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Phosphatidylinositol 3-Kinase in Interleukin 1 Signaling

PHYSICAL INTERACTION WITH THE INTERLEUKIN 1 RECEPTOR AND REQUIREMENT IN NFκB AND AP-1 ACTIVATION*
  • Shrikanth A.G. Reddy
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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  • Jianyi H. Huang
    Affiliations
    Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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  • Warren S.-L. Liao
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2556; Fax: 713-790-0329;
    Affiliations
    Department of Biochemistry and Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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  • Author Footnotes
    * This work is supported by National Institutes of Health Grant AR38858 (to W. S.-L. L).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported by a postdoctoral training grant from the National Institute of Child Health and Human Development.
Open AccessPublished:November 14, 1997DOI:https://doi.org/10.1074/jbc.272.46.29167
      The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFκB and activator protein-1 (AP-1) are poorly defined. We present evidence here that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI 3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibited IL-1-induced activation of both NFκB and AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFκB-dependent gene expression. In this regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate NFκB. Our results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFκB and AP-1.
      The biological processes of growth, differentiation, and immunity are dependent on the highly regulated action of transcription factor families such as NFκB and activator protein-1 (AP-1).
      The abbreviations used are: AP-1, activator protein 1; IL-1, interleukin 1; IL-1RI, type I IL-1 receptor; TNF, tumor necrosis factor; IRAK, IL-1 receptor-associated protein kinase; TRAF, TNF receptor-associated factor; PI 3-kinase, phosphatidylinositol 3-kinase; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C.
      1The abbreviations used are: AP-1, activator protein 1; IL-1, interleukin 1; IL-1RI, type I IL-1 receptor; TNF, tumor necrosis factor; IRAK, IL-1 receptor-associated protein kinase; TRAF, TNF receptor-associated factor; PI 3-kinase, phosphatidylinositol 3-kinase; CAT, chloramphenicol acetyltransferase; PKC, protein kinase C.
      These two transcription factors participate not only in normal physiology but in diseased conditions as well. Extracellular stimuli such as interleukin-1 (IL-1), tumor necrosis factor (TNF), viruses, and UV light are among the known potent inducers of NFκB (
      • Baldwin Jr., A.S.
      ). Of these, IL-1, which is a major proinflammatory cytokine, is responsible for mediating numerous host responses, including fever, activation of lymphocytes, and the induction of acute-phase proteins (
      • O'Neill L.A.J.
      ). Elevated levels of IL-1 have been associated with various pathological conditions, including rheumatoid arthritis (
      • O'Neill L.A.J.
      ). Although several biological activities of IL-1 have been characterized, the molecular mechanisms by which its signals are transduced from the plasma membrane to affect gene transcription in the nucleus remain to be elucidated.
      One of the most prominent IL-1-inducible signals, one which requires the type I IL-1 receptor (IL-1RI), involves the rapid and dramatic activation of NFκB and AP-1 and results in the induction of discrete sets of genes (
      • O'Neill L.A.J.
      ). Since IL-1RI shares no significant homology with conserved protein kinase domains, it is unlikely to have any intrinsic protein kinase activity (
      • Sims J.E.
      • Acres R.B.
      • Grubin C.E.
      • McMahan C.J.
      • Wignall J.M.
      • March C.J.
      • Dover S.K.
      ) and may need to recruit specific cytoplasmic proteins to transmit its signals. One such protein, recruited to the receptor in response to IL-1 stimulation, is theIL-1 receptor-associated proteinkinase (IRAK), which bears significant homology to theDrosophila protein Pelle (

      271, 1128–1131Cao, Z., Henzel, W. J., and Gao, X. (1996) 271,1128–1131.

      ). Although there is no evidence linking IRAK directly to NFκB activation, studies on the function of Pelle have demonstrated its importance in the activation of Dorsal, the mammalian equivalent of NFκB. Recently, IRAK has been shown to physically associate with a protein belonging to the TNFreceptor-associated factor family called TRAF6 (
      • Cao Z.
      • Xiaong J.
      • Takeuchi M.
      • Kurama T.
      • Goeddel D.V.
      ). In contrast to TNF, which recruits TRAF2 to activate NFκB, IL-1-induced activation of NFκB is mediated by TRAF6 (
      • Cao Z.
      • Xiaong J.
      • Takeuchi M.
      • Kurama T.
      • Goeddel D.V.
      ). However, the mechanisms by which the recruitment of TRAFs to the IL-1 and TNF receptors leads to the activation of NFκB are not understood.
      NFκB is normally inactive and kept sequestered in the cytoplasm by its interaction with the inhibitory subunit IκB (
      • Baldwin Jr., A.S.
      ). Upon stimulation, IκB is rapidly phosphorylated, ubiquitinated, and then degraded, resulting in the release and subsequent nuclear translocation of active NFκB (
      • Baldwin Jr., A.S.
      ). In addition to the TRAFs, several other factors appear to play a role in NFκB activation. Although some interact with the TRAFs (
      • Hsu H.
      • Huang J.
      • Shu H.-B.
      • Baichwal V.
      • Goeddel D.V.
      ), others such as Raf kinase, tyrosine kinases, reactive oxygen intermediates, and sphingomyelinases have also been reported (
      • Baldwin Jr., A.S.
      ).
      AP-1 is predominantly a heterodimeric complex of c-Fos and c-Jun proteins, and its activation is mainly due to induction of c-Fos synthesis and the phosphorylation of both c-Jun and c-Fos (
      • Lewin B.
      ). Recent studies have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) may up-regulate c-Fos synthesis (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ) and stimulate the Jun N-terminal kinase pathway (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Williams L.T.
      ), which might then lead to the phosphorylation and activation of c-Jun. Other studies have implicated PI 3-kinase in epidermal growth factor-induced AP-1 activation (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takanayagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S-i.
      • Mizuno K.
      • Hirai S-i.
      • Kazlauskas A.
      • Ohno S.
      ). PI 3-kinase consists of a catalytic subunit (p110) associated with a regulatory polypeptide (p85) (
      • Fry M.J.
      ). Ligand-dependent interactions between the SH2 domains of the p85 subunit and the phosphotyrosine-containing YXXM motif present on several cytokine/growth factor receptors have been reported (
      • Fry M.J.
      ). The phosphorylated lipid products generated by this enzyme may act as second messengers to activate protein kinases such as the Akt gene product (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) or certain isoforms of protein kinase C (
      • 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.
      ). Its reported interactions with Ras (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ) and its potential ability to activate several signaling pathways (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Williams L.T.
      ) underscore the importance of PI 3-kinase in various cellular functions.
      We have demonstrated that the acute-phase gene serum amyloid A 1 and 3 (
      • Huang J.H.
      • Rienhoff Jr., H.Y.
      • Liao W.S.-L.
      ,
      • Li X.
      • Liao W.S.-L.
      ,
      • Li X.
      • Liao W.S.-L.
      ) are highly inducible by cytokines such as IL-1 and shown that NFκB is critical for their expression (
      • Li X.
      • Liao W.S.-L.
      ,
      • Li X.
      • Liao W.S.-L.
      ,
      • Lu S.-Y.
      • Rodriguez M.
      • Liao W.S.-L.
      ). In this study, we investigated the signaling events that lead to NFκB and AP-1 activation in IL-1-stimulated cells. Our results indicate a prominent role for PI 3-kinase in the activation of both NFκB and AP-1.

      DISCUSSION

      We have presented evidence to indicate that the interaction of PI 3-kinase with IL-1RI is one of the early events in IL-1-stimulated cells and that PI 3-kinase may be indispensable for the activation of NFκB and AP-1 by IL-1. Our results show that wortmannin blocked the activation of both transcription factors by IL-1. Interestingly, wortmannin, which is a fungal metabolite, has been shown in experimental animals to exert anti-inflammatory or immunosuppressive effects (
      • Ui M.
      • Okada T.
      • Hazeki K.
      • Hazeki O.
      ,
      • Gunther R.
      • Kishore P.N.
      • Abbas H.K.
      • Mirocha C.J.
      ). It remains to be determined whether these effects can be accounted for, at least in part, by its inhibitory effects on NFκB activation.
      Since wortmannin blocked the activation of NFκB and AP-1 by IL-1, it was predicted that IL-1 would stimulate PI 3-kinase activity. Indeed, IL-1 stimulated PI 3-kinase activity very potently and with rapid kinetics of activation. The activation kinetics of NFκB and AP-1 have been well established and are slower in comparison to those for PI 3-kinase in IL-1-treated cells, consistent with a role for this enzyme in their activation. PI 3-kinase has been attributed with both lipid kinase and protein kinase activities, and since wortmannin inhibits both, it is difficult to assess at this time the relative importance of each activity in IL-1 signaling.
      Wortmannin has recently been shown to inhibit phospholipase A2 at concentrations that earlier were thought to be selective for PI 3-kinase (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.O.
      ). We therefore used a dominant negative mutant, p85DN, of the regulatory subunit of PI 3-kinase to verify the importance of this enzyme in the activation of NFκB and AP-1. p85DN has been used previously to confirm a role for PI 3-kinase in various cellular functions (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stevens L.
      • Jackson T.R.
      • Hawkins P.T.
      • Dhand R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ). Consistent with our wortmannin experiments, overexpression of p85DN strongly inhibited the activation of NFκB- and AP-1- dependent gene expression by IL-1. PI 3-kinase therefore appears to be essential for their activation by IL-1.
      The cytoplasmic domain of IL-1RI protein contains a sequence (Y496EKM) which fits the YXXM motif that has been demonstrated to mediate direct physical interaction between receptor proteins and PI 3-kinase. Our coimmunoprecipitation studies demonstrate that the IL-1RI and PI 3-kinase do interact and that this association is induced very rapidly by IL-1. Although these data strongly suggest that the interaction between p85 PI 3-kinase and IL-1RI is direct, the presence of an accessory factor that links these two proteins cannot be ruled out. Interestingly, this Y496EKM motif is within the 50-amino acid (477–527) region in the IL-1RI cytoplasmic domain found to be essential for IL-1 signal transduction (
      • Heguy A.
      • Baldari C.T.
      • Macchia G.
      • Telford J.L.
      • Melli M.
      ). Similar sequence motifs are also found in the mouse IL-1RI and the Drosophila Toll protein, which is equivalent to human IL-1RI.
      Various studies have indicated that for the YXXM motifs to be capable of binding to p85 PI 3-kinase, the tyrosine residues must be phosphorylated (Ref.
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.-J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      , see references in Ref.
      • Kapeller R.
      • Cantley L.C.
      ). Several receptor and nonreceptor tyrosine kinases that could potentially phosphorylate these sites and lie upstream of PI 3-kinase in various signaling pathways have been implicated (
      • Fry M.J.
      ). In one study, a sequence in the insulin receptor substrate-1 containing a PI 3-kinase binding motif and several flanking amino acid residues was evaluated as a substrate for different nonreceptor tyrosine kinases (
      • Garcia P.
      • Shoelson S.E.
      • George S.T.
      • Hinds D.A.
      • Goldberg A.R.
      • Miller W.T.
      ). Using synthetic peptides in protein kinase assays, it was determined that the tyrosine kinases differed in their tolerance for various amino acid substitutions, and in particular, an aspartate immediately N-terminal to the tyrosine was indispensable. In this context, it may be noted that in addition to other acidic amino acids, there is an aspartic acid (Asp495) immediately adjacent to the Y496EKM sequence on the IL-1RI. This suggests that the Y496EKM sequence on the IL-1RI may be a good substrate for tyrosine kinases and may facilitate direct interaction between IL-1RI and PI 3-kinase. An IL-1-inducible tyrosine kinase activity has been reported but not identified (
      • Iwasaki T.
      • Uehara Y.
      • Graves L.
      • Rachie N.
      • Bomsztyk K.
      ). The involvement of tyrosine kinase activities in the steps leading to NFκB activation has been reported for various inducers (see references in Ref.
      • Baldwin Jr., A.S.
      ).
      Overexpression of p110 was sufficient to induce the AP-1/CAT reporter and to increase nuclear c-Fos protein levels as potently as IL-1. These results are consistent with recent reports that epidermal growth factor-induced activation of AP-1 requires PI 3-kinase (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takanayagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S-i.
      • Mizuno K.
      • Hirai S-i.
      • Kazlauskas A.
      • Ohno S.
      ). In sharp contrast to the AP-1 reporter, we observed that although overexpression of p110 activated the NFκB/CAT gene in a dose-dependent manner, the activation was much less than what we normally observe with IL-1. Addition of IL-1 to p110-overexpressing cells, however, resulted in a synergistic activation of the NFκB/CAT reporter. It appears therefore that whereas PI 3-kinase is necessary for IL-1-induced activation of NFκB, its overexpression is not sufficient to fully activate this transcription factor. To identify signaling molecules that can cooperate with PI 3-kinase, we expressed the IRAK in various cotransfection experiments. We show evidence here that overexpression of IRAK is sufficient to activate the NFκB reporter in a dose-dependent manner. To address the possibility that IRAK and PI 3-kinase cooperate to activate NFκB, we followed two approaches. We cotransfected various doses of IRAK with the p110-expression vector and the NFκB/CAT reporter. A clear synergism with p110 could be observed at lower doses of IRAK. In the second approach, since IRAK synergized with IL-1, we used the dominant negative mutant p85DN to test whether PI 3-kinase may be involved. Cotransfection of p85DN inhibited the synergism between IRAK and IL-1, lowering the CAT activity levels to those activated by IRAK in the absence of IL-1. In addition to providing further evidence for the involvement of PI 3-kinase in IL-1-induced activation of NFκB, these data support the idea that to mediate NFκB activation, PI 3-kinase cooperates with molecules such as IRAK that possibly lie on other signaling pathways (Fig. 7). Similar observations have been made for TNF- and CD40l-induced activation of NFκB (
      • Cheng G.
      • Baltimore D.
      ). While TRAF2 overexpression is sufficient to activate NFκB, it was recently shown that TRAF2 may need a coactivator protein TRAF family member-associated NFκB activator. In contrast to TRAF2, overexpression of TRAF family member-associated NFκB activator alone was unable to activate NFκB. The function of PI 3-kinase in transcription factor activation may not be limited to NFκB and AP-1, for it has been implicated recently in the activation of STAT3 (
      • Pfeffer L.M.
      • Mullersman J.E.
      • Pfeffer S.R.
      • Murti A.
      • Shi W.
      • Yang C.H.
      ). Whereas the tyrosine phosphorylation of STAT3 plays a prominent role in its activation, phosphorylation of this factor on a specific serine residue is important and may require PI 3-kinase.
      Figure thumbnail gr7
      Figure 7A schematic model for IL-1-induced activation of NFκB and AP-1. IL-1 induces physical interaction between its type I receptor and the p85 subunit of PI 3-kinase and stimulates PI 3-kinase activity. PI 3-kinase is required for the activation of NFκB by IL-1 but may need to cooperate with other IL-1-inducible signals such as IRAK. PI 3-kinase alone is necessary and sufficient to mediate IL-1-induced activation of AP-1.
      The mechanisms by which activated PI 3-kinase may ultimately result in the activation of NFκB and AP-1 are unclear. However, several signaling molecules have been shown to affect directly or indirectly the pathway leading to the activation of these transcription factors. For example, two atypical forms of protein kinase C, aPKCζ (
      • Diaz-Meco M.T.
      • Berra E.
      • Municio M.M.
      • Sanz L.
      • Lozano L.
      • Dominguez I.
      • Diaz-Golpe V.
      • Lain De Lera M.T.
      • Alcami J.
      • Paya C.V.
      • Arenzana-Seisdedos F.
      • Virelizier J.-L.
      • Moscat J.
      ) and aPKCλ (
      • Akimoto K.
      • Takahashi R.
      • Moriya S.
      • Nishioka N.
      • Takanayagi J.
      • Kimura K.
      • Fukui Y.
      • Osada S-i.
      • Mizuno K.
      • Hirai S-i.
      • Kazlauskas A.
      • Ohno S.
      ), have been shown to be involved in NFκB and AP-1 activation, respectively. Since the phosphorylated lipid products of PI 3-kinase activate various isoforms of PKC in vitro, including aPKCζ (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ), PKC may lie downstream of PI 3-kinase. aPKCζ activity was recently reported to be stimulated by IL-1 in rat renal mesangial cells (
      • Rzymkiewicz D.M.
      • Tetsuka T.
      • Daphna-Iken D.
      • Srivastava S.
      • Morrison A.R.
      ). Small G-proteins and the protein kinase mitogen-activated protein kinase-extracellular signal-regulated kinase kinase kinase 1 (MEKK1) are among the other signaling molecules that have been implicated in the activation of NFκB. Physical interaction between the small GTPase Cdc42 and PI 3-kinase may result in the stimulation of PI 3-kinase activity (
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ,
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Cdc42 appears to be required for the activation of NFκB by TNF (
      • Perona R.
      • Montaner S.
      • Saniger L.
      • Sanchez-Perez I.
      • Bravo R.
      • Lacal J.C.
      ), and our results suggest that the mechanism may involve interactions with PI 3-kinase. Finally, MEKK1, a key mediator of the Jun N-terminal kinase pathway, has recently been implicated as an upstream regulator of the 700-kDa IκB kinase (
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ). Since overexpression of PI 3-kinase activates the Jun N-terminal kinase pathway (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Williams L.T.
      ), it would be of interest to determine if it is involved in the up-regulation of MEKK1 activity in IL-1-stimulated cells. It is not known whether any of these proteins, Cdc42, PKCs, or MEKK1, interact with IRAK and if they do, whether those interactions would account for the synergism between PI 3-kinase and IRAK in NFκB activation.
      Since our results suggested that PI 3-kinase may functionally interact with IRAK to activate NFκB, we are currently investigating the molecular basis of such interactions. It is hoped that in addition to providing greater insights into the mechanisms employed by IL-1 for NFκB activation, these studies would also reveal the identity of the downstream targets of both IRAK and PI 3-kinase.

      Acknowledgments

      We are grateful to Drs. M. Kasuga and W. Ogawa for the p85DN construct, A. Klippel and L. T. Williams for the p110α expression vector, R. Legerski for the HeLa cDNA library, C. Reynolds and the Biological Response Modifiers Program, NCI, National Institutes of Health, for IL-1β, P. J. Chiao for the IκB expression vector, and S. Singh for constructive comments.

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