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• W2085495891 abstract "The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-ζ. In addition to primary monocytes, activation of PKC-ζ in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-ζ, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ε and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-ζ from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase.Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-ζ. Furthermore, activation of PKC-ζ failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-ζ activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-ζ is activated downstream of PI 3-kinase in monocytes in response to LPS. The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-ζ. In addition to primary monocytes, activation of PKC-ζ in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-ζ, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide ε and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-ζ from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase. Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-ζ. Furthermore, activation of PKC-ζ failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-ζ activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-ζ is activated downstream of PI 3-kinase in monocytes in response to LPS. Bacterial lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; PKC, protein kinase C; PI 3-kinase, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; MBP, myelin basic protein; PMA, phorbol 12-myristate 13-acetate; PS,l-α-phosphatidyl-l-serine; PI,l-α-phosphatidylinositol; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; FCS, fetal calf serum; FPLC, fast performance liquid chromatography. is one of the most potent agonists known that contributes to the activation of mononuclear phagocytes. Monocyte activation in response to LPS results in the production of an array of cytokines such as tumor necrosis factor-α, interleukin-1, and interleukin-6, in addition to other inflammatory mediators. In the extreme, the inflammatory response to LPS is an important contributor to septic shock which may occur during infection with Gram-negative bacilli (1Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1335) Google Scholar, 2Watson R.W.G. Redmond H.P. Bouchier-Hayes D. J. Leukocyte Biol. 1994; 56: 95-103Crossref PubMed Scopus (84) Google Scholar). Although there is an extensive body of knowledge about functional changes in monocytes induced by LPS, there is relatively less known about the signaling pathways used by LPS to bring about these changes. Recently, it has become clear that monocyte responses to LPS involve specific cell surface receptors leading to the activation of pathways containing both tyrosine and serine/threonine protein kinases (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 4Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T.E. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar, 5Weinstein S.L. Gold M.R. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4148-4152Crossref PubMed Scopus (302) Google Scholar, 6Stefanová I. Corcoran M.L. Horak E.M. Wahl L.M. Bolen J.B. Horak I.D. J. Biol. Chem. 1993; 268: 20725-20728Abstract Full Text PDF PubMed Google Scholar). The initial events in at least one dominant LPS signaling pathway are dependent upon the glycophosphatidylinositol-linked membrane molecule, CD14 (7Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3420) Google Scholar). Binding of the complex of LPS and LPS-binding protein to CD14 results in the activation of multiple src family protein tyrosine kinases, and this appears to involve the physical association of p53/56 lyn with the receptor complex (6Stefanová I. Corcoran M.L. Horak E.M. Wahl L.M. Bolen J.B. Horak I.D. J. Biol. Chem. 1993; 268: 20725-20728Abstract Full Text PDF PubMed Google Scholar). It has also been shown that LPS-mediated, CD14-dependent activation of p53/p56 lyn leads to its association with an activated form of the lipid kinase, PI 3-kinase (8Herrera-Velit P. Reiner N.E. J. Immunol. 1996; 156: 1157-1165PubMed Google Scholar). Activation of PI 3-kinase results in the production of PIP3, which is known to be an activator of the PKC isoforms ζ, ε, and δ (9Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 10Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar). This is of interest in the context of LPS signaling since evidence has recently been provided to show that a PKC activity is increased in LPS-treated monocytes (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 4Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T.E. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar). Notably, this activity appears to be related to one or more PKC isoforms, the activation of which is sustained in the absence of phosphatidylserine, Ca2+, and diacylglycerol (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar). This latter finding suggests the possibility that LPS may activate one of the aPKC isoforms, either PKC-ζ, PKC-ι, or both. This subfamily of PKC isoforms differs from cPKC (α, βI, βII, γ) and nPKC (δ, ε, η, θ) subfamily members in that aPKC isoforms are neither receptors for phorbol esters nor are regulated by Ca2+ or diacylglycerols (11Konno Y. Ohno S. Akita Y. Kawasaki H. Suzuki K. J. Biochem. ( Tokyo ). 1989; 106: 673-678Crossref PubMed Scopus (63) Google Scholar, 12Selbie L.A. Schmitz-Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar, 13Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar, 14Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2368) Google Scholar). Rather, aPKCs exhibit activator-independent activity which is increased upon exposure to novel lipids such as PIP3 and ceramides (9Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 15Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar, 16Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar). In light of the findings indicating that incubation of monocytes with LPS leads to the activation of both PI 3-kinase and a PKC with unusual properties, the objectives of the present study were to identify this PKC isoform and examine its regulation. The results presented show that PKC-ζ is rapidly activated in LPS-treated human monocytes, and this occurs downstream of activated PI 3-kinase. These findings are consistent with a model in which LPS activates p53/p56 lyn leading to increased PI 3-kinase activity and activation of PKC-ζ through the production of PIP3. Anti-PKC-ζ antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Upstate Biotechnology (Lake Placid, NY), and Life Technologies, Inc. (Burlington, Ontario, Canada). These antibodies gave similar results and were used interchangeably. Histone III-S, bovine myelin basic protein from bovine brain (MBP), protamine sulfate, protein kinase inhibitor (rabbit sequence), phorbol 12-myristate 13-acetate (PMA),l-α-phosphatidyl-l-serine (PS),l-α-phosphatidylinositol (PI), andl-α-phosphatidylinositol 4,5-diphosphate, were purchased from Sigma. Purified PIP3 was a gift from Dr. C.-S. Chen (University of Kentucky) and was prepared as described previously (17Wang D.-S. Chen C.-S. J. Org. Chem. 1996; 61: 5905-5910Crossref Scopus (56) Google Scholar). LY294002 and microcystin were from Calbiochem. Monoclonal anti-PKC-α antibody was from Santa Cruz Biotechnology. Peptide ε and anti-PI 3-kinase antibody were from Upstate Biotechnology (Lake Placid, NY). Mono Q columns and protein G-Sepharose were from Pharmacia Biotech Inc. Horseradish peroxidase-conjugated goat anti-rabbit antibodies, protein A-agarose, and electrophoresis reagents and supplies were purchased from Bio-Rad. U937 and THP-1 cell lines were from the American Type Culture Collection (Rockville, MD). LipofectAMINE was from Life Technologies Inc. Iscove's methyl cellulose, RPMI 1640, Hank's balanced salt solution, and penicillin/streptomycin were from Stem Cell Technologies (Vancouver, British Columbia). [γ-32P]ATP, enhanced chemiluminescence reagents, and enhanced chemiluminescence film were from Amersham Int. (Oakville, Ontario, Canada). Lipopolysaccharide (Escherichia coli O127:B8) was from Difco. Human AB+ serum was provided by The Canadian Red Cross (Vancouver, British Columbia). Unless specified otherwise, all other reagents were of the highest quality available. Fractions of peripheral blood enriched in white blood cells were obtained from the Cell Separator Unit (Vancouver Hospital and Health Sciences Center). Monocytes were enriched (85–95% pure) by adherence as described previously (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar). Monolayers of adherent cells in RPMI 1640 were treated with LPS (solubilized in RPMI + 10% AB+ human serum, final serum concentration, 0.1%) rinsed with ice-cold phosphate-buffered saline, snap frozen using liquid nitrogen, and stored at −70 °C prior to analysis. Cell lysates for column chromatography were prepared by lysing cells on ice (20 min) in fast performance liquid chromatography (FPLC) extraction buffer (1% Nonidet P-40, 12.5 mm MOPS, pH 7.5, 12.5 mmβ-glycerophosphate, 2 mm EGTA, 1.0 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride (PMSF), 100 nm microcystin, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin). Lysates were centrifuged at 16,000 × g to remove insoluble material and were filtered through a 0.2-μm filter. Protein concentrations were determined with Bio-RadDC protein assay using bovine serum albumin as standard. Cells lysates for analysis of PI 3-kinase activity were prepared as described previously (8Herrera-Velit P. Reiner N.E. J. Immunol. 1996; 156: 1157-1165PubMed Google Scholar) in 20 mm Tris, pH 8.0, 1% Triton X-100, 137 mm NaCl, 10% glycerol, 2 mm EDTA, 1 mm Na3VO4, 5 mm NaF, 100 nm microcystin, 1 mm PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin. Monocyte cell lines were maintained in complete RPMI supplemented with 10% heat-inactivated fetal calf serum (FCS). Twelve to 15 h prior to incubation with LPS, cells were rendered quiescent in RPMI without FCS at a concentration of 5 × 105 cells/ml. Following stimulation with LPS, cells were lysed immediately, and the detergent-soluble material was frozen at −70 °C until further analysis. PI 3-kinase activity was measured in U937 cells essentially as described previously (8Herrera-Velit P. Reiner N.E. J. Immunol. 1996; 156: 1157-1165PubMed Google Scholar). Cell lysates (1–3 mg of protein) were loaded onto a Mono Q FPLC column pre-equilibrated in buffer A (12.5 mm MOPS, pH 7.5, 12.5 mmβ-glycerophosphate, 2 mm EGTA, and 0.5 mmNa3VO4). Proteins were resolved with a 20-ml linear gradient of 0–0.8 m NaCl in buffer A at a flow rate of 0.5 ml/min. Fractions of 0.25 ml were collected, and aliquots were assayed for protein kinase activity or immunoreactivity as described below. Quiescent THP-1 cells were either untreated or were incubated with LPS for the times indicated. When the effects of PI 3-kinase inhibitors were being studied, cells were incubated with either 32 μm LY294002 or 100 nm wortmannin for 20 min prior to addition of LPS. Following treatment, cells were immediately lysed at 4 °C for 30 min in lysis buffer (1% Triton X-100, 50 mm HEPES, pH 7.4, 150 mm NaCl with protease and phosphatase inhibitors used at the same concentrations as described above). Lysates were precleared with protein A-agarose and PKC-ζ was immunoprecipitated with rabbit polyclonal anti-PKC-ζ (5 μg per sample, Upstate Biotechnology). Kinase activity was measured in the immunoprecipitates using MBP as substrate as described previously (18Gómez J. Garcı́a A. Borlado L.R. Bonay P. Martı́nez-A C. Silva A. Fresno M. Carrera A.C. Eicher-Streiber C. Rebollo A. J. Immunol. 1997; 158: 1516-1522PubMed Google Scholar). Quantitation of kinase activity was done by scintillation counting of the band corresponding to MBP. Incorporation of radioactivity into MBP in the absence of lysate was used as background and was subtracted from radioactivity present in the immunoprecipitates. Aliquots (5 μl) of column fractions were assayed for phosphotransferase activity using various substrates as described previously (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar). In brief, assays were performed in a final volume of 25 μl of kinase assay buffer containing 12.5 mmMOPS, pH 7.5, 12.5 mm β-glycerophosphate, 2 mm EGTA, 0.5 mm Na3VO4, 2 mm dithiothreitol, 5 mm MgCl2, 4 μm cAMP-dependent protein kinase inhibitor peptide, [γ-32P]ATP (40 μm), and various substrates at the concentrations indicated below. Reactions were allowed to proceed for 10 min at 30 °C at room temperature and were terminated by spotting 23 μl of the mixture on phosphocellulose filter squares. Filters were washed six times in 0.85% (v/v)O-phosphoric acid, and bound radioactivity was determined by scintillation counting. Cell lysates (1 mg), prepared from LPS-treated U937 cells, were incubated with either 7.5 μg of rabbit anti-protein kinase C-ζ antibody or 7.5 μg of monoclonal anti-PKC-α antibody for 2–3 h at 4 °C. Immune complexes were then incubated for 1 h with either protein A-agarose or protein G-Sepharose. Solid phase complexes were washed 2 times with FPLC extraction buffer by centrifugation at 14,000 ×g for 1 min, and the supernatant fraction was subjected to a second immunoprecipitation with the same antibodies. Immunoadsorbed supernatants were fractionated by Mono Q chromatography as described above, and fractions were analyzed by immunoblotting and protein kinase assays. pSRα-based mammalian expression plasmids, containing the entire coding regions of either wild-type bovine p85α or mutant bovine p85α (Δp85α), were kindly provided by Masato Kasuga (Kobe University School of Medicine, Kobe, Japan). The mutant has a deletion of 35 amino acids from residues 479–513 of bovine p85α and the insertion of two other amino acids (Ser-Arg) in the deleted position. This alteration prevents the association of mutant p85α with the p110 catalytic subunit. However, mutant p85α is able to compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant (19Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (418) Google Scholar). U937 cells were grown to a density of 4–8 × 105 cells/ml in RPMI 1640 media supplemented with 10% heat-inactivated FCS. Cells were washed and resuspended in 800 μl of antibiotic-free RPMI (without FCS). Transfection was done using LipofectAMINE according to the protocol supplied by the manufacturer. pSRα plasmids containing either wild-type or mutant p85α were cotransfected along with pMC1neo-poly(A), a plasmid encoding resistance to the antibiotic, G418 sulfate. DNA-liposome complexes were added to the cells followed by a 5-h incubation after which the cultures were supplemented with RPMI containing 10% FCS, penicillin, and gentamicin. Expression of foreign DNA was allowed to proceed for 2 days followed by the addition of 350 μg/ml G418. After 4 days in G418, the cells were suspended in Iscove's methyl cellulose supplemented with 2-mercaptoethanol, FCS, bovine serum albumin, G418, and glutamine. The cells were incubated at 37 °C for 10 days and colonies were picked and resuspended in 400 μl of RPMI + 10% FCS supplemented with G418. Thereafter cells were maintained in medium containing 350 μg/ml G418. Following incubation of adherent monocytes with LPS, cells were fractionated essentially as described previously (20Knutson K.L. Hoenig M. Endocrinology. 1994; 135: 881-886Crossref PubMed Scopus (54) Google Scholar). In brief, monocytes were scraped into hypotonic fractionation buffer (10 mm Tris, pH 7.4, 4.5 mm EDTA, 2.5 mm EGTA, 2.3 mm2-mercaptoethanol, 1.0 mm Na3VO4, 1 mm PMSF, 100 nm microcystin, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) and lysed for 20 min at 4 °C while rotating. Lysates were then centrifuged at 100,000 × g for 30 min to separate cytosolic from particulate fractions. The resulting pellets were extracted in fractionation buffer containing 1% Nonidet P-40 for 20 min and centrifuged (16,000 × g, 20 min, 4 °C) to separate the detergent-insoluble and -soluble material. The resulting supernatant was taken to represent a membrane fraction. Twenty to 40 μg of the cytosolic and membrane fractions were then subjected to immunoblotting using anti-PKC-ζ antibodies. Following incubation of peripheral blood monocytes with LPS, cell lysates were fractionated by Mono Q chromatography. As shown in Fig. 1 A, one major peak of MBP kinase activity eluted between 370 and 410 mm NaCl. A second, minor peak was also frequently observed eluting at ∼300 mm NaCl. This latter peak had previously been identified as p42 and p44 mitogen-activated protein kinases (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar). Compared with lysates prepared from untreated cells, the mean increase in activity of the major peak was 2.5 ± 0.7 (mean ± S.E.,n = 6) fold. Since previous data had suggested that this activity was a lipid-independent isoform of PKC (3Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar), the possible presence of PKC-ζ was analyzed by immunoblotting fractions with an isoform-specific antibody. Immunoreactivity for PKC-ζ was observed in the fractions corresponding to the peak of kinase activity (Fig.1 B). The antibody recognized a protein of ∼80–85 kDa which was sometimes resolved into a doublet or triplet of closely migrating proteins. The specificity of antibody reactivity with this ∼80-kDa protein complex was confirmed by peptide competition. Thus, as shown in Fig. 1 B, an excess of the PKC-ζ peptide used as immunogen to raise the antibody specifically abrogated recognition of these bands, whereas nonspecific reactivity with other proteins was not eliminated. Activation of PKC-ζ in response to LPS was also observed in two human, promonocytic cell lines, THP-1 and U937 (Fig.2 A). The MBP kinase activities exhibited similar elution profiles, and immunoreactivity for PKC-ζ corresponded with the peaks of activity (Fig. 2 B). The effect of LPS onin vivo PKC-ζ activity was also examined in a parallel system by immune complex kinase assay. PKC-ζ was immunoprecipitated from lysates of control or LPS-treated THP-1 cells, and kinase activity was measured using MBP as substrate. As shown in Fig. 2 C, LPS treatment resulted in a rapid and transient increase in kinase activity that was apparent as early as 5 min, was maximal at 10 min, and was nearly back to base line by 30 min. Immunodepletion was used to examine further whether the peak of MBP kinase activity was accounted for by PKC-ζ. Prior to fractionation on Mono Q, lysates prepared from LPS-treated U937 cells were either untreated or immunoadsorbed with either of two anti-PKC-ζ antibodies. Anti-PKC-α was used as a control for specificity. As shown in Fig.3 A, immunoabsorption of lysates with anti-PKC-ζ antibodies resulted in a significant reduction in peak activity when compared with fractions from either unadsorbed lysates or to lysates immunoadsorbed with anti-PKC-α antibody. In fact, peak MBP kinase activity was reduced by treatment with anti-PKC-ζ to a level essentially equivalent to that observed in fractions from control (non-LPS-treated) cells. Immunoadsorption of MBP kinase activity also resulted in removal of PKC-ζ immunoreactivity from Mono Q fractions (Fig. 3 B) and was observed with anti-PKC-ζ antibodies from two different sources (data not shown).Figure 1A, LPS-induced activation of serine/threonine kinases in human monocytes incubated with LPS (100 ng/ml) or medium alone (final serum concentration, 0.1%). Following incubation (15 min), cells were lysed in FPLC extraction buffer, and detergent-soluble lysates were fractionated by Mono Q anion-exchange chromatography, and fractions were assayed for MBP kinase activity as described under “Materials and Methods.” The data shown are from one experiment and are representative of results obtained in >6 independent experiments. B, detection of PKC-ζ immunoreactivity in fractions corresponding to the major peak eluting from the column at 390–420 mm NaCl. Aliquots of the fractions (125 μl) were analyzed for the presence of PKC-ζ by immunoblotting with anti-PKC-ζ antibody. Specific recognition of the 80–85-kDa protein by anti-PKC-ζ antibody was determined by immunoblotting in the absence or presence of peptide used to raise the antibody. Bands that were eliminated by preadsorbing the antibody with the competing peptide were deemed specific. The results shown are from one of three similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Immunoadsorption of the major peak of MBP kinase activity with anti-PKC-ζ antibodies. U937 cells were either stimulated with 1 μg/ml LPS or medium alone (final serum concentration, 0.1%), and detergent-soluble lysates were prepared for immunoadsorption as described under “Material and Methods.” Lysates from medium control and LPS-stimulated cells were either untreated or subjected to immunoadsorption with either of two rabbit anti-PKC-ζ antibodies (Life Technologies, Inc. (ζ1) or Santa Cruz (ζ2)) or anti-PKC-α (α) antibodies (specificity control). A, 1 mg of lysate from each sample was then fractionated by Mono Q chromatography, and MBP kinase activities were measured as described under “Materials and Methods.” The data shown are from one of three independent experiments that gave similar results. B, aliquots of fractions from LPS-treated samples were analyzed by immunoblotting with anti-PKC-ζ to confirm depletion of PKC-ζ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Translocation of PKC from the cytosol to the membrane fraction is commonly used as a marker of activation of the kinase. Following incubation of normal human monocytes with LPS, cytosolic and membrane fractions were prepared and analyzed by immunoblotting with anti-PKC-ζ antibody. Fig. 4 shows that after exposure to LPS for 10 min, levels of PKC-ζ increased significantly in the membrane fraction. Because of the abundance of cytosolic PKC-ζ, and since only a fraction of this was translocated to the membrane, there was no apparent decrease in cytosolic PKC-ζ. Since PKC-ζ is known to behave differently from other PKC family members, experiments were done to biochemically characterize the putative PKC-ζ. Aliquots of Mono Q fractions containing PKC-ζ were analyzed for activity using multiple substrates as shown in Fig. 5 A. Of the different substrates tested, the kinase phosphorylated MBP (0.2 mg/ml), peptide ε (84 μm), and S6 peptide (0.1 mm) with similar efficiency. In comparison, activities toward Kemptide (0.2 mg/ml), histone (0.2 mg/ml), and protamine sulfate (0.2 mg/ml) were lower. This profile of substrate preferences is consistent with previous reports for PKC-ζ (10Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 13Ways D.K. Cook P.P. Webster C. Parker P.J. J. Biol. Chem. 1992; 267: 4799-4805Abstract Full Text PDF PubMed Google Scholar). Cofactor requirements for PKC-ζ have also been found to be different than those of other PKC isoforms (10Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 15Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar). Fig. 5 B shows the activity of the kinase toward MBP in the presence or absence of various PKC activators and cofactors. PIP3, an activator of PKC-ζ, enhanced the activity of the kinase in Mono Q fractions prepared from control cells (Fig. 5 B) but not in those prepared from LPS-treated cells (data not shown). Unlike PIP3, neither arachidonic acid (alone or with diacylglycerol) nor phosphatidylserine significantly enhanced the activity of the kinase when compared with its activity detected in the absence of added lipids (Fig. 5 B). In contrast to these findings with PKC-ζ, when partially purified cPKC was tested in the presence of phosphatidylserine and diacylglycerol, arachidonic acid (50 μm) was observed to further enhance its activity (data not shown). PMA, a known activator of several PKC isoforms, was also tested in the presence of PS and Ca2+. The addition of PS, Ca2+, and PMA together resulted in an approximate 35% increase in activity when compared with activity in the absence of cofactors (Fig. 5 C). In contrast, fractions corresponding to cPKC (i.e. 15–20) showed a robust activation in response to the combination of PMA, PS, and Ca2+. Recent evidence has suggested a role for PI 3-kinase metabolites in a signaling cascade leading to the activation of PKC (9Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar). To examine the potential involvement of PI 3-kinase in LPS-induced activation of PKC-ζ, cells were incubated with the PI 3-kinase inhibitors, wortmannin or LY294002, prior to the addition of LPS. As shown in Fig. 6, when used at concentrations known to be relatively selective for inhibition of PI 3-kinase, both inhibitors markedly attenuated activation of PKC-ζ induced by LPS. The requirement for PI 3-kinase for activation of PKC-ζ was also examined in cells transfected with a dominant negative mutant of p85 (Δp85). Stable transfection with Δp85 resulted in a significant reduction in both basal and LPS-stimulated PI 3-kinase activity (Fig.7, A and B). In contrast, cells transfected with wild-type p85 showed increased PI 3-kinase activity in response to LPS stimulation. To assess the effects of Δp85 on activation of PKC-ζ by LPS, lysates of transfected cells were analyzed using Mono Q chromatography. As shown in Fig. 8activation of PKC-ζ by LPS was abrogated in cells transfected with Δp85, and cells expressing wild-type p85 showed enhanced PKC-ζ activity in response to LPS. Fig. 8 C demonstrates that PKC-ζ was expressed in Δp85 transfected cells and PKC-ζ immunoreactivity correlated with the peak kinase activity.Figure 8Attenuation of LPS-induced activation of PKC-ζ by a dominant negative mutant of the p85 subunit (Δp85) of PI 3-kinas" @default.
• W2085495891 created "2016-06-24" @default.
• W2085495891 creator A5031005364 @default.
• W2085495891 creator A5039788418 @default.
• W2085495891 creator A5083218249 @default.
• W2085495891 date "1997-06-01" @default.
• W2085495891 modified "2023-10-15" @default.
• W2085495891 title "Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes" @default.
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