) with a 0–60% (v/v) acetonitril. The radioactive peak was collected, and the N-terminal amino acid sequence of the32P-labeled peptide was determined by automatic Edman degradation using the 476A protein sequencer (Applied Biosystems). The pBlue-wtPI-TPα, pBlue-PI-TPα(S166A) and pBlue-PI-TPα(S166D) constructs were used to generate Myc-tagged PI-TPα fusion proteins. The pBlue-PI-TPα constructs contained a SacI site upstream of the translational start codon, an NcoI restriction site around the translational start codon, and a BamHI restriction site downstream of the translational stop codon. The SacI andNcoI sites were used to insert the linker encoding the Myc-tagged into the coding sequence. The linker consisted of two oligonucleotide primers carrying a SacI and anNcoI sticky end. The oligonucleotides used were: 5′-CATGGAACAAAAACTTATTTCTGAAGAAGATCTGC-3′ and 5′-CATGGCAGATCTTCTTCAGAAATAAGTTTTTGTTCCATGAGCT-3′. The underlined nucleotides of primer 2 represent theNcoI sticky end, and the bold nucleotides represent theSacI sticky end; the remaining bases are complementary to primer 1. The primers (10 μm each) were annealed at 60 °C for 15 min. After cooling to room temperature, the resulting linker was ligated into the pBlue-PI-TPα constructs that were previously digested with SacI and NcoI. The ensuing constructs were digested with SacI andBamHI, and the resulting DNA encoding Myc-tagged PI-TPα were ligated into the corresponding sites of the pBK-CMV expression vector. The obtained constructs were denoted as pBK-CMV-Myc-wtPI-TPα, pBK-CMV-Myc-PI-TPα(S166A), and pBK-CMV-Myc-PI-TPα(S166D). Expression of these constructs yields PI-TPα fused to an N-terminal peptide containing the 9E10 epitope (the peptide EQKLISEEDL) of the human c-Myc protein (Myc-tagged). NIH3T3 cells were seeded 24 h prior to transfection at 1 × 104 cells/cm2. Cells were transfected with 2 μg of the pBK-CMV-Myc-PI-TPα constructs using the FuGENE6 Transfection Reagent kit according to the manufacturer's instruction (Roche Molecular Biochemicals). The following day the cells were reseeded at approximately 5 × 103 cells/cm2. After another 24 h, G418 (0.4 mg/ml) was added for selection of G418-resistant cells. Fresh medium containing G418 was added every 4 days, and resistant clones were identified after 3 weeks of growth. The localization of endogenous PI-TPα, Myc-tagged wtPI-TPα, Myc-tagged PI-TPα(S166A), and Myc-tagged PI-TPα(S166D) in serum-starved (semi-quiescent) NIH3T3 cells was determined before and after stimulation with PMA or platelet derived growth factor (PDGF) as described in Ref. 15.Snoek G.T. Westerman J. Wouters F.S. Wirtz K.W.A. Biochem. J. 1993; 291: 649-656Crossref PubMed Scopus (20) Google Scholar. Briefly, NIH3T3 cells were made semi-quiescent by replacing the growth medium with Dulbecco's modified Eagle's medium containing 0.5% newborn calf serum. After 2 days the cells were incubated for 15 min at 37 °C with PMA (50 ng/ml) or PDGF (20 ng/ml) and fixed with methanol (endogenous PI-TPα) or paraformaldehyde (Myc-tagged PI-TPαs). Endogenous PI-TPα was visualized by indirect immunofluorescence using a polyclonal antibody directed against PI-TPα and goat-anti-rabbit-Cy3 as the second antibody. Myc-tagged PI-TPαs were visualized using a mouse monoclonal antibody directed against the Myc-tagged and goat anti-mouse tetramethyl rhodamine isothiocyanate as the second antibody. The effect of PMA and PDGF on the production of lysoPI in NIH3T3 cells before and after stimulation with PMA or PDGF was determined as described in Ref. 12.Snoek G.T. Berrie C.P. Geijtenbeek T.B. van der Helm H.A. Cadee J.A. Iurisci C. Corda D. Wirtz K.W.A. J. Biol. Chem. 1999; 274: 35393-35399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar. Briefly, NIH3T3 cells were cultured in a 6-well plate to 80% confluency. The cell cultures were incubated for 48 h with 2 μCi of [3H]-myo-inositol in HEPES-buffered DF medium without inositol containing 2% dialyzed newborn calf serum. Cultures were washed twice with phosphate-buffered saline and incubated for 10 min at 37 °C with DF medium (without inositol) containing 0.3% bovine serum albumin and 10 mm LiCl. Subsequently, PMA (50 ng/ml) or PDGF (20 ng/ml) was added, and the incubation was continued for another 15 min. The cells were washed twice with phosphate-buffered saline and scraped in 1 ml of −20 °C methanol. The [3H]inositol phospholipids were extracted and analyzed as described previously (23.Falasca M. Corda D. Eur. J. Biochem. 1994; 221: 383-389Crossref PubMed Scopus (70) Google Scholar). The PKC-dependent phosphorylation of the charge isomers PI-TPαI and II from bovine brain was determined in vitroas a function of concentration. From the Lineweaver-Burk plot it was calculated that the V max of the phosphorylation of PI-TPαI was 2.0 nmol/min and that of PI-TPαII 6.0 nmol/min (Fig.1 A). The K mof either reaction was comparable (0.65 and 0.72 μm, respectively). This implies that the affinity of rat brain PKC for both isomers is the same, yet that PI-TPα containing a PC molecule is phosphorylated at a faster rate than the protein containing a PI molecule. To confirm these results we have done similar experiments on mouse recPI-TPα. RecPI-TPα contains a PG molecule that can be readily exchanged for either PI or PC (16.Geijtenbeek T.B. de Groot E. van Baal J. Brunink F. Westerman J. Snoek G.T. Wirtz K.W.A. Biochim. Biophys. Acta. 1994; 1213: 309-318Crossref PubMed Scopus (27) Google Scholar). Phosphorylation of the charge isomers of recPI-TPα by PKC confirmed that the PC-containing protein was phosphorylated at a faster rate than the PI-containing protein (Fig. 1 B). In addition, phosphorylation of the PG-containing recPI-TPα was comparable with that of the PC-containing protein (data not shown). In comparison with the bovine PI-TPαs the mouse recPI-TPαs were better substrates, withV max values about 1 order of magnitude higher and K m values 1 order of magnitude lower (0.1 μm). Two-dimensional analysis of tryptic32P-labeled peptides showed that PI-TPαI, PI-TPαII and recPI-TPα have one major phosphopeptide in common as well as a minor one. The phosphopeptide map of PI-TPαII showed three additional minor spots (Fig. 2). Phosphoamino acid analysis of the in vitro phosphorylated proteins demonstrated that all three PI-TPαs are mainly phosphorylated on serine (Fig. 3). The immunoprecipitated PI-TPα from PMA-stimulated 32P-labeled Swiss mouse 3T3 cells was p
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