Phosphorylation-dependent Regulation of Phospholipase D2 by Protein Kinase Cδ in Rat Pheochromocytoma PC12 Cells
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m108343200
ISSN1083-351X
AutoresJung Min Han, Jae Ho Kim, Byoung Dae Lee, Sang Do Lee, Yong Kim, Yon Woo Jung, Sukmook Lee, Wonhwa Cho, Motoi Ohba, Toshio Kuroki, Pann‐Ghill Suh, Sung Ho Ryu,
Tópico(s)Receptor Mechanisms and Signaling
ResumoMany studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31–8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKCδ by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKCδ using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKCδ kinase activity in PC12 cells. PKCδ co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylatedin vitro by PKCδ were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKCδ inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKCδ was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKCδ-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells. Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31–8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKCδ by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKCδ using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKCδ kinase activity in PC12 cells. PKCδ co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylatedin vitro by PKCδ were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKCδ inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKCδ was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKCδ-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells. phospholipase D l-1-tosylamido-2-phenylethyl chloromethyl ketone phorbol 12-myristate 13-acetate Dulbecco's modified Eagle's medium tetramethylrhodamine B isothiocyanate Phospholipase D (PLD)1 catalyzes phosphatidylcholine hydrolysis to generate phosphatidic acid. Phosphatidic acid can be further metabolized to lysophosphatidic acid and diacylglycerol by phospholipase A2 (PLA2) and phosphatidic acid phosphohydrolase, respectively (1Exton J.H. Physiol. Rev. 1997; 77: 303-320Crossref PubMed Scopus (387) Google Scholar). These reactions are involved in receptor-mediated signal transductions and several cellular processes, such as membrane trafficking, cell growth and differentiation, cytoskeletal reorganization, respiratory burst, and apoptosis (1Exton J.H. Physiol. 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Various agonists such as platelet-derived growth factor, epidermal growth factor, bradykinin, angiotensin II, thrombin, and carbachol activate PLD in many cell types, whereas PKC mediates agonist-induced PLD activation (24Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (336) Google Scholar, 25Billah M.M. Curr. Opin. Immunol. 1993; 5: 114-123Crossref PubMed Scopus (151) Google Scholar, 26Lee Y.H. Kim H.S. Pai J.-K. Ryu S.H. Suh P.-G. J. Biol. Chem. 1994; 269: 26842-26847Abstract Full Text PDF PubMed Google Scholar, 27Yeo E.-J. Exton J.H. J. Biol. Chem. 1995; 270: 3980-3988Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). PLD1 was directly associated with, and activated by PKCα in the presence of PMA in NIH 3T3 cells and in PLD1-transfected COS-7 cells (28Lee T.G. Park J.B. Lee S.D. Hong S. Kim J.H. Kim Y. Yi K.S. Bae S. Hannun Y.A. Obeid L.M. Suh P.-G. Ryu S.H. Biochim. Biophys. Acta. 1997; 1347: 199-204Crossref PubMed Scopus (65) Google Scholar). In previous studies, we described the phosphorylation-dependent activation of PLD1 by PKCα in 3Y1 fibroblast cells and in PLD1-transfected COS-7 cells (29Kim Y. Han J.M. Park J.B. Lee S.D. Oh Y.S. Chung C. Lee T.G. Kim J.H. Park S.K. Yoo J.S. Suh P.-G. Ryu S.H. Biochemistry. 1999; 38: 10344-10351Crossref PubMed Scopus (69) Google Scholar) and PKCα-mediated PLD1 phosphorylation with the activation occurring in caveolin-enriched microdomains within the plasma membrane (30Kim Y. Han J.M. Han B.R. Lee K.A. Kim J.H. Lee B.D. Jang I.H. Suh P.-G. Ryu S.H. J. Biol. Chem. 2000; 275: 13621-13627Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, the details of the regulation of PLD2 by PKC are not well understood, although recently, it was reported that PLD2 could be activated in different cell types in response to PMA (31Sciorra V.A. Rudge S.A. Prestwick G.D. Frohman M.A. Engebrecht J. Morris A.J. EMBO J. 1999; 18: 5911-5921Crossref PubMed Scopus (147) Google Scholar, 32Czarny M. Lavie Y. Fiucci G. Liscovitch M. J. Biol. Chem. 1999; 274: 2717-2724Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 33Gibbs T.C. Meier K.E. J. Cell. Physiol. 2000; 182: 77-87Crossref PubMed Scopus (48) Google Scholar). The aim of this study was to examine the involvement of PKC in PMA-induced PLD2 stimulation in PC12 cells and to investigate whether PKC-mediated PLD2 activation is phosphorylation-dependent. In this report, we show that PLD2 can be activated in PC12 cells by PMA treatment. Moreover, for the first time, we were able to demonstrate that PLD2 becomes phosphorylated in response to PMA treatment and that PKCδ mediates the phosphorylation-dependent activation of PLD2 in PC12 cells. PMA, tetracycline, TPCK-trypsin, sodium cholate, and anti-actin monoclonal antibody were purchased from Sigma. Silica Gel 60 thin layer chromatography (TLC) plates and cellulose TLC plates were from Merck (Darmstadt, Germany). Phenylmethylsulfonyl fluoride, leupeptin, and aprotinin were from Roche Molecular Biochemicals (Mannheim, Germany). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Kirkegaard and Perry Laboratories, Inc. (Parker Ford, PA). [γ-32P]ATP (300 Ci/mmol), [32P]orthophosphate, and [3H]myristic acid (54 Ci/mmol) were from PerkinElmer Life Sciences (Boston, MA). The chemiluminescence kit (ECL) was from Amersham International (Buckinghamshire, UK). Immobilized protein A was from Pierce (Rockford, IL). Dulbecco's modified Eagle's medium was from Invitrogen (Grand Island, NY). Fetal calf serum was from HyClone (Logan, UT). GF109203X, Ro-31–8220, Go6976, and rottlerin were from Calbiochem (San Diego, CA). Anti-PKCδ and anti-PKCα monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). PC12 Tet-Off cells were purchased from CLONTECH (Palo Alto, CA). Rat pheochromocytoma PC12 Tet-Off cells were cultured at 37 °C in a humidified 5% CO2 atmosphere in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated equine serum and 5% (v/v) heat-inactivated fetal calf serum (4Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y. Suh P.-G. Ryu S.H. J. Neurochem. 2000; 75: 1053-1059Crossref PubMed Scopus (51) Google Scholar). Adenovirus expression vectors for wild type and the dominant-negative type of PKCδ have been described previously (34Kuroki T. Kashiwagi M. Ishino K. Huh N.H. Ohba M. J. Invest. Dermatol. Symp. Proc. 1999; 4: 153-157Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 35Ohba M. Ishino K. Kashiwagi M. Kawabe S. Chida K. Huh N.H. Kuroki T. Mol. Cell. Biol. 1998; 18: 5199-5207Crossref PubMed Google Scholar). Subconfluent PC12 cells in 6-well plates were infected with wild type (WT-PKCδ AdV) or dominant negative PKCδ adenovirus (DN-PKCδ AdV) for 12 h at different multiplicities of infection (m.o.i.) in 0.1% serum containing DMEM. After removing the virus, cells were cultured for an additional 24 h in DMEM supplemented with 10% heat-inactivated equine serum and 5% fetal calf serum. Cells were then incubated for 12 h in 0.5% fetal calf serum containing DMEM. PLD activity was assayed by measuring the formation of phosphatidylbutanol (PBtOH), the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol as previously described (4Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y. Suh P.-G. Ryu S.H. J. Neurochem. 2000; 75: 1053-1059Crossref PubMed Scopus (51) Google Scholar). PC12 cells were subcultured in 6-well tissue culture plates at 1 × 106 cells/well in the presence or absence of 0.5 μg/ml tetracycline. The cells were loaded with [3H]myristic acid (3 μCi/ml) for 3 h and then treated with 100 nm PMA in the presence of 0.4% 1-butanol (v/v) for the indicated times at 37 °C. To inhibit PKC, 5 μm GF109203X, 5 μm Ro-31-8220, 0.5 μm Go6976, or 15 μm rottlerin were administered for 15 min before the PMA treatment. After the incubation, the medium was aspirated, and 0.4 ml of ice-cold methanol was added to the cells. The cell debris was scraped into an Eppendorf tube, and chloroform and 0.1 n HCl were added, resulting in a final chloroform, methanol, 0.1 n HCl ratio of 1/1/1 (v/v/v). After vortexing, the tubes were centrifuged at 15,000 ×g for 1 min, and the organic phase was harvested, dried, and spotted onto a Silica Gel 60 TLC plate which was then developed with chloroform/methanol/acetic acid (9/1/1, v/v/v). The amounts of labeled PBtOH and total lipids were determined using a Fuji BAS-2000 image analyzer (Tokyo, Japan). To detect PLD2 and PKC, the proteins were separated by SDS-PAGE on 8% acrylamide gels, transferred to nitrocellulose membranes, and blotted with anti-PLD (16Kim J.H. Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.-G. Ryu S.H. FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar) and anti-PKC antibodies. PC12 cells were incubated in the presence or absence of 100 nm PMA at 37 °C for 5 min and then disrupted in hypotonic buffer (20 mm Tris/HCl, pH 7.5, 1 mmMgCl2, 10 mm NaCl, 0.5 mmphenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 5 μg/ml aprotinin) containing 1% Triton X-100 and 1% sodium cholate by sonication. After centrifugation, the membrane extract was immunoprecipitated using anti-PLD antibody, as previously described (30Kim Y. Han J.M. Han B.R. Lee K.A. Kim J.H. Lee B.D. Jang I.H. Suh P.-G. Ryu S.H. J. Biol. Chem. 2000; 275: 13621-13627Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). PKCδ was expressed in baculovirus-infected Sf9 cells and purified, as described previously (36Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17551Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) with slight modifications. Monolayers of Sf9 cells (2 × 107 cells/150-mm dish) were infected at a multiplicity of infection (m.o.i.) of 10. PKCδ was purified using HiTrap Q followed by phenyl-Superose FPLC. Direct interaction between PLD2 and binding protein was analyzed by the method, as described previously (19Park J.B. Kim J.H. Kim Y. Ha S.H. Kim J.H. Yoo J.S. Du G. Frohman M.A. Suh P.-G. Ryu S.H. J. Biol. Chem. 2000; 275: 21295-21301Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) with slight modifications. Recombinant PLD2 was expressed in baculovirus-infected Sf9 cells and purified, as described previously (16Kim J.H. Lee S.D. Han J.M. Lee T.G. Kim Y. Park J.B. Lambeth J.D. Suh P.-G. Ryu S.H. FEBS Lett. 1998; 430: 231-235Crossref PubMed Scopus (89) Google Scholar). Purified PLD2 was incubated with anti-PLD antibody immobilized on protein A-Sepharose in incubation buffer (20 mm Tris/HCl, pH 7.5, 1 mm MgCl2, 10 mm NaCl, 1% Triton X-100, and 1% sodium cholate). After incubating the immune complex (100 ng of purified PLD2, coupled to immunoaffinity resin) with 50 ng of PKCδ in the absence or presence of 100 nm PMA at 37 °C under in vitro binding conditions (30 mm Tris/HCl, pH 7.0, 6 mmMgCl2, 0.25 mm EGTA, 0.4 mmCaCl2, and 0.1% Triton X-100), the samples were pelleted and washed 5 times with washing buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, and 1% Triton X-100). The existence of PKCδ in the precipitate was determined by immunoblotting with anti-PKCδ antibody. Initially, 1 × 107 PC12 Tet-Off cells/100-mm dish were incubated with 5 mCi of [32P]orthophosphate in 5 ml of phosphate-free DMEM for 5 h at 37 °C. After the cells had been washed twice with serum-free medium, they were treated with 100 nm PMA for 5 min. To inhibit PKC, the cells were pretreated with 5 μmRo-31–8220, 0.5 μm Go6976, or 15 μmrottlerin for 15 min followed by 100 nm PMA for 5 min. The cells were then washed with ice-cold hypotonic buffer containing phosphatase inhibitors (30 mm NaF, 1 mmNa3VO4, and 30 mmNa4O7P2) and lysed in 1 ml of hypotonic buffer containing 1% Triton X-100, 1% sodium cholate, and phosphatase inhibitors (30 mm NaF, 1 mmNa3VO4, and 30 mmNa4O7P2). After centrifugation (15,000 × g for 15 min), equal amounts of soluble extract were incubated with 2 μg of anti-PLD antibody and 30 μl of immobilized protein A. The immunoprecipitated proteins were then separated by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to a photographic film for autoradiography. Immune complex (100 ng of purified PLD2, coupled to immunoaffinity resin) and 50 ng of PKCδ were incubated in phosphorylation buffer (30 mm Tris/HCl, pH 7.0, 6 mm MgCl2, 0.25 mm EGTA, 0.4 mm CaCl2, 0.1% Triton X-100, 0.12 mm ATP, and 2 μCi of [γ-32P]ATP (3,000 Ci/mmol)) in the presence of 100 nm PMA for 15 min. The reaction mixture was then electrophoresed through an 8% SDS-polyacrylamide gel, and the dried gel exposed to a photographic film for autoradiography. Two-dimensional phosphopeptide mapping was performed as described previously, with slight modification (29Kim Y. Han J.M. Park J.B. Lee S.D. Oh Y.S. Chung C. Lee T.G. Kim J.H. Park S.K. Yoo J.S. Suh P.-G. Ryu S.H. Biochemistry. 1999; 38: 10344-10351Crossref PubMed Scopus (69) Google Scholar). Immunoprecipitates of in vivo or in vitro 32P-labeled PLD2 were resolved by 8% SDS-PAGE and then transferred to a nitrocellulose membrane. The PLD2 was localized by autoradiography, excised from the membrane, and washed with deionized water. The piece of membrane corresponding to the PLD2 band was then incubated with 0.5% polyvinylpyrrolidone in 100 mm acetic acid at 37 °C for 30 min, washed with water, and then with fresh 50 mm ammonium bicarbonate. Tryptic digestion was achieved by incubating the piece of membrane in 150 μl of 50 mmammonium bicarbonate with 10 μg TPCK-treated trypsin at 37 °C for 6 h, followed by the addition of another 10 μg of TPCK-treated trypsin and a second 6-h incubation at 37 °C. The tryptic digest was lyophilized, oxidized with performic acid, re-lyophilized, and then dissolved in 20 μl of pH 1.9 buffer (glacial acetic acid, formic acid (88%), H2O; 78:25:8973 v/v/v). Phosphotryptic peptides were separated on cellulose TLC plates by electrophoresis at pH 1.9 in the first dimension. In the second dimension TLC was performed inn-butanol:pyridine:glacial acetic acid:H2O (75:50:15:60, v/v/v/v). For phosphoamino acid analysis, the piece of membrane corresponding to the PLD2 band was dissolved in 6 n HCl and hydrolyzed for 1 h at 110 °C. The HCl was then removed by lyophilization, and the pellet dissolved in pH 3.5 buffer (glacial acetic acid:pyridine:H2O; 50:5:945, v/v/v). A mixture of phosphoserine, phosphothreonine, and phosphotyrosine (1 μg of each) was then added. The 32P-labeled phosphoamino acids were separated by electrophoresis on 20 × 20-cm cellulose TLC plates. 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 identified by autoradiography. Immunocytochemical analysis was performed as described previously (30Kim Y. Han J.M. Han B.R. Lee K.A. Kim J.H. Lee B.D. Jang I.H. Suh P.-G. Ryu S.H. J. Biol. Chem. 2000; 275: 13621-13627Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, PC12 cells were grown on coverslips. After treatment with 100 nm PMA, cells were fixed in 3.7% (w/v) paraformaldehyde for 10 min at 37 °C, washed with phosphate-buffered saline, and then incubated in blocking buffer (1% goat serum in phosphate-buffered saline containing 0.2% Triton X-100) at 4 °C for 1 h. Subsequently cells were incubated with 2 μg/ml anti-PLD antibody and 2 μg/ml anti-PKCδ antibody overnight at 4 °C. After washing with phosphate-buffered saline containing 0.05% Triton X-100, cells were incubated with secondary antibodies: TRITC-conjugated anti-mouse antibody and fluorescein isothiocyanate-conjugated anti-rabbit antibody. After washing with phosphate-buffered saline containing 0.05% Triton X-100, the slides were mounted and examined under a fluorescence microscope (Nikon, Inc., Melville, NY). It has been reported that PMA, an activator of PKC, stimulated PLD activity in PC12 cells (37Kanoh H. Kanaho Y. Nozawa Y. J. Neurochem. 1992; 59: 1786-1794Crossref PubMed Scopus (30) Google Scholar) and that PLD2 was prominently expressed in PC12 cells (33Gibbs T.C. Meier K.E. J. Cell. Physiol. 2000; 182: 77-87Crossref PubMed Scopus (48) Google Scholar). We confirmed the presence of PLD2 in PC12 cells using anti-PLD antibody (data not shown). Therefore, to investigate whether PLD2 is activated in PC12 cells upon PMA treatment, we used a PLD2-inducible PC12 Tet-Off cell line (4Lee S.D. Lee B.D. Han J.M. Kim J.H. Kim Y. Suh P.-G. Ryu S.H. J. Neurochem. 2000; 75: 1053-1059Crossref PubMed Scopus (51) Google Scholar). In this case, recombinant human PLD2 protein is expressed under the control of an inducible tetracycline-regulated promoter. The removal of tetracycline from the culture medium thus results in increased expression of PLD2, which was verified by Western blot analysis using anti-PLD antibody, as shown in Fig.1 C. Treatment of the cells with 100 nm PMA stimulated endogenous PLD activity (+Tet) in PC12 cells, while the induction of heterologous PLD2 expression (−Tet) led to a further increase in PMA-induced PLD activity, indicating that the PLD2 activity had increased in response to PMA. The effect of PMA on the PLD2 activity was both time- and concentration-dependent. Activation of PLD2 by 100 nm PMA occurred within 5 min, and the PLD activity was saturated at 100 nm PMA in the presence or absence of tetracycline (Fig. 1, A and B). These data show that PLD2 can be activated by PMA in PC12 cells in time- and concentration-dependent manner. To confirm that PLD1 is also activated by PMA treatment, we created an inducible PC12 Tet-Off cell line, which overexpressed PLD1 in the absence of tetracycline. On checking the PMA-induced PLD1 activity in these PC12 cells, we found that the activity of PLD1 was also enhanced by the PMA treatment (data not shown). Next, we determined whether the PMA-induced PLD2 activation in PC12 cells was PKC-dependent. We used the PKC inhibitors GF109203X and Ro-31–8220 to detect any effect on PLD activity after PMA stimulation. As shown in Fig. 2, in the PLD2-overexpressing PC12 cells (−Tet) basal activity had increased, and furthermore, maximal PMA-induced PLD activation had also significantly increased compared with the control PC12 cells (+Tet), which do not overexpress PLD2. This indicated that the overexpressed PLD2 was responsive to PMA treatment. Inhibition of PKC by pretreating of the cells with GF109203X and Ro-31–8220, specific inhibitors of PKC, abrogated the PMA-induced PLD activity in the presence or absence of tetracycline. It has been reported that PC12 cells express PKCα, β, γ, δ, η, and ζ (43O'Driscoll K.R. Teng K.K. Fabbro D. Greene L.A. Weinstein I.B. Mol. Biol. Cell. 1995; 6: 449-458Crossref PubMed Scopus (60) Google Scholar), and that PMA activates all PKC isozymes except ζ. To determine which isozyme of PKC was involved in the PMA-induced PLD2 activation in PC12 cells, we used two different PKC isozyme-specific inhibitors, classical PKC isozymes-specific Go6976 and PKCδ-specific rottlerin. As shown in Fig. 2, Go6976 had a slight inhibitory effect on the PMA-induced PLD2 activity (∼14%), while rottlerin strongly inhibited the PMA-induced PLD2 activity (∼69%), suggesting that PKCδ might be mainly involved in the PMA-induced activation of PLD2 in PC12 cells. To examine whether PLD2 is phosphorylated upon PMA treatment, we looked for PMA-dependent phosphorylation of PLD2 in PC12 cells. Lysates from [32P]orthophosphate-loaded control PC12 cells (+Tet and −Tet) and PC12 cells (+Tet and −Tet) treated with 100 nmPMA for 5 min were immunoprecipitated with anti-PLD antibody. As seen in Fig. 3 A, in the PLD2-overexpressing PC12 (− Tet) cells, PLD2 became basally phosphorylated during the initial incubation with [32P]orthophosphate, but more PLD2 became phosphorylated after PMA treatment. However, in (+Tet) cells, the amount of endogenous PLD2 was negligible (Fig. 1 C), we could not detect phosphorylation of endogenous PLD2 in (+Tet) cells (data not shown). To determine whether PKC mediated the PMA-dependent PLD2 phosphorylation in PC12 cells, we inhibited PKC activity by pretreating the cells with Ro-31–8220. We found that the intensity of the PMA-induced PLD2 phosphorylation was reduced to basal level by the Ro-31–8220 pretreatment, suggesting that PKC is indeed involved in the PMA-induced PLD2 phosphorylation. Next, we further checked which isozyme of PKC was involved in the PMA-induced PLD2 phosphorylation using the PKC isozyme-specific inhibitors Go6976 and rottlerin. As shown in Fig. 3 A, Go6976 slightly attenuated, but rottlerin significantly inhibited the PMA-induced PLD2 phosphorylation, suggesting that the PMA-induced PLD2 phosphorylation may be mainly mediated by PKCδ. Direct analysis of PLD2 phosphoamino acids proved that serine and threonine residues were phosphorylated. Pretreatment of rottlerin greatly reduced the PMA-induced serine/threonine phosphorylation of PLD2 (Fig. 3 B). Next, we eluted32P-labeled PLD2 species, digested it with trypsin, and subjected it to two-dimensional phosphopeptide mapping (Fig.3 C). Multiple phosphopeptides were resolved from PLD2 before and after PMA treatment. Seventeen phosphopeptides (1–17) were either enhanced or created by the PMA treatment and pretreatment with rottlerin significantly reduced PMA-induced phosphopeptides, suggesting that 17 phosphopeptides from PLD2 after PMA treatment were the exclusive result of PKCδ kinase activity. We previously demonstrated that bradykinin activates PLD2 via PKCδ (44Lee S.D. Lee B.D. Ki
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