Abstract
The transcription factor PDX-1 (pancreatic and duodenal homeobox-1) is essential for pancreatic development and the maintainence of expression of islet beta-cell-specific genes. In an previous study [Rafiq, Kennedy and Rutter (1998) J. Biol. Chem. 273, 23241-23247] we demonstrated that PDX-1 may be activated at elevated glucose concentrations by translocation from undefined binding sites in the cytosol and nuclear membrane into the nucleoplasm. In the present study, we show that PDX-1 interacts directly and specifically in vitro with the nuclear import receptor family member, importin beta1, and that this interaction is mediated by the PDX-1 homeodomain (amino acids 146-206). Demonstrating the functional importance of the PDX-1-importin beta1 interaction, microinjection of MIN6 beta-cells with anti-(importin beta1) antibodies blocked both the nuclear translocation of PDX-1, and the activation by glucose (30 mM versus 3 mM) of the pre-proinsulin promoter. However, treatment with extracts from pancreatic islets incubated at either low or high glucose concentrations had no impact on the ability of PDX-1 to interact with importin beta1 in vitro. Furthermore, importin beta1 also interacted with SREBP1c (sterol-regulatory-element-binding protein 1c) in vitro, and microinjection of importin beta1 antibodies blocked the activation by glucose of SREBP1c target genes. Since the subcellular distribution of SREBP1c is unaffected by glucose, these findings suggest that a redistribution of importin beta1 is unlikely to explain the glucose-stimulated nuclear uptake of PDX-1. Instead, we conclude that the uptake of PDX-1 into the nucleoplasm, as glucose concentrations increase, may be mediated by release of the factor both from sites of retention in the cytosol and from non-productive complexes with importin beta1 at the nuclear membrane.
Full Text
The Full Text of this article is available as a PDF (238.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andreolas Chrysovalantis, da Silva Xavier Gabriela, Diraison Frederique, Zhao Chao, Varadi Aniko, Lopez-Casillas Fernando, Ferré Pascal, Foufelle Fabienne, Rutter Guy A. Stimulation of acetyl-CoA carboxylase gene expression by glucose requires insulin release and sterol regulatory element binding protein 1c in pancreatic MIN6 beta-cells. Diabetes. 2002 Aug;51(8):2536–2545. doi: 10.2337/diabetes.51.8.2536. [DOI] [PubMed] [Google Scholar]
- Brown M. S., Goldstein J. L. Sterol regulatory element binding proteins (SREBPs): controllers of lipid synthesis and cellular uptake. Nutr Rev. 1998 Feb;56(2 Pt 2):S1–S75. doi: 10.1111/j.1753-4887.1998.tb01680.x. [DOI] [PubMed] [Google Scholar]
- Brown M. S., Goldstein J. L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997 May 2;89(3):331–340. doi: 10.1016/s0092-8674(00)80213-5. [DOI] [PubMed] [Google Scholar]
- Chakrabarti Swarup K., James Joshua C., Mirmira Raghavendra G. Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding. J Biol Chem. 2002 Feb 1;277(15):13286–13293. doi: 10.1074/jbc.M111857200. [DOI] [PubMed] [Google Scholar]
- Chook Y. M., Blobel G. Karyopherins and nuclear import. Curr Opin Struct Biol. 2001 Dec;11(6):703–715. doi: 10.1016/s0959-440x(01)00264-0. [DOI] [PubMed] [Google Scholar]
- Cingolani G., Lashuel H. A., Gerace L., Müller C. W. Nuclear import factors importin alpha and importin beta undergo mutually induced conformational changes upon association. FEBS Lett. 2000 Nov 10;484(3):291–298. doi: 10.1016/s0014-5793(00)02154-2. [DOI] [PubMed] [Google Scholar]
- Cingolani Gino, Bednenko Janna, Gillespie Matthew T., Gerace Larry. Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta. Mol Cell. 2002 Dec;10(6):1345–1353. doi: 10.1016/s1097-2765(02)00727-x. [DOI] [PubMed] [Google Scholar]
- Cuif M. H., Porteu A., Kahn A., Vaulont S. Exploration of a liver-specific, glucose/insulin-responsive promoter in transgenic mice. J Biol Chem. 1993 Jul 5;268(19):13769–13772. [PubMed] [Google Scholar]
- De Vit M. J., Waddle J. A., Johnston M. Regulated nuclear translocation of the Mig1 glucose repressor. Mol Biol Cell. 1997 Aug;8(8):1603–1618. doi: 10.1091/mbc.8.8.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diraison Frédérique, Dusserre Eric, Vidal Hubert, Sothier Monique, Beylot Michel. Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity. Am J Physiol Endocrinol Metab. 2002 Jan;282(1):E46–E51. doi: 10.1152/ajpendo.2002.282.1.E46. [DOI] [PubMed] [Google Scholar]
- Elrick L. J., Docherty K. Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1. Diabetes. 2001 Oct;50(10):2244–2252. doi: 10.2337/diabetes.50.10.2244. [DOI] [PubMed] [Google Scholar]
- Foretz M., Pacot C., Dugail I., Lemarchand P., Guichard C., Le Lièpvre X., Berthelier-Lubrano C., Spiegelman B., Kim J. B., Ferré P. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose. Mol Cell Biol. 1999 May;19(5):3760–3768. doi: 10.1128/mcb.19.5.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fukuda M., Gotoh Y., Nishida E. Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 1997 Apr 15;16(8):1901–1908. doi: 10.1093/emboj/16.8.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furuno T., Hirashima N., Onizawa S., Sagiya N., Nakanishi M. Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK kinase after antigen stimulation in RBL-2H3 cells. J Immunol. 2001 Apr 1;166(7):4416–4421. doi: 10.4049/jimmunol.166.7.4416. [DOI] [PubMed] [Google Scholar]
- Gotoh M., Maki T., Kiyoizumi T., Satomi S., Monaco A. P. An improved method for isolation of mouse pancreatic islets. Transplantation. 1985 Oct;40(4):437–438. doi: 10.1097/00007890-198510000-00018. [DOI] [PubMed] [Google Scholar]
- Guillemain G., Loizeau M., Pinçon-Raymond M., Girard J., Leturque A. The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells. J Cell Sci. 2000 Mar;113(Pt 5):841–847. doi: 10.1242/jcs.113.5.841. [DOI] [PubMed] [Google Scholar]
- Guillemain Ghislaine, Muñoz-Alonso Maria J., Cassany Aurélia, Loizeau Martine, Faussat Anne-Marie, Burnol Anne-Françoise, Leturque Armelle. Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells. Biochem J. 2002 May 15;364(Pt 1):201–209. doi: 10.1042/bj3640201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Görlich D., Henklein P., Laskey R. A., Hartmann E. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. EMBO J. 1996 Apr 15;15(8):1810–1817. [PMC free article] [PubMed] [Google Scholar]
- Görlich D., Kutay U. Transport between the cell nucleus and the cytoplasm. Annu Rev Cell Dev Biol. 1999;15:607–660. doi: 10.1146/annurev.cellbio.15.1.607. [DOI] [PubMed] [Google Scholar]
- Görlich D., Vogel F., Mills A. D., Hartmann E., Laskey R. A. Distinct functions for the two importin subunits in nuclear protein import. Nature. 1995 Sep 21;377(6546):246–248. doi: 10.1038/377246a0. [DOI] [PubMed] [Google Scholar]
- Henderson B. R., Percipalle P. Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta. J Mol Biol. 1997 Dec 19;274(5):693–707. doi: 10.1006/jmbi.1997.1420. [DOI] [PubMed] [Google Scholar]
- Hessabi B., Ziegler P., Schmidt I., Hessabi C., Walther R. The nuclear localization signal (NLS) of PDX-1 is part of the homeodomain and represents a novel type of NLS. Eur J Biochem. 1999 Jul;263(1):170–177. doi: 10.1046/j.1432-1327.1999.00481.x. [DOI] [PubMed] [Google Scholar]
- Holland Andrew M., Hale Michael A., Kagami Hideaki, Hammer Robert E., MacDonald Raymond J. Experimental control of pancreatic development and maintenance. Proc Natl Acad Sci U S A. 2002 Sep 9;99(19):12236–12241. doi: 10.1073/pnas.192255099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoppe T., Rape M., Jentsch S. Membrane-bound transcription factors: regulated release by RIP or RUP. Curr Opin Cell Biol. 2001 Jun;13(3):344–348. doi: 10.1016/s0955-0674(00)00218-0. [DOI] [PubMed] [Google Scholar]
- Imamoto N., Shimamoto T., Kose S., Takao T., Tachibana T., Matsubae M., Sekimoto T., Shimonishi Y., Yoneda Y. The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile. FEBS Lett. 1995 Jul 24;368(3):415–419. doi: 10.1016/0014-5793(95)00699-a. [DOI] [PubMed] [Google Scholar]
- Jäkel S., Görlich D. Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 1998 Aug 3;17(15):4491–4502. doi: 10.1093/emboj/17.15.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaffman A., O'Shea E. K. Regulation of nuclear localization: a key to a door. Annu Rev Cell Dev Biol. 1999;15:291–339. doi: 10.1146/annurev.cellbio.15.1.291. [DOI] [PubMed] [Google Scholar]
- Kawaguchi Takumi, Osatomi Kiyoshi, Yamashita Hiromi, Kabashima Tsutomu, Uyeda Kosaku. Mechanism for fatty acid "sparing" effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase. J Biol Chem. 2001 Nov 27;277(6):3829–3835. doi: 10.1074/jbc.M107895200. [DOI] [PubMed] [Google Scholar]
- Kennedy H. J., Viollet B., Rafiq I., Kahn A., Rutter G. A. Upstream stimulatory factor-2 (USF2) activity is required for glucose stimulation of L-pyruvate kinase promoter activity in single living islet beta-cells. J Biol Chem. 1997 Aug 15;272(33):20636–20640. doi: 10.1074/jbc.272.33.20636. [DOI] [PubMed] [Google Scholar]
- Khokhlatchev A. V., Canagarajah B., Wilsbacher J., Robinson M., Atkinson M., Goldsmith E., Cobb M. H. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell. 1998 May 15;93(4):605–615. doi: 10.1016/s0092-8674(00)81189-7. [DOI] [PubMed] [Google Scholar]
- Kim J. B., Spotts G. D., Halvorsen Y. D., Shih H. M., Ellenberger T., Towle H. C., Spiegelman B. M. Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain. Mol Cell Biol. 1995 May;15(5):2582–2588. doi: 10.1128/mcb.15.5.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim Seung K., MacDonald Raymond J. Signaling and transcriptional control of pancreatic organogenesis. Curr Opin Genet Dev. 2002 Oct;12(5):540–547. doi: 10.1016/s0959-437x(02)00338-6. [DOI] [PubMed] [Google Scholar]
- Kishi Akio, Nakamura Takaaki, Nishio Yoshihiko, Maegawa Hiroshi, Kashiwagi Atsunori. Sumoylation of Pdx1 is associated with its nuclear localization and insulin gene activation. Am J Physiol Endocrinol Metab. 2002 Dec 17;284(4):E830–E840. doi: 10.1152/ajpendo.00390.2002. [DOI] [PubMed] [Google Scholar]
- Kuersten S., Ohno M., Mattaj I. W. Nucleocytoplasmic transport: Ran, beta and beyond. Trends Cell Biol. 2001 Dec;11(12):497–503. doi: 10.1016/s0962-8924(01)02144-4. [DOI] [PubMed] [Google Scholar]
- Lu M., Miller C., Habener J. F. Functional regions of the homeodomain protein IDX-1 required for transactivation of the rat somatostatin gene. Endocrinology. 1996 Jul;137(7):2959–2967. doi: 10.1210/endo.137.7.8770920. [DOI] [PubMed] [Google Scholar]
- Luo X. C., Kim K. H. An enhancer element in the house-keeping promoter for acetyl-CoA carboxylase gene. Nucleic Acids Res. 1990 Jun 11;18(11):3249–3254. doi: 10.1093/nar/18.11.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melloul D., Marshak S., Cerasi E. Regulation of insulin gene transcription. Diabetologia. 2002 Mar;45(3):309–326. doi: 10.1007/s00125-001-0728-y. [DOI] [PubMed] [Google Scholar]
- Miyazaki J., Araki K., Yamato E., Ikegami H., Asano T., Shibasaki Y., Oka Y., Yamamura K. Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology. 1990 Jul;127(1):126–132. doi: 10.1210/endo-127-1-126. [DOI] [PubMed] [Google Scholar]
- Moede T., Leibiger B., Pour H. G., Berggren P., Leibiger I. B. Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1. FEBS Lett. 1999 Nov 19;461(3):229–234. doi: 10.1016/s0014-5793(99)01446-5. [DOI] [PubMed] [Google Scholar]
- Moore J. D., Yang J., Truant R., Kornbluth S. Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1. J Cell Biol. 1999 Jan 25;144(2):213–224. doi: 10.1083/jcb.144.2.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muslin A. J., Xing H. 14-3-3 proteins: regulation of subcellular localization by molecular interference. Cell Signal. 2000 Dec;12(11-12):703–709. doi: 10.1016/s0898-6568(00)00131-5. [DOI] [PubMed] [Google Scholar]
- Nagoshi E., Imamoto N., Sato R., Yoneda Y. Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor, occurs through the direct interaction of importin beta with HLH-Zip. Mol Biol Cell. 1999 Jul;10(7):2221–2233. doi: 10.1091/mbc.10.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagoshi E., Yoneda Y. Dimerization of sterol regulatory element-binding protein 2 via the helix-loop-helix-leucine zipper domain is a prerequisite for its nuclear localization mediated by importin beta. Mol Cell Biol. 2001 Apr;21(8):2779–2789. doi: 10.1128/MCB.21.8.2779-2789.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osborne T. F. Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action. J Biol Chem. 2000 Oct 20;275(42):32379–32382. doi: 10.1074/jbc.R000017200. [DOI] [PubMed] [Google Scholar]
- Peshavaria M., Cissell M. A., Henderson E., Petersen H. V., Stein R. The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells. Mol Endocrinol. 2000 Dec;14(12):1907–1917. doi: 10.1210/mend.14.12.0563. [DOI] [PubMed] [Google Scholar]
- Pouli A. E., Emmanouilidou E., Zhao C., Wasmeier C., Hutton J. C., Rutter G. A. Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera. Biochem J. 1998 Jul 1;333(Pt 1):193–199. doi: 10.1042/bj3330193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prieve M. G., Guttridge K. L., Munguia J. E., Waterman M. L. The nuclear localization signal of lymphoid enhancer factor-1 is recognized by two differentially expressed Srp1-nuclear localization sequence receptor proteins. J Biol Chem. 1996 Mar 29;271(13):7654–7658. doi: 10.1074/jbc.271.13.7654. [DOI] [PubMed] [Google Scholar]
- Quimby B. B., Corbett A. H. Nuclear transport mechanisms. Cell Mol Life Sci. 2001 Nov;58(12-13):1766–1773. doi: 10.1007/PL00000816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rafiq I., Kennedy H. J., Rutter G. A. Glucose-dependent translocation of insulin promoter factor-1 (IPF-1) between the nuclear periphery and the nucleoplasm of single MIN6 beta-cells. J Biol Chem. 1998 Sep 4;273(36):23241–23247. doi: 10.1074/jbc.273.36.23241. [DOI] [PubMed] [Google Scholar]
- Rafiq I., da Silva Xavier G., Hooper S., Rutter G. A. Glucose-stimulated preproinsulin gene expression and nuclear trans-location of pancreatic duodenum homeobox-1 require activation of phosphatidylinositol 3-kinase but not p38 MAPK/SAPK2. J Biol Chem. 2000 May 26;275(21):15977–15984. doi: 10.1074/jbc.275.21.15977. [DOI] [PubMed] [Google Scholar]
- Rutter G. A., Burnett P., Rizzuto R., Brini M., Murgia M., Pozzan T., Tavaré J. M., Denton R. M. Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5489–5494. doi: 10.1073/pnas.93.11.5489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rutter G. A. Insulin secretion: feed-forward control of insulin biosynthesis? Curr Biol. 1999 Jun 17;9(12):R443–R445. doi: 10.1016/s0960-9822(99)80277-2. [DOI] [PubMed] [Google Scholar]
- Rutter G. A., Kennedy H. J., Wood C. D., White M. R., Tavaré J. M. Real-time imaging of gene expression in single living cells. Chem Biol. 1998 Nov;5(11):R285–R290. doi: 10.1016/s1074-5521(98)90287-3. [DOI] [PubMed] [Google Scholar]
- Rutter G. A., White M. R., Tavaré J. M. Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells. Curr Biol. 1995 Aug 1;5(8):890–899. doi: 10.1016/s0960-9822(95)00179-5. [DOI] [PubMed] [Google Scholar]
- Rutter Guy A., Tavaré Jeremy M., Palmer D. Gail. Regulation of Mammalian Gene Expression by Glucose. News Physiol Sci. 2000 Jun;15(NaN):149–154. doi: 10.1152/physiologyonline.2000.15.3.149. [DOI] [PubMed] [Google Scholar]
- Stoffers D. A., Zinkin N. T., Stanojevic V., Clarke W. L., Habener J. F. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet. 1997 Jan;15(1):106–110. doi: 10.1038/ng0197-106. [DOI] [PubMed] [Google Scholar]
- Vaulont S., Vasseur-Cognet M., Kahn A. Glucose regulation of gene transcription. J Biol Chem. 2000 Oct 13;275(41):31555–31558. doi: 10.1074/jbc.R000016200. [DOI] [PubMed] [Google Scholar]
- Váradi Anikó, Rutter Guy A. Green fluorescent protein calcium biosensors. Calcium imaging with GFP cameleons. Methods Mol Biol. 2002;183:255–264. doi: 10.1385/1-59259-280-5:255. [DOI] [PubMed] [Google Scholar]
- Wang Haiyan, Wollheim Claes B. ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells. J Biol Chem. 2002 Jun 26;277(36):32746–32752. doi: 10.1074/jbc.M201635200. [DOI] [PubMed] [Google Scholar]
- Webb G. C., Akbar M. S., Zhao C., Steiner D. F. Expression profiling of pancreatic beta cells: glucose regulation of secretory and metabolic pathway genes. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5773–5778. doi: 10.1073/pnas.100126597. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xiao Z., Liu X., Lodish H. F. Importin beta mediates nuclear translocation of Smad 3. J Biol Chem. 2000 Aug 4;275(31):23425–23428. doi: 10.1074/jbc.C000345200. [DOI] [PubMed] [Google Scholar]