Phosphatidylinositol 3-Kinase-dependent Activation of Protein Kinase C-ζ in Bacterial Lipopolysaccharide-treated Human Monocytes*

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.

clear phagocytes. Monocyte activation in response to LPS results in the production of an array of cytokines such as tumor necrosis factor-␣, 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 (1,2).
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 (3)(4)(5)(6). The initial events in at least one dominant LPS signaling pathway are dependent upon the glycophosphatidylinositol-linked membrane molecule, CD14 (7). 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 (6). It has also been shown that LPS-mediated, CD14dependent activation of p53/p56 lyn leads to its association with an activated form of the lipid kinase, PI 3-kinase (8).
Activation of PI 3-kinase results in the production of PIP 3 , which is known to be an activator of the PKC isoforms , ⑀, and ␦ (9,10). 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 (3,4). Notably, this activity appears to be related to one or more PKC isoforms, the activation of which is sustained in the absence of phosphatidylserine, Ca 2ϩ , and diacylglycerol (3). This latter finding suggests the possibility that LPS may activate one of the aPKC isoforms, either PKC-, PKC-, or both. This subfamily of PKC isoforms differs from cPKC (␣, ␤I, ␤II, ␥) and nPKC (␦, ⑀, , ) subfamily members in that aPKC isoforms are neither receptors for phorbol esters nor are regulated by Ca 2ϩ or diacylglycerols (11)(12)(13)(14). Rather, aPKCs exhibit activator-independent activity which is increased upon exposure to novel lipids such as PIP 3 and ceramides (9,15,16).
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-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-through the production of PIP 3 .

MATERIALS AND METHODS
Reagents-Anti-PKC-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-␣-phosphatidyl-L-serine (PS), L-␣-phosphatidylinositol (PI), and L-␣-phosphatidylinositol 4,5-diphosphate, were purchased from Sigma. Purified PIP 3 was a gift from Dr. C.-S. Chen (University of Kentucky) and was prepared as described previously (17). LY294002 and microcystin were from Calbiochem. Monoclonal anti-PKC-␣ antibody was from Santa Cruz Biotechnology. Peptide ⑀ 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 antirabbit 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). Lipo-fectAMINE 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).
[␥-32 P]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 ϩ serum was provided by The Canadian Red Cross (Vancouver, British Columbia). Unless specified otherwise, all other reagents were of the highest quality available.
Isolation of Monocytes, Cell Treatment, and Processing-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 (3). Monolayers of adherent cells in RPMI 1640 were treated with LPS (solubilized in RPMI ϩ 10% ABϩ human serum, final serum concentration, 0.1%) rinsed with ice-cold phosphate-buffered saline, snap frozen using liquid nitrogen, and stored at Ϫ70°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 mM ␤-glycerophosphate, 2 mM EGTA, 1.0 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 nM microcystin, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin). Lysates were centrifuged at 16,000 ϫ g to remove insoluble material and were filtered through a 0.2-m filter. Protein concentrations were determined with Bio-Rad DC protein assay using bovine serum albumin as standard.
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 ϫ 10 5 cells/ml. Following stimulation with LPS, cells were lysed immediately, and the detergentsoluble material was frozen at Ϫ70°C until further analysis.
Anion-exchange Chromatography-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 mM ␤-glycerophosphate, 2 mM EGTA, and 0.5 mM Na 3 VO 4 ). 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.
PKC-Immune Complex Kinase Assay-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 M 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-was immunoprecipitated with rabbit polyclonal anti-PKC-(5 g per sample, Upstate Biotechnology). Kinase activity was measured in the immunoprecipitates using MBP as substrate as described previously (18). 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.
Protein Kinase Assays-Aliquots (5 l) of column fractions were assayed for phosphotransferase activity using various substrates as described previously (3). In brief, assays were performed in a final volume of 25 l of kinase assay buffer containing 12.5 mM MOPS, pH 7.5, 12.5 mM ␤-glycerophosphate, 2 mM EGTA, 0.5 mM Na 3 VO 4 , 2 mM dithiothreitol, 5 mM MgCl 2 , 4 M cAMP-dependent protein kinase inhibitor peptide, [␥-32 P]ATP (40 M), 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 l 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.
Immunoadsorption of Kinase Activity-Cell lysates (1 mg), prepared from LPS-treated U937 cells, were incubated with either 7.5 g of rabbit anti-protein kinase C-antibody or 7.5 g of monoclonal anti-PKC-␣ 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 ϫ 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.
Transfection of U937 Cells-pSR␣-based mammalian expression plasmids, containing the entire coding regions of either wild-type bo-FIG. 1. A, 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 Ͼ6 independent experiments. B, detection of PKCimmunoreactivity in fractions corresponding to the major peak eluting from the column at 390 -420 mM NaCl. Aliquots of the fractions (125 l) were analyzed for the presence of PKC-by immunoblotting with anti-PKCantibody. Specific recognition of the 80 -85-kDa protein by anti-PKCantibody 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. vine p85␣ or mutant bovine p85␣ (⌬p85␣), 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 p85␣ and the insertion of two other amino acids (Ser-Arg) in the deleted position. This alteration prevents the association of mutant p85␣ with the p110 catalytic subunit. However, mutant p85␣ is able to compete with native p85 for binding to essential signaling proteins and behaves as a dominant negative mutant (19). U937 cells were grown to a density of 4 -8 ϫ 10 5 cells/ml in RPMI 1640 media supplemented with 10% heat-inactivated FCS. Cells were washed and resuspended in 800 l of antibiotic-free RPMI (without FCS). Transfection was done using LipofectAMINE according to the protocol supplied by the manufacturer. pSR␣ plasmids containing either wild-type or mutant p85␣ 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 g/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 l of RPMI ϩ 10% FCS supplemented with G418. Thereafter cells were maintained in medium containing 350 g/ml G418.
Translocation Assay-Following incubation of adherent monocytes with LPS, cells were fractionated essentially as described previously (20). 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 mM 2-mercaptoethanol, 1.0 mM Na 3 VO 4 , 1 mM PMSF, 100 nM microcystin, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin) and lysed for 20 min at 4°C while rotating. Lysates were then centrifuged at 100,000 ϫ 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 ϫ 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 g of the cytosolic and membrane fractions were then subjected to immunoblotting using anti-PKCantibodies.

Lipopolysaccharide-induced Activation of Protein Kinase C-
in Monocytes-Following incubation of peripheral blood monocytes with LPS, cell lysates were fractionated by Mono Q chromatography. As shown in Fig. 1A, one major peak of MBP kinase activity eluted between 370 and 410 mM NaCl. A second, minor peak was also frequently observed eluting at ϳ300 mM NaCl. This latter peak had previously been identified as p42 and p44 mitogen-activated protein kinases (3). Compared with lysates prepared from untreated cells, the mean increase in activity of the major peak was 2.5 Ϯ 0.7 (mean Ϯ S.E., n ϭ 6) fold. Since previous data had suggested that this activity was a lipid-independent isoform of PKC (3), the possible presence of PKCwas analyzed by immunoblotting fractions with an isoform-specific antibody. Immunoreactivity for PKCwas observed in the fractions corresponding to the peak of kinase activity (Fig. 1B). The antibody recognized a protein of ϳ80 -85 kDa which was sometimes resolved into a doublet or triplet of closely migrating proteins. The specificity of antibody reactivity with this ϳ80-kDa protein complex was confirmed by pep- FIG. 2. LPS-induced activation of MBP kinase activity in promonocytic cell lines. THP-1 or U937 cells were rendered quiescent in serum-free RPMI for 12-15 h and subsequently stimulated (15 min) with 1 g/ml LPS or medium as described under "Materials and Methods." A, cells were lysed and subjected to Mono Q chromatography. MBP kinase activity in aliquots (5 l) of each fraction from each cell line was determined as described in Fig. 1. B, detection of PKCimmunoreactivity in fractions corresponding to the major peak was done as described in the legend to Fig. 1. Results are from one of three similar experiments. C, THP-1 cells were treated with LPS for the indicated times, and PKCwas immunoprecipitated with polyclonal anti-PKCantibodies (Upstate Biotechnology). In vitro kinase activities in the immunoprecipitates were measured using MBP as substrate as described under "Materials and Methods." tide competition. Thus, as shown in Fig. 1B, an excess of the PKCpeptide used as immunogen to raise the antibody specifically abrogated recognition of these bands, whereas nonspecific reactivity with other proteins was not eliminated.
Activation of PKCin response to LPS was also observed in two human, promonocytic cell lines, THP-1 and U937 (Fig. 2A). The MBP kinase activities exhibited similar elution profiles, and immunoreactivity for PKCcorresponded with the peaks of activity (Fig. 2B). The effect of LPS on in vivo PKCactivity was also examined in a parallel system by immune complex kinase assay. PKCwas immunoprecipitated from lysates of control or LPS-treated THP-1 cells, and kinase activity was measured using MBP as substrate. As shown in Fig. 2C, 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-. Prior to fractionation on Mono Q, lysates prepared from LPS-treated U937 cells were either untreated or immunoadsorbed with either of two anti-PKCantibodies. Anti-PKC-␣ was used as a control for specificity. As shown in Fig. 3A, immunoabsorption of lysates with anti-PKCantibodies resulted in a significant reduction in peak activity when compared with fractions from either unadsorbed lysates or to lysates immunoadsorbed with anti-PKC-␣ antibody. In fact, peak MBP kinase activity was reduced by treatment with anti-PKCto a level essentially equivalent to that observed in fractions from control (non-LPStreated) cells. Immunoadsorption of MBP kinase activity also resulted in removal of PKCimmunoreactivity from Mono Q fractions (Fig. 3B) and was observed with anti-PKCantibodies from two different sources (data not shown). 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-PKCantibody. Fig. 4 shows that after exposure to LPS for 10 min, levels of PKCincreased significantly in the membrane fraction. Because of the abundance of cytosolic PKC-, and since only a fraction of this was translocated to the membrane, there was no apparent decrease in cytosolic PKC-.
Biochemical Characterization of PKC--Since PKC-is known to behave differently from other PKC family members, experiments were done to biochemically characterize the putative PKC-. Aliquots of Mono Q fractions containing PKCwere analyzed for activity using multiple substrates as shown in Fig. 5A. Of the different substrates tested, the kinase phosphorylated MBP (0.2 mg/ml), peptide ⑀ (84 M), 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- (10,13).
Cofactor requirements for PKChave also been found to be different than those of other PKC isoforms (10, 15). Fig. 5B shows the activity of the kinase toward MBP in the presence or absence of various PKC activators and cofactors. PIP 3 , an ac-tivator of PKC-, enhanced the activity of the kinase in Mono Q fractions prepared from control cells (Fig. 5B) but not in those prepared from LPS-treated cells (data not shown). Unlike PIP 3 , 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. 5B). In contrast to these findings with PKC-, when partially purified cPKC was tested in the presence of phosphatidylserine and diacylglycerol, arachidonic acid (50 M) 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 Ca 2ϩ . The addition of PS, Ca 2ϩ , and PMA together resulted in an approximate 35% increase in activity when compared with activity in the absence of cofactors (Fig. 5C). In contrast, fractions corresponding to cPKC (i.e. [15][16][17][18][19][20] showed a robust activation in response to the combination of PMA, PS, and Ca 2ϩ . Activation of PKC-Is Phosphatidylinositol 3-Kinasedependent-Recent evidence has suggested a role for PI 3-kinase metabolites in a signaling cascade leading to the activation of PKC (9). To examine the potential involvement of PI 3-kinase in LPS-induced activation of PKC-, 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 PKCinduced by LPS.
The requirement for PI 3-kinase for activation of PKCwas FIG. 6. Abrogation of LPS-induced activation of PKC-by the PI 3-kinase inhibitors, wortmannin or LY294002. A, human monocytes were preincubated with 50 nM wortmannin (n ϭ 4), 16 M LY294002 (n ϭ 2), or vehicle for 15 min. Cells were then treated with 100 ng/ml LPS or medium alone for an additional 15 min. Detergent lysates were prepared and fractionated by Mono Q chromatography. MBP kinase activities were determined in fractions (5 l) from each cell preparation as described in Fig. 1. B, THP-1 cells were preincubated with either 100 nM wortmannin, 32 M LY294002, or vehicle for 20 min prior to addition of LPS (1 g/ml, 10 min). Lysates were immunoprecipitated with anti-PKCantibodies, and MBP kinase activity was measured in the immunoprecipitates as described in the legend to Fig. 2C.

FIG. 7. PI 3-kinase activity in U937 cells transfected with either wild-type bovine p85␣ or dominant negative mutant,
⌬p85␣. Cells were stimulated with 1 g/ml LPS or medium alone followed by detergent lysis. A, lysates were immunoprecipitated with anti-PI 3-kinase antibody, and phosphatidylinositol kinase activity was assayed as described under "Materials and Methods." Radioactivity observed at the origin (Ori) reflects residual, water-soluble 32 P-labeled material in the samples, the amount of which is not relevant to the results. B, spots corresponding to PIP were cut and analyzed by scintillation counting. Activity is expressed as a percentage of control (untreated) cells transfected with wild-type p85␣ (Wp85). Results presented are from one of two independent experiments with similar results. also examined in cells transfected with a dominant negative mutant of p85 (⌬p85). Stable transfection with ⌬p85 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 ⌬p85 on activation of PKCby LPS, lysates of transfected cells were analyzed using Mono Q chromatography. As shown in Fig. 8 activation of PKCby LPS was abrogated in cells transfected with ⌬p85, and cells expressing wild-type p85 showed enhanced PKCactivity in response to LPS. Fig. 8C demonstrates that PKCwas expressed in ⌬p85 transfected cells and PKCimmunoreactivity correlated with the peak kinase activity. DISCUSSION Previous studies demonstrated that a PKC with unusual properties is activated in human monocytes following exposure to LPS (3,4). Important questions arising from these studies are the identity of this PKC isoform and its mechanism of activation. Several considerations suggested the possibility that this LPS-activated isoform may be PKC-. PKC-is a member of a subfamily of atypical PKCs in that it is not activated by diacylglycerols, and it does not require Ca 2ϩ for activity. Rather, recent findings indicate that the activity of PKCis enhanced in vitro by PIP 3 . This, together with the previous observations that LPS induces the accumulation of PIP 3 in monocytes (8), suggested the hypothesis that LPS may activate PKCin a PI 3-kinase-dependent manner. Multiple lines of evidence presented in this paper support this argument.
Analysis of extracts of LPS-treated peripheral blood monocytes, U937 cells, and THP-1 cells showed principal peaks of enhanced MBP kinase activity eluting from Mono Q between 370 and 410 mM NaCl (Figs. 1A and 2A). These peaks of kinase activity were detected in the absence of exogenous lipids and were also independent of Ca 2ϩ . These characteristics are consistent with PKC-, and immunoblotting of Mono Q fractions indicated the presence of PKCin these samples (Figs. 1B and  2B). In addition, immunoprecipitation of PKCdirectly from lysates of LPS-treated cells showed that the activity of the enzyme was increased when compared with the activity observed in immunoprecipitates obtained from control cells (Fig.  2C). Furthermore, the LPS-enhanced MBP kinase activity could be removed by immunoadsorption with anti-PKC- (Fig.  3). Prior analyses of PKC expression in blood monocytes, U937 cells, and HL-60 cells indicate the presence of PKC-␣, -␤ I , -␤ II , -⑀, -, and isoforms (13,(21)(22)(23). In U937 cells, PKC-␣, -␤, and -⑀ elute from Mono Q at or below 320 mM NaCl, whereas PKCelutes at ϳ460 mM NaCl (13). The somewhat earlier elution of PKC-(370 -410 mM NaCl) observed in the present study most likely reflects procedural differences related to detergent solubilization and variations in the elution buffers.
Two of the anti-PKCantibodies (Santa Cruz Biotechnology and Life Technologies, Inc.) used in this study were raised against a peptide (SEFEGFEYINPLLLSAEESV) corresponding to amino acids 573-592 present in the COOH terminus of the kinase. This exact sequence is not found in any of the other known PKC isoforms (24). The closely related PKCcontains a similar COOH-terminal sequence differing at only two amino acids (SEFEGFEYINPLLMSAEECV) and can be detected with antibodies to the COOH terminus of PKC-. However, it is unlikely that the major immunoreactive band detected in this study is PKC-since this isoform is known to migrate at 65 kDa (12), consistently lower than the ϳ80 -85-kDa protein observed in this study. The third antibody (Upstate Biotechnology) used in the present study is reported not to cross-react with either PKC-␣, PKC-, or PKC-(according to information provided by the manufacturer). It has also been reported that antibodies directed against the COOH terminus of PKC-react with a Ca 2ϩ and phorbol ester-sensitive PKC isoform (25). However, the kinase that is the subject of this report is only weakly activated by a combination of PMA, Ca 2ϩ , and PS and has sustained activity in the absence of exogenous lipids. These findings preclude the notion that it may be a member of either the cPKC or nPKC subfamilies.
Membrane translocation of PKC has been used extensively as a measure of its activation (14). The finding that PKCtranslocates to the membrane following exposure to LPS provides additional evidence for its activation. Given that PIP 3 increases the activity of PKC-in vitro (Ref. 9 and Fig. 5) and that activation of PKC-in vivo requires activation of PI 3-kinase ( Figs. 6 and 8), a possible mechanism for redistribution of PKC-is through direct binding to PIP 3 in the membrane compartment. Although membrane translocation of PKC-induced by PIP 3 in vivo has not been reported, it has been shown that PKC-translocates to the membrane transiently following tetanization of hippocampal slices (26). This translocation correlated with the cytosolic accumulation of PKM-, a 51-kDa catalytic subunit of the holoenzyme which suggested activation-induced proteolysis. These findings raise the interesting possibility that PKM-may be generated by LPS in monocytes and that this may be responsible for the increased kinase activity detected. However, two lines of evidence argue against LPS-induced generation of PKM-. First, levels of the ϳ80 -85-kDa PKC-were not observed to be decreased in Mono Q fractions prepared from LPS-treated cells. Second, smaller anti-PKC-immunoreactive proteins were not observed in response to LPS (data not shown).
PKC isoforms have been observed to display different substrate specificity profiles, and to some extent the results obtained are influenced by the specific assay conditions (13,15,27). In the present study, it was found that MBP, peptide ⑀, and S6 peptide were equally efficient substrates for monocyte PKC-. In contrast, protamine sulfate, histone, and Kemptide were relatively poor substrates (Fig. 5). These findings are consistent with previous studies in which PKC-phosphorylated MBP and peptide substrates with equal efficiency (10) but showed lower activity against histone and Kemptide (13,28). Although the data contrast somewhat with other results suggesting that peptide ⑀ may be a more efficient substrate than MBP (15,28), these comparisons are complicated by species differences as well as different assay conditions. Activator and cofactor requirements for PKC isoforms have served as the basis for a general classification scheme for these kinases. Members of the cPKC and nPKC subgroups have generally been found to require the presence of PS and diacylglycerol for activation and exhibit little activity in the absence of these factors (29). The cPKC members also require the presence of Ca 2ϩ . In contrast, human PKC-exhibits activator-and cofactor-independent activity which is not increased by the addition of PS (13,27). In the present study, neither arachidonic acid nor phosphatidylserine significantly enhanced the activity of the kinase when compared with the activity detected in the absence of added lipids (Fig. 5). The biochemical characteristics of LPS-activated monocyte PKC-reported in this paper (Fig. 5) are consistent with previous studies (13,27). The findings presented contrast somewhat, however, with studies of PKC-in other species. For example, rat and bovine homologues display activator-and cofactor-independent activity which is significantly enhanced by the addition of PS (reviewed in Ref. 30).
The mechanisms leading to the activation of PKC-in cells in response to external stimuli are not fully understood. The involvement of PI 3-kinase in the regulation of nPKC and aPKC isoforms has been suggested by studies showing a stimulatory effect of the PI 3-kinase products PI 3,4-P 2 and PIP 3 in vitro (9,10). The role of PI 3-kinase in LPS-induced activation of monocyte PKC-was examined using two different approaches. The first approach involved the use of two structurally unrelated PI 3-kinase inhibitors. LPS-induced activation of PKC-was abrogated by both wortmannin and LY294002 (Fig.  6). The effects of wortmannin are considered to be relatively specific for PI 3-kinase at concentrations similar to those used in this study (50 -100 nM). However, the compound has been shown to inhibit phospholipase A 2 with an IC 50 similar to that previously reported for PI 3-kinase (31). On the other hand, the structurally unrelated compound, LY294002, has been shown to have inhibitory effects on PI 3-kinase by a different mechanism (32). Moreover, LY294002 shows no inhibitory effects on other lipid kinases or on several protein kinases, including PKC and mitogen-activated protein kinase (32). Therefore, the findings that both compounds exhibit inhibitory effects on LPS-induced activation of PKCsupport the argument that PI 3-kinase is involved in the regulation of PKC-. Further support for this argument was provided by experiments in which a dominant negative mutant of PI 3-kinase (⌬p85) expressed in U937 cells completely blocked LPS-induced activation of PKC- (Fig. 8). It has previously been shown that incubation of monocytes with LPS activates PI 3-kinase leading to increased cellular levels of PIP 3 (8). Thus, the most likely mechanism for the attenuation of activation of PKCby either wortmannin, LY294002, or ⌬p85 is inhibition of the formation of PIP 3 in vivo. Moreover, in the present study it was observed that the addition of PIP 3 in vitro to specific Mono Q fractions from control cells led to enhanced enzyme activity (Fig. 5B). This observation is consistent with a previous report showing direct activation of purified bovine PKCby PIP 3 (9). The related finding that PKCfrom LPS-treated cells could not be further activated in vitro by PIP 3 most likely reflects the fact that the enzyme was maximally activated in vivo in response to LPS. An important question arising from these observations is how PKCmaintains its activation following exposure to PIP 3 . One possibility is that PIP 3 induces a change in the phosphorylation state of the PKCwhich sustains its activity until it becomes dephosphorylated by a cellular phosphatase.
In summary, this report provides evidence indicating that LPS activates PKCin primary human monocytes and in human promonocytic cell lines. This activation requires LPSinduced activation of PI 3-kinase and is sustained in the absence of exogenous lipids. Thus, the results are consistent with a model in which LPS activates p53/p56 lyn leading to increased PI 3-kinase activity regulating PKCthrough the production of PIP 3 .