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

Open AccessPublished:June 27, 1997DOI:https://doi.org/10.1074/jbc.272.26.16445
      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)
      The 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.
      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 (
      • Rietschel 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.
      ,
      • Watson R.W.G.
      • Redmond H.P.
      • Bouchier-Hayes D.
      ).
      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 (
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ,
      • Shapira L.
      • Takashiba S.
      • Champagne C.
      • Amar S.
      • Van Dyke T.E.
      ,
      • Weinstein S.L.
      • Gold M.R.
      • DeFranco A.L.
      ,
      • Stefanová I.
      • Corcoran M.L.
      • Horak E.M.
      • Wahl L.M.
      • Bolen J.B.
      • Horak I.D.
      ). The initial events in at least one dominant LPS signaling pathway are dependent upon the glycophosphatidylinositol-linked membrane molecule, CD14 (
      • Wright S.D.
      • Ramos R.A.
      • Tobias P.S.
      • Ulevitch R.J.
      • Mathison J.C.
      ). 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 (
      • Stefanová I.
      • Corcoran M.L.
      • Horak E.M.
      • Wahl L.M.
      • Bolen J.B.
      • Horak I.D.
      ). 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 (
      • Herrera-Velit P.
      • Reiner N.E.
      ).
      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 (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ). 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 (
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ,
      • Shapira L.
      • Takashiba S.
      • Champagne C.
      • Amar S.
      • Van Dyke T.E.
      ). 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 (
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ). 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 (
      • Konno Y.
      • Ohno S.
      • Akita Y.
      • Kawasaki H.
      • Suzuki K.
      ,
      • Selbie L.A.
      • Schmitz-Peiffer C.
      • Sheng Y.
      • Biden T.J.
      ,
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Nishizuka Y.
      ). Rather, aPKCs exhibit activator-independent activity which is increased upon exposure to novel lipids such as PIP3 and ceramides (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Nakanishi H.
      • Exton J.H.
      ,
      • Lozano J.
      • Berra E.
      • Municio M.M.
      • Diaz-Meco M.T.
      • Dominguez I.
      • Sanz L.
      • Moscat J.
      ).
      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.

      DISCUSSION

      Previous studies demonstrated that a PKC with unusual properties is activated in human monocytes following exposure to LPS (
      • Liu M.K.
      • Herrera-Velit P.
      • Brownsey R.W.
      • Reiner N.E.
      ,
      • Shapira L.
      • Takashiba S.
      • Champagne C.
      • Amar S.
      • Van Dyke T.E.
      ). 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-ζ. PKC-ζ is a member of a subfamily of atypical PKCs in that it is not activated by diacylglycerols, and it does not require Ca2+ for activity. Rather, recent findings indicate that the activity of PKC-ζ is enhanced in vitro by PIP3. This, together with the previous observations that LPS induces the accumulation of PIP3 in monocytes (
      • Herrera-Velit P.
      • Reiner N.E.
      ), suggested the hypothesis that LPS may activate PKC-ζ in 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. 1 A and 2 A). These peaks of kinase activity were detected in the absence of exogenous lipids and were also independent of Ca2+. These characteristics are consistent with PKC-ζ, and immunoblotting of Mono Q fractions indicated the presence of PKC-ζ in these samples (Figs. 1 B and2 B). In addition, immunoprecipitation of PKC-ζ directly 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. 2 C). Furthermore, the LPS-enhanced MBP kinase activity could be removed by immunoadsorption with anti-PKC-ζ (Fig. 3). Prior analyses of PKC expression in blood monocytes, U937 cells, and HL-60 cells indicate the presence of PKC-α, -βI, -βII, -ε, -ζ, and θ isoforms (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Chang Z.-L.
      • Beezhold D.H.
      ,
      • Kiley S.C.
      • Parker P.J.
      ,
      • Baier G.
      • Telford D.
      • Giampa L.
      • Coggeshall K.M.
      • Baier-Bitterlich G.
      • Isakov N.
      • Altman A.
      ). In U937 cells, PKC-α, -β, and -ε elute from Mono Q at or below 320 mm NaCl, whereas PKC-ζ elutes at ∼460 mm NaCl (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ). The somewhat earlier elution of PKC-ζ (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-PKC-ζ antibodies (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 (
      • Nishida E.
      • Gotoh Y.
      ). The closely related PKC-ι contains a similar COOH-terminal sequence differing at only two amino acids (SEFEGFEYINPLLMSAEECV) and can be detected with antibodies to the COOH terminus of PKC-ζ. However, it is unlikely that the major immunoreactive band detected in this study is PKC-ι since this isoform is known to migrate at 65 kDa (
      • Selbie L.A.
      • Schmitz-Peiffer C.
      • Sheng Y.
      • Biden T.J.
      ), consistently lower than the ∼80–85-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-α, PKC-ι, or PKC-λ (according to information provided by the manufacturer). It has also been reported that antibodies directed against the COOH terminus of PKC-ζ react with a Ca2+ and phorbol ester-sensitive PKC isoform (
      • Batlle E.
      • Fabre M.
      • De Herreros A.G.
      ). However, the kinase that is the subject of this report is only weakly activated by a combination of PMA, Ca2+, 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 (
      • Nishizuka Y.
      ). The finding that PKC-ζ translocates to the membrane following exposure to LPS provides additional evidence for its activation. Given that PIP3 increases the activity of PKC-ζ in vitro (Ref.
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      and Fig. 5) and that activation of PKC-ζ in vivo requires activation of PI 3-kinase (Figs. 6and 8), a possible mechanism for redistribution of PKC-ζ is through direct binding to PIP3 in the membrane compartment. Although membrane translocation of PKC-ζ induced by PIP3 in vivo has not been reported, it has been shown that PKC-ζ translocates to the membrane transiently following tetanization of hippocampal slices (
      • Sacktor T.C.
      • Osten P.
      • Valsamis H.
      • Jiang X.
      • Naik M.U.
      • Sublette E.
      ). This translocation correlated with the cytosolic accumulation of PKM-ζ, a 51-kDa catalytic subunit of the holoenzyme which suggested activation-induced proteolysis. These findings raise the interesting possibility that PKM-ζ 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-ζ. First, levels of the ∼80–85-kDa PKC-ζ were not observed to be decreased in Mono Q fractions prepared from LPS-treated cells. Second, smaller anti-PKC-ζ 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 (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Nakanishi H.
      • Exton J.H.
      ,
      • Kochs G.
      • Hummel R.
      • Meyer D.
      • Hug H.
      • Marmé D.
      • Sarre T.F.
      ). In the present study, it was found that MBP, peptide ε, and S6 peptide were equally efficient substrates for monocyte PKC-ζ. In contrast, protamine sulfate, histone, and Kemptide were relatively poor substrates (Fig. 5). These findings are consistent with previous studies in which PKC-ζ phosphorylated MBP and peptide substrates with equal efficiency (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ) but showed lower activity against histone and Kemptide (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Boutin J.A.
      ). Although the data contrast somewhat with other results suggesting that peptide ε may be a more efficient substrate than MBP (
      • Nakanishi H.
      • Exton J.H.
      ,
      • Boutin J.A.
      ), 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 (
      • Newton A.C.
      ). The cPKC members also require the presence of Ca2+. In contrast, human PKC-ζ exhibits activator- and cofactor-independent activity which is not increased by the addition of PS (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Kochs G.
      • Hummel R.
      • Meyer D.
      • Hug H.
      • Marmé D.
      • Sarre T.F.
      ). 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-ζ reported in this paper (Fig. 5) are consistent with previous studies (
      • Ways D.K.
      • Cook P.P.
      • Webster C.
      • Parker P.J.
      ,
      • Kochs G.
      • Hummel R.
      • Meyer D.
      • Hug H.
      • Marmé D.
      • Sarre T.F.
      ). The findings presented contrast somewhat, however, with studies of PKC-ζ 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.
      • Zhou G.
      • Wooten M.W.
      • Coleman E.S.
      ).
      The mechanisms leading to the activation of PKC-ζ 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-P2 and PIP3 in vitro(
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ). The role of PI 3-kinase in LPS-induced activation of monocyte PKC-ζ 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-ζ 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 A2 with an IC50 similar to that previously reported for PI 3-kinase (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.O.
      ). On the other hand, the structurally unrelated compound, LY294002, has been shown to have inhibitory effects on PI 3-kinase by a different mechanism (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). Moreover, LY294002 shows no inhibitory effects on other lipid kinases or on several protein kinases, including PKC and mitogen-activated protein kinase (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). Therefore, the findings that both compounds exhibit inhibitory effects on LPS-induced activation of PKC-ζ support the argument that PI 3-kinase is involved in the regulation of PKC-ζ. Further support for this argument was provided by experiments in which a dominant negative mutant of PI 3-kinase (Δp85) expressed in U937 cells completely blocked LPS-induced activation of PKC-ζ (Fig. 8). It has previously been shown that incubation of monocytes with LPS activates PI 3-kinase leading to increased cellular levels of PIP3 (
      • Herrera-Velit P.
      • Reiner N.E.
      ). Thus, the most likely mechanism for the attenuation of activation of PKC-ζ by either wortmannin, LY294002, or Δp85 is inhibition of the formation of PIP3 in vivo. Moreover, in the present study it was observed that the addition of PIP3 in vitroto specific Mono Q fractions from control cells led to enhanced enzyme activity (Fig. 5 B). This observation is consistent with a previous report showing direct activation of purified bovine PKC-ζ by PIP3 (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). The related finding that PKC-ζ from LPS-treated cells could not be further activated in vitro by PIP3 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 PKC-ζ maintains its activation following exposure to PIP3. One possibility is that PIP3 induces a change in the phosphorylation state of the PKC-ζ which sustains its activity until it becomes dephosphorylated by a cellular phosphatase.
      In summary, this report provides evidence indicating that LPS activates PKC-ζ in primary human monocytes and in human promonocytic cell lines. This activation requires LPS-induced 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 PKC-ζ through the production of PIP3.

      Acknowledgments

      We thank Dr. Vince Duronio and Dr. Graeme Dougherty for helpful discussions, Dr. Keith Humphries for providing pMC1neo-poly(A), Dr. Masato Kasuga for providing Δp85 and Wp85 constructs, and Dr. C.-S. Chen for the gift of PIP3.

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