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Molecular Medicine

, Volume 21, Issue 1, pp 154–166 | Cite as

Protein-Binding Function of RNA-Dependent Protein Kinase Promotes Proliferation through TRAF2/RIP1/NF-κB/c-Myc Pathway in Pancreatic β cells

  • LiLi Gao
  • Wei Tang
  • ZhengZheng Ding
  • DingYu Wang
  • XiaoQiang Qi
  • HuiWen Wu
  • Jun Guo
Research Article

Abstract

Double-stranded RNA-dependent protein kinase (PKR), an intracellular pathogen recognition receptor, is involved both in insulin resistance in peripheral tissues and in downregulation of pancreatic β-cell function in a kinase-dependent manner, indicating PKR as a core component in the progression of type 2 diabetes. PKR also acts as an adaptor protein via its protein-binding domain. Here, the PKR protein-binding function promoted β-cell proliferation without its kinase activity, which is associated with enhanced physical interaction with tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. In addition, the transcription of the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-dependent survival gene c-Myc was upregulated significantly and is necessary for proliferation. Upregulation of the PKR protein-binding function induced the NF-κB pathway, as observed by dose-dependent degradation of IκBα, induced nuclear translocation of p65 and elevated NF-κB-dependent reporter gene expression. NF-κB-dependent reporter activity and β-cell proliferation both were suppressed by TRAF2-siRNA, but not by TRAF6-siRNA. TRAF2-siRNA blocked the ubiquitination of receptor-interacting serine/threonine-protein kinase 1 (RIP1) induced by PKR protein binding. Furthermore, R/P1-siRNA inhibited β-cell proliferation. Proinflammatory cytokines (TNFα) and glucolipitoxicity also promoted the physical interaction of PKR with TRAF2. Collectively, these data indicate a pivotal role for PKR’s protein-binding function on the proliferation of pancreatic β cells through TRAF2/RIP1/NF-κB/c-Myc pathways. Therapeutic opportunities for type 2 diabetes may arise when its kinase catalytic function, but not its protein-binding function, is downregulated.

Notes

Acknowledgments

We thank Charles Tom Dever for providing plasmids encoding PKR-K296R, GyrB-PKR, GyrB-PKR-K296H and pSG5. The work was supported by grants from the National Natural Science Foundation of China (no. 81170714), the Natural Science Foundation of Jiangsu Province (BK20131110) and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Supplementary material

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References

  1. 1.
    Garcia MA, et al. (2006) Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol. Mol. Biol. Rev. 70:1032–60.CrossRefGoogle Scholar
  2. 2.
    Bennett RL, et al. (2012) The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G(1) arrest. Cell Cycle. 11:407–17.CrossRefGoogle Scholar
  3. 3.
    Zhang S, et al. (2014) Activation of the PKR/eIF2alpha signaling cascade inhibits replication of Newcastle disease virus. Virol. J. 11:62.CrossRefGoogle Scholar
  4. 4.
    Marchal JA, et al. (2014) The impact of PKR activation: from neurodegeneration to cancer. FASEB J. 28:1965–74.CrossRefGoogle Scholar
  5. 5.
    Barber GN, Jagus R, Meurs EF, Hovanessian AG, Katze MG. (1995) Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase. J. Biol. Chem. 270:17423–8.CrossRefGoogle Scholar
  6. 6.
    Yim HC, Williams BR. (2014) Protein kinase R and the inflammasome. J.Interferon Cytokine Res. 34:447–54.CrossRefGoogle Scholar
  7. 7.
    He Y, Franchi L, Nunez G. (2013) The protein kinase PKR is critical for LPS-induced iNOS production but dispensable for inflammasome activation in macrophages. Eur. J. Immunol. 43:1147–52.CrossRefGoogle Scholar
  8. 8.
    Nakamura T, et al. (2010) Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Cell. 140:338–48.CrossRefGoogle Scholar
  9. 9.
    Carvalho-Filho MA, et al. (2012) Double-stranded RNA-activated protein kinase is a key modulator of insulin sensitivity in physiological conditions and in obesity in mice. Endocrinology. 153:5261–74.CrossRefGoogle Scholar
  10. 10.
    Carvalho BM, et al. (2013) Modulation of double-stranded RNA-activated protein kinase in insulin sensitive tissues of obese humans. Obesity (Silver Spring). 21:2452–7.CrossRefGoogle Scholar
  11. 11.
    Nakamura T, Arduini A, Baccaro B, Furuhashi M, Hotamisligil GS. (2014) Small-molecule inhibitors of PKR improve glucose homeostasis in obese diabetic mice. Diabetes. 63:526–34.CrossRefGoogle Scholar
  12. 12.
    Chen SS, et al. (2014) Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic beta-cells. Biochem. Biophys. Res. Commun. 443:814–20.CrossRefGoogle Scholar
  13. 13.
    Lundh M, Scully SS, Mandrup-Poulsen T, Wagner BK. (2013) Small-molecule inhibition of inflammatory beta-cell death. Diabetes Obes. Metab. 15 Suppl 3:176–84.CrossRefGoogle Scholar
  14. 14.
    Gil J, et al. (2004) TRAF family proteins link PKR with NF-kappa B activation. Mol. Cell. Biol. 24:4502–12.CrossRefGoogle Scholar
  15. 15.
    Oganesyan G, et al. (2006) Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response. Nature. 439:208–11.CrossRefGoogle Scholar
  16. 16.
    Sadler AJ, Williams BR. (2007) Structure and function of the protein kinase R. Curr. Top. Microbiol. Immunol. 316:253–92.PubMedGoogle Scholar
  17. 17.
    Li S, et al. (2006) Molecular basis for PKR activation by PACT or dsRNA. Proc. Natl. Acad. Sci. U. S. A. 103:10005–10.CrossRefGoogle Scholar
  18. 18.
    Bradley JR, Pober JS. (2001) Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene. 20:6482–91.CrossRefGoogle Scholar
  19. 19.
    Xie P. (2013) TRAF molecules in cell signaling and in human diseases. J. Mol. Signal 8:7.CrossRefGoogle Scholar
  20. 20.
    Ogolla PS, et al. (2013) The protein kinase double-stranded RNA-dependent (PKR) enhances protection against disease cause by a non-viral pathogen. PLoS Pathog. 9:e1003557.CrossRefGoogle Scholar
  21. 21.
    Jackson-Bernitsas DG, et al. (2007) Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in human head and neck squamous cell carcinoma. Oncogene. 26:1385–97.CrossRefGoogle Scholar
  22. 22.
    Ung TL, Cao C, Lu J, Ozato K, Dever TE. (2001) Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR. EMBO J. 20:3728–37.CrossRefGoogle Scholar
  23. 23.
    Wang Y, et al. (2013) Elevated toll-like receptor 3 inhibits pancreatic beta-cell proliferation through G1 phase cell cycle arrest. Mol. Cell. Endocrinol. 377:112–22.CrossRefGoogle Scholar
  24. 24.
    Institute of Laboratory Animal Resources (U.S.), Committee on Care and Use of Laboratory Animals. (1985) Guide for the Care and Use of Laboratory Animals. Rev. 1985. Bethesda (MD): NIH. 83 pp. (NIH publication; no. 85-23).Google Scholar
  25. 25.
    Han X, Sun Y, Scott S, Bleich D. (2001) Tissue inhibitor of metalloproteinase-1 prevents cytokine-mediated dysfunction and cytotoxicity in pancreatic islets and beta-cells. Diabetes. 50:1047–55.CrossRefGoogle Scholar
  26. 26.
    Kang HC, Bae YH. (2009) Transfection of rat pancreatic islet tissue by polymeric gene vectors. Diabetes Technol. Ther. 11:443–9.CrossRefGoogle Scholar
  27. 27.
    Gu L, et al. (2013) Early activation of nSMase2/ceramide pathway in astrocytes is involved in ischemia-associated neuronal damage via inflammation in rat hippocampi. J. Neuroinflammation. 10:109.CrossRefGoogle Scholar
  28. 28.
    Meng ZX, et al. (2009) Activation of liver X receptors inhibits pancreatic islet beta cell proliferation through cell cycle arrest. Diabetologia. 52:125–35.CrossRefGoogle Scholar
  29. 29.
    Hu W, et al. (2009) Double-stranded RNA-dependent protein kinase-dependent apoptosis induction by a novel small compound. J. Pharmacol. Exp. Ther. 328:866–72.CrossRefGoogle Scholar
  30. 30.
    Hu Y, Conway TW. (1993) 2-Aminopurine inhibits the double-stranded RNA-dependent protein kinase both in vitro and in vivo. J. Interferon. Res. 13:323–8.CrossRefGoogle Scholar
  31. 31.
    Ackermann AM, Gannon M. (2007) Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J. Mol. Endocrinol. 38:193–206.CrossRefGoogle Scholar
  32. 32.
    Wang HL, et al. (2014) Mangiferin facilitates islet regeneration and beta-cell proliferation through upregulation of cell cycle and beta-cell regeneration regulators. Int. J. Mol. Sci. 15:9016–35.CrossRefGoogle Scholar
  33. 33.
    Takada Y, et al. (2007) Genetic deletion of PKR abrogates TNF-induced activation of IkappaBalpha kinase, JNK, Akt and cell proliferation but potentiates p44/p42 MAPK and p38 MAPK activation. Oncogene. 26:1201–12.CrossRefGoogle Scholar
  34. 34.
    Karslioglu E, et al. (2011) cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication. Mol. Endocrinol. 25:1760–72.CrossRefGoogle Scholar
  35. 35.
    Gao Y, et al. (2012) PERK is required in the adult pancreas and is essential for maintenance of glucose homeostasis. Mol. Cell. Biol. 32:5129–39.CrossRefGoogle Scholar
  36. 36.
    Bourgarel-Rey V, et al. (2001) Involvement of nuclear factor kappaB in c-Myc induction by tubulin polymerization inhibitors. Mol. Pharmacol. 59:1165–70.CrossRefGoogle Scholar
  37. 37.
    Visconti R, et al. (1997) Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression. Oncogene. 15:1987–94.CrossRefGoogle Scholar
  38. 38.
    Kataoka Y, Murley JS, Khodarev NN, Weichselbaum RR, Grdina DJ. (2002) Activation of the nuclear transcription factor kappaB (NFkappaB) and differential gene expression in U87 glioma cells after exposure to the cytoprotector amifostine. Int. J. Radiat. Oncol. Biol. Phys. 53:180–9.CrossRefGoogle Scholar
  39. 39.
    Ghashghaeinia M, et al. (2011) The NFκB pathway inhibitors Bay 11-7082 and parthenolide induce programmed cell death in anucleated erythrocytes. Cell. Physiol. Biochem. 27:45–54.CrossRefGoogle Scholar
  40. 40.
    Liu SY, et al. (2011) Lipopolysaccharide-enhanced early proliferation of insulin secreting NIT-1 cell is associated with nuclear factor-kappaB- mediated inhibition of caspase 3 cleavage. Chin. Med J. (Engl.) 124:3652–6.Google Scholar
  41. 41.
    Xu H, You M, Shi H, Hou Y. (2014) Ubiquitin-mediated NFkappaB degradation pathway. Cell Mol. Immunol. 2014, Oct 27 [Epub ahead of print].Google Scholar
  42. 42.
    Schmid JA, Birbach A. (2008) IkappaB kinase beta (IKKbeta/IKK2/IKBKB)—a key molecule in signaling to the transcription factor NF-kappaB. Cytokine Growth Factor Rev. 19:157–65.CrossRefGoogle Scholar
  43. 43.
    Takeuchi M, Rothe M, Goeddel DV. (1996) Anatomy of TRAF2. Distinct domains for nuclear factor-kappaB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271:19935–42.CrossRefGoogle Scholar
  44. 44.
    Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. (2006) Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell. 22:245–57.CrossRefGoogle Scholar
  45. 45.
    Habelhah H. (2010) Emerging complexity of protein ubiquitination in the NF-kappaB pathway. Genes Cancer. 1:735–47.CrossRefGoogle Scholar
  46. 46.
    Poitout V. (2008) Glucolipotoxicity of the pancreatic beta-cell: myth or reality? Biochem. Soc. Trans. 36:901–4.CrossRefGoogle Scholar
  47. 47.
    Somesh BP, et al. (2013) Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity. BMC Cell. Biol. 14:31.CrossRefGoogle Scholar
  48. 48.
    Kim JW, Yoon KH. (2011) Glucolipotoxicity in pancreatic beta-Cells. Diabetes Metab. J. 35:444–50.CrossRefGoogle Scholar
  49. 49.
    Poitout V, Robertson RP. (2008) Glucolipotoxicity: fuel excess and beta-cell dysfunction. Endocr. Rev. 29:351–66.CrossRefGoogle Scholar
  50. 50.
    Garcia-Elorriaga G, et al. 2012. Pro-inflammatory cytokines related to severity and mortality in type 2 diabetes patients with soft tissue infection. [Article in Spanish] Rev. Med. Inst. Mex. Seguro Soc. 50:237–41.PubMedGoogle Scholar
  51. 51.
    Esser N, Legrand-Poels S, Piette J, Scheen AJ, Paquot N. (2014) Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. Diabetes Res. Clin. Pract. 105:141–50.CrossRefGoogle Scholar
  52. 52.
    Kong X, et al. (2014) Glucagon-like peptide 1 stimulates insulin secretion via inhibiting RhoA/ROCK signaling and disassembling glucotoxicity-induced stress fibers. Endocrinology. 155:4676–85.CrossRefGoogle Scholar
  53. 53.
    Chang-Chen KJ, Mullur R, Bernal-Mizrachi E. (2008) Beta-cell failure as a complication of diabetes. Rev. Endocr. Metab. Disord. 9:329–43.CrossRefGoogle Scholar
  54. 54.
    Hayes HL, et al. (2013) Pdx-1 activates islet alpha-and beta-cell proliferation via a mechanism regulated by transient receptor potential cation channels 3 and 6 and extracellular signal-regulated kinases 1 and 2. Mol. Cell. Biol. 33:4017–29.CrossRefGoogle Scholar
  55. 55.
    Chen G, Liu C, Xue Y, Mao X, Xu K. (2011) Molecular mechanism of pancreatic beta-cell adaptive proliferation: studies during pregnancy in rats and in vitro. Endocrine. 39:118–27.CrossRefGoogle Scholar
  56. 56.
    Ishii T, Kwon H, Hiscott J, Mosialos G, Koromilas AE. (2001) Activation of the I kappa B alpha kinase (IKK) complex by double-stranded RNA-binding defective and catalytic inactive mutants of the interferon-inducible protein kinase PKR. Oncogene. 20:1900–12.CrossRefGoogle Scholar
  57. 57.
    Bonnet MC, Weil R, Dam E, Hovanessian AG, Meurs EF. (2000) PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex. Mol. Cell. Biol. 20:4532–42.CrossRefGoogle Scholar
  58. 58.
    Green TJ, Montagnani C. (2013) Poly I:C induces a protective antiviral immune response in the Pacific oyster (Crassostrea gigas) against subsequent challenge with Ostreid herpesvirus (OsHV-1 muvar). Fish Shellfish Immunol. 35:382–8.CrossRefGoogle Scholar
  59. 59.
    Hsu H, Huang J, Shu HB, Baichwal V, Goeddel DV. (1996) TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity. 4:387–96.CrossRefGoogle Scholar
  60. 60.
    Li X, et al. (1999) Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol. Cell. Biol. 19:4643–52.CrossRefGoogle Scholar
  61. 61.
    Kim SH, Gunnery S, Choe JK, Mathews MB. (2002) Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR. Oncogene. 21:8741–8.CrossRefGoogle Scholar
  62. 62.
    Kim SH, Forman AP, Mathews MB, Gunnery S. (2000) Human breast cancer cells contain elevated levels and activity of the protein kinase, PKR. Oncogene. 19:3086–94.CrossRefGoogle Scholar
  63. 63.
    Kunkeaw N, et al. (2013) Cell death/proliferation roles for nc886, a non-coding RNA, in the protein kinase R pathway in cholangiocarcinoma. Oncogene. 32:3722–31.CrossRefGoogle Scholar
  64. 64.
    Bretones G, Delgado MD, Leon J. (2015) Myc and cell cycle control. Biochim. Biophys. Acta. 1849:506–16.CrossRefGoogle Scholar
  65. 65.
    Gil J, Alcami J, Esteban M. (1999) Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB. Mol. Cell. Biol. 19:4653–63.CrossRefGoogle Scholar
  66. 66.
    Liuwantara D, et al. (2006) Nuclear factor-kappaB regulates beta-cell death: a critical role for A20 in beta-cell protection. Diabetes. 55:2491–501.CrossRefGoogle Scholar
  67. 67.
    Zhou R, et al. (2013) Blockage of progesterone receptor effectively protects pancreatic islet beta cell viability. Steroids. 78:987–95.CrossRefGoogle Scholar
  68. 68.
    Humphrey RK, et al. (2013) Lysine 63-linked ubiquitination modulates mixed lineage kinase-3 interaction with JIP1 scaffold protein in cytokine-induced pancreatic beta cell death. J. Biol. Chem. 288:2428–40.CrossRefGoogle Scholar
  69. 69.
    Lin WJ, et al. (2011) Crucial role for TNF receptor-associated factor 2 (TRAF2) in regulating NFkappaB2 signaling that contributes to autoimmunity. Proc. Natl. Acad. Sci. U. S. A. 108:18354–9.CrossRefGoogle Scholar
  70. 70.
    Hupalowska A, Pyrzynska B, Miaczynska M. (2012) APPL1 regulates basal NF-kappaB activity by stabilizing NIK. J. Cell Sci. 125:4090–102.CrossRefGoogle Scholar
  71. 71.
    Jin HR, Jin X, Dat NT, Lee JJ. (2011) Cucurbitacin B suppresses the transactivation activity of RelA/p65. J. Cell. Biochem. 112:1643–50.CrossRefGoogle Scholar
  72. 72.
    Doppler H, Liou GY, Storz P. (2013) Downregulation of TRAF2 mediates NIK-induced pancreatic cancer cell proliferation and tumorigenicity. PloS One. 8:e53676.CrossRefGoogle Scholar
  73. 73.
    Sun LL, et al. (2014) Suppressive role of miR-502-5p in breast cancer via downregulation of TRAF2. Oncol. Rep. 31:2085–92.CrossRefGoogle Scholar
  74. 74.
    Mahul-Mellier AL, et al. (2012) De-ubiquitinating protease USP2a targets RIP1 and TRAF2 to mediate cell death by TNF. Cell Death Differ. 19:891–9.CrossRefGoogle Scholar
  75. 75.
    O’Donnell MA, Hase H, Legarda D, Ting AT. (2012) NEMO inhibits programmed necrosis in an NFkappaB-independent manner by restraining RIP1. PloS One. 7:e41238.CrossRefGoogle Scholar
  76. 76.
    Ozcan U, et al. (2004) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science. 306:457–61.CrossRefGoogle Scholar
  77. 77.
    Tran K, et al. (2014) Identification of small molecules that protect pancreatic beta cells against endoplasmic reticulum stress-induced cell death. ACS Chem. Biol. 9:2796–806.CrossRefGoogle Scholar
  78. 78.
    Pelengaris S, Khan M. (2003) The many faces of c-MYC. Arch. Biochem. Biophys. 416:129–36.CrossRefGoogle Scholar
  79. 79.
    Pelengaris S, Khan M, Evan GI. (2002) Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell. 109:321–34.CrossRefGoogle Scholar

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Authors and Affiliations

  • LiLi Gao
    • 1
    • 2
  • Wei Tang
    • 3
  • ZhengZheng Ding
    • 1
    • 2
  • DingYu Wang
    • 1
    • 2
  • XiaoQiang Qi
    • 1
    • 2
  • HuiWen Wu
    • 4
  • Jun Guo
    • 1
    • 2
    • 5
  1. 1.Key Laboratory of Human Functional Genomics of Jiangsu ProvinceNanjing Medical UniversityNanjingRepublic of China
  2. 2.Department of Biochemistry and Molecular BiologyNanjing Medical UniversityNanjingPeople’s Republic of China
  3. 3.Department of EndocrinologyThe Affiliated Jiangyin Hospital of Southeast University Medical CollegeJiangyinPeople’s Republic of China
  4. 4.Laboratory Center for Basic Medical SciencesNanjing Medical UniversityNanjingPeople’s Republic of China
  5. 5.Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular BiologyNanjing Medical UniversityNanjingRepublic of China

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