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Molecular and Cellular Biochemistry

, Volume 300, Issue 1–2, pp 113–127 | Cite as

Investigation of interleukin 1β-mediated regulation of NF-κB activation in colonic cells reveals divergence between PKB and PDK-transduced events

  • Kuljit Parhar
  • Sharlene Eivemark
  • Kiran Assi
  • Antonio Gómez-Muñoz
  • Arthur Yee
  • Baljinder Salh
Original Paper

Abstract

Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1β-induced NF-κB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-κB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1β-induced NF-κB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1β-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-κB activation. This was supported using biochemical analysis in which immunoprecipitated IKKγ from IL-1β-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-κB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKβ.

Keywords

PDK PKB NF-κB IL-1β TPCK 

Abbreviations

IL-1β

interleukin 1 beta

IL-1R

interleukin 1 receptor

NF-κB

nuclear factor kappa B

MyD88

myeloid differentiation primary response gene 88

IRAK 1

IL-1 receptor associated kinase 1

Traf 6

TNF-receptor associated factor 6

IKK

I kappa kinase

PKB

protein kinase B

PDK

phosphoinositide-dependent kinase 1

PI3K

phosphatidylinositol 3 kinase

ILK

integrin-linked kinase

IECs

intestinal epithelial cells

CBP

CREB binding protein

TAK1

TGFβ-activated kinase

TAD

transactivation domain

References

  1. 1.
    Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:675–680PubMedCrossRefGoogle Scholar
  2. 2.
    Cario E, Rosenberg IM, Brandwein SL, Beck PL, Reinecker HC, Podolsky DK (2000) Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J Immunol 164:966–972PubMedGoogle Scholar
  3. 3.
    Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Arditi M (2001) Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol 167:1609–1616PubMedGoogle Scholar
  4. 4.
    Jobin C, Sartor RB (2000) The I kappa B/NF-kappa B system: a key determinant of mucosal inflammation and protection. Am J Physiol Cell Physiol 278:C451–C462PubMedGoogle Scholar
  5. 5.
    Dwinell MB, Lugering N, Eckmann L, Kagnoff MF (2001) Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells. Gastroenterology 120:49–59PubMedCrossRefGoogle Scholar
  6. 6.
    Yang SK, Eckmann L, Panja A, Kagnoff MF (1997) Differential and regulated expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells. Gastroenterology 113:1214–1223PubMedCrossRefGoogle Scholar
  7. 7.
    Li Q, Verma IM (2002) NF-kappaB regulation in the immune system. Nat Rev Immunol 2:725–734PubMedCrossRefGoogle Scholar
  8. 8.
    Chen LF, Mu Y, Greene WC (2002) Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J 21:6539–6548PubMedCrossRefGoogle Scholar
  9. 9.
    Zhong H, May MJ, Jimi E, Ghosh S (2002) The phosphorylation status of nuclear NF-kappaB determines its association with CBP/p300 or HDAC-1. Mol Cell 9:625–636PubMedCrossRefGoogle Scholar
  10. 10.
    Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, Okumura K, Nakano H (2003) Phosphorylation of serine 276 is essential for p65 NF-kappaB subunit-dependent cellular responses. Biochem Biophys Res Commun 300:807–812PubMedCrossRefGoogle Scholar
  11. 11.
    Zhong H, Voll RE, Ghosh S (1998) Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661–671PubMedCrossRefGoogle Scholar
  12. 12.
    Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, Haegeman G (2003) Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J 22:1313–1324PubMedCrossRefGoogle Scholar
  13. 13.
    Duran A, Diaz-Meco MT, Moscat J (2003) Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation. EMBO J 22:3910–3918PubMedCrossRefGoogle Scholar
  14. 14.
    Bird TA, Schooley K, Dower SK, Hagen H, Virca GD (1997) Activation of nuclear transcription factor NF-kappaB by interleukin-1 is accompanied by casein kinase II-mediated phosphorylation of the p65 subunit. J Biol Chem 272:32606–32612PubMedCrossRefGoogle Scholar
  15. 15.
    Bae JS, Jang MK, Hong S, An WG, Choi YH, Kim HD, Cheong J (2003) Phosphorylation of NF-kappa B by calmodulin-dependent kinase IV activates anti-apoptotic gene expression. Biochem Biophys Res Commun 305:1094–1098PubMedCrossRefGoogle Scholar
  16. 16.
    Sakurai H, Suzuki S, Kawasaki N, Nakano H, Okazaki T, Chino A, Doi T, Saiki I (2003) Tumor necrosis factor-α-induced IKK phosphorylation of NF-κB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway. J Biol Chem 278:36916–36923PubMedCrossRefGoogle Scholar
  17. 17.
    Haller D, Russo MP, Sartor RB, Jobin C (2002) IKK beta and phosphatidylinositol 3-kinase/Akt participate in non-pathogenic gram-negative enteric bacteria-induced RelA phosphorylation and NF-kappa B activation in both primary and intestinal epithelial cell lines. J Biol Chem 277:38168–38178PubMedCrossRefGoogle Scholar
  18. 18.
    Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr (2001) Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276:18934–18940PubMedCrossRefGoogle Scholar
  19. 19.
    Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W (1999) IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain. J Biol Chem 274:30353–30356PubMedCrossRefGoogle Scholar
  20. 20.
    Sizemore N, Leung S, Stark GR (1999) Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphorylation and activation of the NF-kappaB p65/RelA subunit. Mol Cell Biol 19:4798–4805PubMedGoogle Scholar
  21. 21.
    Madrid LV, Wang CY, Guttridge DC, Schottelius AJ, Baldwin AS, Mayo MW (2000) Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB. Mol Cell Biol 20:1626–1638PubMedCrossRefGoogle Scholar
  22. 22.
    Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, Baldwin AS (2003) A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression. Nature 423:659–663PubMedCrossRefGoogle Scholar
  23. 23.
    Yamamoto Y, Verma UN, Prajapati S, Kwak YT, Gaynor RB (2003) Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature 423:655–659PubMedCrossRefGoogle Scholar
  24. 24.
    Delhase M, Hayakawa M, Chen Y, Karin M (1999) Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science 284:309–313PubMedCrossRefGoogle Scholar
  25. 25.
    Takaesu G, Surabhi RM, Park KJ, Ninomiya-Tsuji J, Matsumoto K, Gaynor RB (2003) TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway. J Mol Biol 326:105–115PubMedCrossRefGoogle Scholar
  26. 26.
    Yang J, Lin Y, Guo Z, Cheng J, Huang J, Deng L, Liao W, Chen Z, Liu Z, Su B (2001) The essential role of MEKK3 in TNF-induced NF-kappaB activation. Nat Immunol 2:620–624PubMedCrossRefGoogle Scholar
  27. 27.
    Leitges M, Sanz L, Martin P, Duran A, Braun U, Garcia JF, Camacho F, Diaz-Meco MT, Rennert PD, Moscat J (2001) Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway. Mol Cell 8:771–780PubMedCrossRefGoogle Scholar
  28. 28.
    Zandi E, Chen Y, Karin M (1998) Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: discrimination between free and NF-kappaB-bound substrate. Science 281:1360–1363PubMedCrossRefGoogle Scholar
  29. 29.
    Lee FS, Peters RT, Dang LC, Maniatis T (1998) MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc Natl Acad Sci USA 95:9319–9324PubMedCrossRefGoogle Scholar
  30. 30.
    Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M (1997) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell 91:243–252PubMedCrossRefGoogle Scholar
  31. 31.
    Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV (1997) IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. Science 278:866–869PubMedCrossRefGoogle Scholar
  32. 32.
    Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A, Rao A (1997) IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 278:860–866PubMedCrossRefGoogle Scholar
  33. 33.
    Scheid MP, Woodgett JR (2003) Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett 546:108–112PubMedCrossRefGoogle Scholar
  34. 34.
    Romashkova JA, Makarov SS (1999) NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:86–90PubMedCrossRefGoogle Scholar
  35. 35.
    Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB (1999) NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82–85PubMedCrossRefGoogle Scholar
  36. 36.
    Storz P, Toker A (2002) 3′-phosphoinositide-dependent kinase-1 (PDK-1) in PI 3-kinase signaling. Front Biosci 7:d886–d902PubMedCrossRefGoogle Scholar
  37. 37.
    Sato S, Fujita N, Tsuruo T (2002) Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3. J Biol Chem 277:39360–39367PubMedCrossRefGoogle Scholar
  38. 38.
    Fujita N, Sato S, Ishida A, Tsuruo T (2002) Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1. J Biol Chem 277:10346–10353PubMedCrossRefGoogle Scholar
  39. 39.
    Kim DW, Hwang JH, Suh JM, Kim H, Song JH, Hwang ES, Hwang IY, Park KC, Chung HK, Kim JM, Park J, Hemmings BA, Shong M (2003) RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1. Mol Endocrinol 17:1382–1394PubMedCrossRefGoogle Scholar
  40. 40.
    Horton RM (1995) PCR-mediated recombination and mutagenesis. SOEing together tailor-made genes. Mol Biotechnol 3:93–99PubMedGoogle Scholar
  41. 41.
    Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105PubMedCrossRefGoogle Scholar
  42. 42.
    Ballif BA, Shimamura A, Pae E, Blenis J (2001) Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-l-phenylalanyl chloromethyl ketone. J Biol Chem 276:12466–12475PubMedCrossRefGoogle Scholar
  43. 43.
    Delhase M, Li N, Karin M (2000) Kinase regulation in inflammatory response. Nature 406:367–368PubMedCrossRefGoogle Scholar
  44. 44.
    Tanaka H, Fujita N, Tsuruo T (2005) 3-Phosphoinositide-dependent protein kinase 1-mediated IκB kinase (IKKB) phosphorylation activates NF-κB signaling. J Biol Chem 280:40965–40973PubMedCrossRefGoogle Scholar
  45. 45.
    Schoellmann G (1962) Biochemistry 2:252–255CrossRefGoogle Scholar
  46. 46.
    Troll W, Klassen A, Janoff A (1970) Tumorigenesis in mouse skin: inhibition by synthetic inhibitors of proteases. Science 169:1211–1213PubMedCrossRefGoogle Scholar
  47. 47.
    Slaga TJ, Klein-Szanto AJ, Fischer SM, Weeks CE, Nelson K, Major S (1980) Studies on mechanism of action of anti-tumor-promoting agents: their specificity in two-stage promotion. Proc Natl Acad Sci USA 77:2251–2254PubMedCrossRefGoogle Scholar
  48. 48.
    Grammer TC, Blenis J (1996) The serine protease inhibitors, tosylphenylalanine chloromethyl ketone and tosyllysine chloromethyl ketone, potently inhibit pp70s6k activation. J Biol Chem 271:23650–23652PubMedCrossRefGoogle Scholar
  49. 49.
    Wu M, Lee H, Bellas RE, Schauer SL, Arsura M, Katz D, FitzGerald MJ, Rothstein TL, Sherr DH, Sonenshine GE (1996) Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells. EMBO J 15:4682–4690PubMedGoogle Scholar
  50. 50.
    Tang ED, Inohara N, Wang CY, Nunez G, Guan KL (2003) Roles for homotypic interactions and transautophosphorylation in IκB Kinase (IKKβ) activation. J Biol Chem 278:38566–38570PubMedCrossRefGoogle Scholar
  51. 51.
    Chen G, Cao P, Goeddel DV (2002) TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90. Mol Cell 9:401–410PubMedCrossRefGoogle Scholar
  52. 52.
    Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR (2000) Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406:86–90PubMedCrossRefGoogle Scholar
  53. 53.
    Buss H, Dorrie A, Schmitz ML, Frank R, Livingstone M, Resch K, Kracht M (2004) Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity. J Biol Chem 279:49571–49574PubMedCrossRefGoogle Scholar
  54. 54.
    Lee KY, D’Acquisto F, Hayden MS, Shim JH, Ghosh S (2005) PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation. Science 308:114–118PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Kuljit Parhar
    • 1
  • Sharlene Eivemark
    • 1
  • Kiran Assi
    • 1
  • Antonio Gómez-Muñoz
    • 1
  • Arthur Yee
    • 1
  • Baljinder Salh
    • 1
    • 2
  1. 1.The Division of Gastroenterology, The Faculty of MedicineThe Jack Bell Research CentreVancouverCanada
  2. 2.Faculty of MedicineUniversity of British ColumbiaVancouverCanada

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