phosphorylation-dependent nucleocytoplasmic shuttling of...

Phosphorylation-Dependent NucleocytoplasmicShuttling of Pancreatic Duodenal Homeobox-1Lucy J. Elrick and Kevin Docherty

Pancreatic duodenal homeobox-1 (PDX-1) is a homeo-domain protein that plays an important role in thedevelopment of the pancreas and in maintaining theidentity and function of the islets of Langerhans. It alsoregulates the expression of the insulin gene in responseto changes in glucose and insulin concentrations. Glu-cose and insulin regulate PDX-1 by way of a signalingpathway involving phosphatidylinositol 3-kinase (PI3-kinase) and SAPK2/p38. Activation of this pathwayleads to phosphorylation of PDX-1 and its movementinto the nucleus. To investigate the intracellular traf-ficking of PDX-1, immunocytochemistry was used tolocalize PDX-1 in the human �-cell line NesPDX-1, inwhich PDX-1 is overexpressed, and in MIN6 �-cells. Inlow-glucose conditions, PDX-1 localized predominantlyto the nuclear periphery, with some staining in thecytoplasm. After stimulation with glucose, PDX-1 waspresent in the nucleoplasm. The translocation of PDX-1to the nucleoplasm was complete within 15 min andoccurred in 5�10 mmol/l glucose. Insulin and sodiumarsenite, an activator of the stress-activated pathway,also stimulated PDX-1 movement from the nuclear pe-riphery to the nucleoplasm. When cells were transferredbetween high glucose� and low glucose�containing me-dium, PDX-1 rapidly shuttled between the nuclear periph-ery and the nucleoplasm. Glucose- and insulin-stimulatedtranslocation of PDX-1 to the nucleoplasm was inhibitedby wortmannin and SB 203580, indicating that a pathwayinvolving PI 3-kinase and SAPK2/p38 was involved; trans-location was unaffected by PD 098959 and rapamycin,suggesting that neither mitogen-activated protein kinasenor p70s6k were involved. Arsenite-stimulated import ofPDX-1 into the nucleus was inhibited by SB 203580 butnot by wortmannin. Export from the nucleoplasm to thenuclear periphery was inhibited by calyculin A and oka-daic acid, suggesting that dephosphorylation of PDX-1was involved. These results demonstrated that PDX-1shuttles between the nuclear periphery and nucleoplasmin response to changes in glucose and insulin concen-trations and that these events are dependent on PI3-kinase, SAPK2/p38, and a nuclear phosphatase(s).Diabetes 50:2244–2252, 2001

Pancreatic duodenal homeobox-1 (PDX-1) is ahomeodomain protein present in �-cells and D-cells of islets of Langerhans. It is also expressedin neuroendocrine cells of the gut. PDX-1 plays

an important role in the development of the pancreas (1).Thus PDX-1�deficient mice are born without a pancreas(2,3), whereas the heterozygote PDX-1 (�/�) mouse de-velops a pancreas but becomes mildly diabetic (4). Pan-creatic agenesis has also been observed in a human withPDX-1 deficiency resulting from a homozygous mutation inthe PDX-1 gene (5). Family members of that individualwho were heterozygote carriers of this mutation devel-oped maturity onset diabetes of the young, an autosomalform of type 2 diabetes (6). Other mutations in the PDX-1gene have been linked to type 2 diabetes (7,8).

In addition to its role in the development of the pan-creas, PDX-1 also binds to sequences within and regulatesthe promoter activity of a number of islet genes, includinginsulin (9), GLUT-2 (10), glucokinase (11), islet amyloidpolypeptide (12–15), and somatostatin (16). Its exact rolein regulating insulin gene transcription is not clear, but itis known not to be essential to the process, as high levelsof insulin mRNA have been observed under conditions inwhich PDX-1 activity is reduced (17) or virtually absent(18,19). There is strong evidence that PDX-1 plays a role inactivating the insulin promoter (20–22) and increasinginsulin mRNA levels (23) in response to glucose. Glucose’seffects on PDX-1 are mediated by way of a cell-signalingpathway that involves phosphatidylinositol 3-kinase (PI3-kinase) and SAPK2/p38 (24), although atypical proteinkinase C�� has also been implicated in the mechanisms bywhich glucose stimulates PDX-1 (25). Insulin can alsoactivate the insulin promoter and PDX-1 DNA-bindingactivity (26,27).

It has been shown previously that glucose stimulates themovement of PDX-1 from the cytoplasm to the nucleus(28,29) and that in low-glucose conditions, PDX-1 mightbe preferentially associated with the nuclear periphery(15,28). The aim of the present study was to investigatenuclear translocation of PDX-1 in further detail by usingimmunocytochemistry. The experiments were performedin the human �-like cell line NES2Y stably transfected withhuman PDX-1 to generate the NesPDX-1 cell line (23) andin MIN6 �-cells. The results demonstrated that in unstimu-lated cells, PDX-1 preferentially associated with the nu-clear periphery, with some staining in the cytoplasm, andthat glucose and insulin affected the shuttling of PDX-1between the nuclear periphery and the nucleoplasm. Move-ment from the nuclear periphery to the nucleoplasm was

From the Department of Molecular and Cell Biology, University of Aberdeen,Institute of Medical Sciences, Aberdeen, U.K.

Address correspondence and reprint requests to Professor Kevin Docherty,Department of Molecular and Cell Biology, University of Aberdeen, Instituteof Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, U.K. E-mail: [email protected].

Received for publication 29 March 2001 and accepted in revised form 3 July2001.

BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; GRP, glu-cokinase regulatory protein; HP-1, heterochromatin protein-1; NFAT, nuclearfactor of activated T-cell; PBS, phosphate-buffered saline; PDX-1, pancreaticduodenal homeobox-1; PI 3-kinase, phosphatidylinositol 3-kinase; USF, up-stream stimulatory factor.

2244 DIABETES, VOL. 50, OCTOBER 2001

inhibited by wortmannin and SB203580, thus supporting theview that PI 3-kinase and SAPK2/p38 were involved. Exportfrom the nucleoplasm to the nuclear periphery was blockedby the phosphatase inhibitors calyculin A and okadaic acid.

RESEARCH DESIGN AND METHODS

Chemicals and reagents. Inhibitors SB 203580, rapamycin, wortmannin, andcalyculin A were purchased from Sigma-Aldrich (Poole, U.K.); PD 098059 andokadaic acid, from Calbiochem (Nottingham, Nottinghamshire, U.K.); andsodium arsenite, from Fisons (Loughborough, Nottinghamshire, U.K.). Anti-PDX-1 antibody was a kind gift from Dr. C. V. Wright (Vanderbilt University,Nashville, TN). Anti�upstream stimulatory factor (USF) antibody was pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti�lamin-B1antibody was from Chemicon International (Harrow, Essex, U.K.). Fluores-cein isothiocyanate (FITC)�labeled anti-rabbit IgG antibody was obtainedfrom Diagnostics Scotland, Law Hospital, (Carluke, Lanarkshire, U.K.) andrhodamine-labeled anti-mouse IgG was obtained from Jackson Immunore-search (Westgrove, PA). All other chemicals were purchased from Sigma-Aldrich.Cell culture. NesPDX-1 cells that overexpress PDX-1 were generated bystably transfecting NES2Y cells with a cDNA encoding PDX-1. NES2Y cellswere derived from islets of Langerhans isolated from the pancreas of a patientwith persistent hyperinsulinemic hypoglycemia of infancy, as previouslydescribed (18). NES2Y cells express PDX-1 at very low levels that can bedetected by reverse transcriptase�polymerase chain reaction, but not byelectrophoretic mobility shift assay, Western blot, or immunocytochemistry.NesPDX-1 cells were maintained in RPMI 1640 medium (Life Technologies,Paisley, Strathclyde, U.K.) containing 11 mmol/l glucose and 2 mmol/l glu-tamine, supplemented with 10% (vol/vol) fetal calf serum (Life Technologies)and 800 �g/ml G418. MIN6 (30) is a pancreatic �-cell line derived fromtransgenic mice expressing the simian virus 40 large T-antigen under thecontrol of the rat insulin gene promoter. MIN6 cells were grown in Dulbecco’smodified Eagle’s medium containing 5 mmol/l glucose supplemented with10% (vol/vol) fetal bovine serum in a humidified atmosphere containing 95%air/5% CO2.Immunocytochemistry. Cells were grown in 35-mm plates (Nunclon, Naper-ville, IL) to 50% confluence. After an overnight incubation in medium

containing 10% fetal calf serum and 0.5 mmol/l glucose, the cells were thenincubated under conditions described in figure legends. Cells were thenwashed four times with phosphate-buffered saline (PBS) and fixed in ice-coldmethanol for 10 min at 4°C. The cells were again washed four times in PBSbefore being blocked for 15 min at room temperature in blocking buffercontaining 6.7% (vol/vol) glycerol, 0.2% (vol/vol) Tween 20, and 2% (wt/vol)bovine serum albumin (BSA) in PBS. Primary antibodies were added to thecells in 1 ml blocking buffer, and cells were incubated overnight at 4°C. Afterfour washes in a solution containing 6.7% glycerol, 0.4% Tween 20, and 2% BSAin PBS, cells were incubated in a 1:400 dilution, in blocking buffer, of anFITC-conjugated anti-rabbit secondary antibody for 1 h in the dark. The cellswere then washed in the same wash buffer for 1–2 h in the dark with gentleagitation, before they were mounted on slides in Vectashield (Vector Labora-tories, Burlingame, CA) mounting medium and coverslips were applied. Theplates were then studied by confocal microscopy with a 40� water immersionobjective. Cells were randomly selected and the confocal image was adjustedto take a section through the entire body of the cell, using a BioRadmicroradiance confocal scanning system. Images selected were representa-tive of 75–100% of cells present on each plate. Specificity of the reaction wasconfirmed by demonstrating no staining with pre-immune serum and second-ary antibody alone or in NES2Y cells, which do not express detectable levelsof PDX-1.

RESULTS

To examine the effect of glucose on the intracellulardistribution of PDX-1, NesPDX-1 cells were incubated inmedium containing low or high glucose concentrationsand subjected to immunocytochemistry using an anti�PDX-1 antibody. In cells incubated in 0.5 mmol/l glucose,as previously shown (15,28), PDX-1 was located predom-inantly around the nuclear periphery, with some stainingwithin the cytoplasm (Fig. 1). After incubation for 30 minin medium containing 20 mmol/l glucose, PDX-1 waslocalized exclusively within the nucleus (Fig. 1); within thenucleus, PDX-1 staining appeared punctate, which may

FIG. 1. Effect of glucose on the intracellular localization of PDX-1. NesPDX-1 cells were preincubated for 16 h in 0.5 mmol/l glucose (A) and thenstimulated for 30 min in medium containing 20 mmol/l glucose (B). Cells were then washed, fixed, and stained with an anti–PDX-1 primaryantibody and fluorescein-coupled secondary antibody (green).

L.J. ELRICK AND K. DOCHERTY

DIABETES, VOL. 50, OCTOBER 2001 2245

have reflected association with subnuclear structures, suchas nuclear speckles that are known to be sites of increasedtranscriptional activity and pre-mRNA splicing. The distri-bution of PDX-1 was similar to that of lamin-B1, a proteinlocalized in the nuclear envelope. The intracellular distri-bution of lamin-B1, however, was unaffected by glucose(data not shown). In addition, glucose had no effect on theintracellular distribution of another transcription factor,USF, that was localized within the nucleus under allconditions (data not shown).

The glucose-dependent movement of PDX-1 from thenuclear periphery/cytoplasm to the nucleoplasm occurredwithin 10 min after treatment with high glucose and wascomplete within 15 min (Fig. 2). The effect of glucose onthe intracellular distribution of PDX-1 was dosage depen-dent. At 0.5 and 3 mmol/l glucose, PDX-1 was predomi-nantly localized to the nuclear periphery. At 5 mmol/lglucose, there was a substantial amount within the nu-cleus, and at 10 mmol/l glucose, the movement of PDX-1was complete, with no further effect observed over a rangeof concentrations up to 30 mmol/l (Fig. 2).

It has been shown previously that insulin and sodiumarsenite (a reagent known to stimulate stress-activatedpathways) can stimulate PDX-1 DNA binding and insulinpromoter activity (24). To investigate their effect on thenuclear trafficking of PDX-1, NesPDX-1 cells were incu-bated for 30 min in 0.5 mmol/l glucose supplemented with20 ng/ml insulin or 1 mmol/l sodium arsenite or in 20mmol/l glucose. Both insulin and arsenite stimulated the

movement of PDX-1 from the nuclear periphery/cytoplasmto the nucleoplasm (Fig. 3).

To determine whether the movement of PDX-1 to thenucleoplasm was reversible, NesPDX-1 cells were incu-bated in 20 mmol/l glucose or 20 ng/ml insulin in thepresence of the protein synthesis inhibitor cyclohexi-mide and then incubated for periods of up to 5 h in 0.5mmol/l glucose. In preliminary experiments, cyclohexi-mide had no effect on the intracellular distribution ofPDX-1 in cells incubated in low or high glucose (datanot shown). Both glucose and insulin stimulated move-ment of PDX-1 from the nuclear periphery/cytoplasm tothe nucleoplasm within 30 min. Subsequent transfer tolow (0.5 mmol/l) glucose resulted in export from thenucleoplasm (Fig. 4).

PDX-1 was further shown to shuttle between the nuclearperiphery/cytoplasm and the nucleoplasm when the con-centration of glucose in the medium was varied (Fig. 5).Thus, after incubation for 1 h in 20 mmol/l glucose,PDX-1 was present in the nucleus. After a 5-h incubationin 0.5 mmol/l glucose, PDX-1 was present at the nuclearperiphery/cytoplasm. When the concentration of glu-cose in the medium was increased to 20 mmol/l, PDX-1was localized back in the nucleoplasm within 30 min(Fig. 5).

The effects of both glucose and insulin on the movementof PDX-1 from the nuclear periphery/cytoplasm to thenucleoplasm were inhibited by the SAPK2/p38 inhibitor SB

FIG. 2. Time course and dosage effects of glucose on the intracellular localization of PDX-1. NesPDX-1 cells were preincubated for 16 h in 0.5 mmol/lglucose and then incubated for the indicated periods in 20 mmol/l glucose (A) or for 30 min in medium containing the indicated concentration ofglucose (B). Cells were then washed, fixed, and stained with an anti–PDX-1 primary antibody and fluorescein-coupled secondary antibody (green).

PDX-1 SHUTTLING IN PANCREATIC �-CELLS


203580 and the PI 3-kinase inhibitor wortmannin. Rapamy-cin, an inhibitor of p70s6k, and PD098059, an inhibitor ofthe mitogen-activated protein kinase pathway, had no ef-fect (Fig. 6). In keeping with the effects of arsenite onPDX-1 DNA binding activity and insulin promoter activity(24), the arsenite-stimulated movement of PDX-1 intothe nucleus was inhibited by SB 203580, but was unaf-fected by wortmannin. The export of PDX-1 from thenucleoplasm was inhibited by calyculin A and okadaicacid, implying that a dephosphorylation event was oc-curring (Fig. 7).

Similar results were observed for endogenous PDX-1 inMIN6 cells. Thus, glucose stimulated the movement ofPDX-1 from the nuclear periphery/cytoplasm with a simi-lar time course (Fig. 8) and dosage response to glucose(3�5 mmol/l; data not shown), as observed for the exog-enous PDX-1 in NesPDX-1 cells. In MIN6 cells, insulin andarsenite also stimulated the intranuclear transport ofPDX-1. As for NesPDX-1 cells, the effects of glucose (Fig.8) and insulin (data not shown) on the movement of PDX-1from the nuclear envelope to the nucleoplasm were inhib-ited by wortmannin and SB203580, but were unaffectedby PD 098059 and rapamycin. The arsenite-stimulatedmovement of PDX-1 to the nucleoplasm was inhibited

by SB203580, but was unaffected by wortmannin (datanot shown). PDX-1 was further shown to shuttle be-tween the nuclear periphery/cytoplasm and nucleoplasm asthe cells were transferred between stimulatory media (i.e.,that containing 20 mmol/l glucose [Fig. 8], insulin, or arse-nite [data not shown]) and nonstimulatory media (i.e.,that contained 0.5 mmol/l glucose) conditions. As forNesPDX-1 cells, okadaic acid and calyculin A (Fig. 8)inhibited the export of PDX-1 from the nucleus in MIN6cells. It should be noted that the morphology of MIN6cells is different from that of NesPDX-1 cells; the formerare smaller, more rounded, and have a tendency to grouptogether.

DISCUSSION

The findings that PDX-1 undergoes phosphorylation inresponse to glucose (22,29) and that wortmannin and SB203580 inhibit its intranuclear translocation are compati-ble with the view that movement of PDX-1 from thenuclear periphery/cytoplasm to the nucleoplasm is trig-gered by phosphorylation. In turn, phosphatase-mediateddephosphorylation of PDX-1 appears to be involved in themovement of PDX-1 from the nucleoplasm to the nuclear

FIG. 3. Effect of insulin and sodium arsenite on the intracellular localization of PDX-1. NesPDX-1 cells were preincubated for 16 h in 0.5 mmol/lglucose and then treated for 30 min in 0.5, 20, or 0.5 mmol/l glucose supplemented with 20 ng/ml insulin (A) or 0.5 mmol/l glucose supplementedwith 1 mmol/l sodium arsenite (B). Cells were then washed, fixed, and stained with an anti–PDX-1 primary antibody and fluorescein-coupledsecondary antibody (green).



periphery. PDX-1 therefore joins a growing number oftranscription factors that function by a regulated nucleartranslocation mechanism to control specific gene expres-sion. These include signal transducers and activators oftranscription, nuclear factors of Ig�B cells, nuclear factorsof activated T-cells (NFATs), p53, and steroid receptors(31). In each case, an activating signal causes a modifica-tion to the protein, which is then rapidly transported to thenucleus. PDX-1 is similar to these other examples as it

undergoes a modification (phosphorylation), but it differsin that its movement is predominantly between the nuclearperiphery and the nucleoplasm rather than the cytoplasmand the nucleus.

The nuclear/cytoplasmic transport of proteins �40kDa is tightly regulated and energy dependent (32). Manytranscription factors shuttle between the nucleus and thecytoplasm by a process that relies on their interaction withsoluble shuttling receptors that recognize specific nuclear

FIG. 4. The effect of glucose and insulin on the movement of PDX-1 from the nuclear periphery to the nucleoplasm is reversible. NesPDX-1 cellswere preincubated for 16 h in 0.5 mmol/l glucose and then for the indicated periods in 20 mmol/l glucose (A) or 20 ng/ml insulin (B). The cellswere then transferred to 0.5 mmol/l glucose and incubated for the indicated periods. Cells were then washed, fixed, and stained with ananti–PDX-1 primary antibody and fluorescein-coupled secondary antibody (green).

FIG. 5. PDX-1 shuttles between the nuclear periphery and the nucleoplasm. NesPDX-1 cells were preincubated for 16 h in medium containing 0.5mmol/l glucose and then for the indicated periods in 20 mmol/l glucose. After 1 h in 20 mmol/l glucose, the cells were incubated for 5 h in 0.5 mmol/lglucose and then for an additional 30 min in 20 mmol/l glucose. At each time point, as indicated, cells were washed, fixed, and stained with ananti–PDX-1 primary antibody and fluorescein-coupled secondary antibody (green).



localization sequences and nuclear export sequences(33). A functional nuclear localization signal within PDX-1(197RRMKWKK) has been identified (34,35). It remains tobe determined whether a nuclear export sequence is in-volved in the movement of PDX-1 from the nucleoplasm tothe nuclear periphery. Potential leucine-rich nuclear ex-port sequences within PDX-1 occur at 82LHHLPAQLALPand 178VELAVMLNL.

In keeping with the findings of a previous report (28), inwhich real-time imaging was used to study the effect ofglucose on the intracellular distribution of an myc-taggedPDX-1 construct in MIN6 cells, these results clearly dem-onstrated that in unstimulated cells, PDX-1 is localizedpredominantly around the nuclear periphery, with somestaining in the cytoplasm. This localization may be theconsequence of a physical interaction between PDX-1 andthe nuclear envelope or sequestration in a storage zone.Current studies are under way to distinguish betweenthese possibilities by investigating interactions betweenPDX-1 and purified turkey erythrocyte nuclear mem-branes. Proteins known to associate with the nuclearenvelope include cyclooxygenase 1, cyclooxygenase 2,5-lipoxygenase, 5-lipoxygenase�activating protein, leuko-triene synthase, microsomal glutathione s-transferase, andthe lamins. In the case of lamins, tethering to the nuclearenvelope depends on palmitoylation and the presence of abasic amino acid–rich nuclear localization sequence (36).

Prenylation occurs at the CaaX motif at the COOH-termi-nus of proteins. PDX-1 does not contain a consensusprenylation sequence nor is there any evidence that itundergoes prenylation. It is possible that the previouslyidentified nuclear localization sequence (34,35) alone maybe responsible for the stable association of PDX-1 with thenuclear envelope.

Acetylation has also been implicated in the tethering ofproteins to the nuclear envelope. Heterochromatin pro-tein-1 (HP-1) proteins are a family of proteins that functionas chromatin modifiers or regulators of gene expression.When microinjected into HeLa cells, recombinant forms ofHP-1 were efficiently transported into the nucleus, but enroute, they transiently associated with the nuclear enve-lope. This transient association was blocked when thecells were treated with the deacetylase inhibitors tricho-statin A or sodium butyrate (37). However, in the case ofPDX-1, acetylation is unlikely to be involved because thereis no exact match to the consensus acetylation motif(GKXXP/GKXP) (38), and neither trichostatin A nor so-dium butyrate affected the association of PDX-1 with thenuclear envelope (data not shown).

A number of kinases and phosphatases have beenimplicated in the control of nuclear shuttling. In the caseof NFATs, the Ca2�-calmodulin�dependent protein phos-phatase calcineurin dephosphorylates NFAT proteins,leading to the unmasking of nuclear localization signals

FIG. 6. The effect of kinase inhibitors on the glucose- and insulin-stimulated movement of PDX-1 from the nuclear periphery to nucleoplasm.NesPDX-1 cells were preincubated for 16 h in 0.5 mmol/l glucose. The indicated final concentrations of kinase inhibitors were added 30 minbefore further treatment. The cells were then incubated for 30 min in medium containing 20 mmol/l glucose or 20 ng/ml insulin and the in-dicated inhibitors. Cells were then washed, fixed, and stained with an anti–PDX-1 primary antibody and fluorescein-coupled secondary antibody(green).



and translocation of NFATs to the nucleus (39). Activationof c-Jun NH2-terminal kinase by overexpression ofMKK7 opposes the effects of calcium by promoting nuclearexclusion of NFAT-4 in calcineurin-stimulated baby hamp-ster kidney cells (40). Casein kinase-1 and MEKK-1inhibit NFAT-4 nuclear translocation (41), whereas glyco-gen synthase kinase-3 plays a role in the nuclear export ofNFAT-3 in neurones (42). Protein kinase-B/Akt has beenshown to phosphorylate the forkhead transcription factorFKHR and trigger its export from the nucleus (43). In thepresent study, the results with wortmannin and SB 203580lend further support to a model whereby PI 3-kinase andSAPK2/p38 regulate PDX-1 location and DNA-binding ac-tivity as well as insulin-promoter activity in response tostimulation by glucose and insulin. It is of interest there-fore that SAPK2/p38 has also been shown to phosphory-late and affect the intracellular location of NFATp in intactlymphocytes (44).

It will be important to determine whether glucosemediates its effects on nuclear shuttling of PDX-1 via se-creted insulin feeding back on the �-cell or through gen-eration of a metabolic intermediate that triggers thesignaling pathway involved. An example of metabolic reg-ulation of nuclear shuttling is provided by glucokinase.

Glucokinase lacks a nuclear localization signal and cannotenter the nucleus by itself. It enters the nucleus as part ofa complex with glucokinase regulatory protein (GRP),which does contain a nuclear localization signal. On theother hand, glucokinase contains a nuclear export sequence,and on release from binding to GRP in response to meta-bolic cues, glucokinase is exported from the nucleus by anactive process (45).

The results reported here are at variance with thosereported by Moede et al. (34). The latter researchers mon-itored the intracellular location of a green fluorescentprotein–tagged PDX-1 in transfected MIN6 �-cells andfailed to show any cycling of PDX-1 between the nucleusand cytoplasm (or nuclear periphery) in response toglucose. The reason for this discrepancy may be related tothe use of a green fluorescence protein-tagged PDX-1 ver-sus the intact expressed protein used in the present studyor to differences in cell culture conditions.

In conclusion, we demonstrated here that glucose andinsulin stimulate shuttling of PDX-1 between the nuclearperiphery and the nucleoplasm by a phosphorylation-dependent process involving PI 3-kinase, SAPK2/p38, anda nuclear phosphatase(s). The results may have importantimplications for a better understanding of the mechanisms

FIG. 7. Effect of phosphatase inhibitors on the export of PDX-1 from the nucleoplasm to the nuclear periphery. NesPDX-1 cells were preincubatedfor 16 h in 0.5 mmol/l glucose and then for 1 h in 20 mmol/l glucose. The cells were then incubated for 5 h in 0.5 mmol/l glucose in the absence(third panels) or presence (fourth panels) of calyculin A (0.1 �mol/l) or okadaic acid (0.1 �mol/l), as indicated. Cells were then washed, fixed,and stained with an anti–PDX-1 primary antibody and fluorescein-coupled secondary antibody (green).



through which nutrients and hormones regulate insulingene expression in the islets of Langerhans.

ACKNOWLEDGMENTS

This work was supported by the Wellcome Trust. L.J.E.was funded by a Biotechnology and Biological ScienceResearch Council studentship. We thank Chris Wright forproviding the anti�PDX-1 antibody.

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FIG. 8. Phosphorylation-dependent intranuclear shuttling of PDX-1 in MIN6 cells. MIN6 cells were preincubated for 16 h in 0.5 mmol/l glucose andthen incubated for the indicated periods in 20 mmol/l glucose (A). B: After a 16-h preincubation in 0.5 mmol/l glucose, the indicatedconcentrations of inhibitors were added 30 min before stimulation with 20 mmol/l glucose for 30 min. C: After 16 h in 0.5 mmol/l glucose, cellswere incubated separately in 20 mmol/l glucose for 30 min, 0.5 mmol/l glucose for 5 h and then 20 mmol/l glucose for 30 min. D: Cells werepreincubated in 0.5 mmol/l glucose and then incubated for 5 h in 0.5 mmol/l glucose in the absence (image 3) or presence (image 4) of calyculinA (0.1 �mol/l), as indicated. Cells were then washed, fixed, and stained with anti–PDX-1 primary antibody and fluorescein-coupled secondaryantibody (green).



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