rapid changes of mrna-binding protein levels following ...¼ss_2009_1237.pdf · rapid changes of...

Rapid Changes of mRNA-binding ProteinLevels following Glucose and3-Isobutyl-1-methylxanthine Stimulation ofInsulinoma INS-1 Cells*!S

Christin Suss‡, Cornelia Czupalla§¶, Christof Winter¶!, Theresia Pursche§¶,Klaus-Peter Knoch‡, Michael Schroeder¶!**, Bernard Hoflack§¶**,and Michele Solimena‡**‡‡§§¶¶

Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression pro-file of insulin-producing pancreatic !-cells mostly throughpost-transcriptional mechanisms. A thorough analysis ofthese changes, however, has not yet been performed. Bycombining two-dimensional differential gel electrophoresisand mass spectrometry, we identified 165 spots, corre-sponding to 78 proteins, whose levels significantly changeafter stimulation of the !-cell model INS-1 cells with 25 mM

glucose " 1 mM IBMX for 2 h. Changes in the expression ofselected proteins were verified by one- and two-dimensionalimmunoblotting. Most of the identified proteins are novel tar-gets of rapid regulation in !-cells. The transcription inhibitoractinomycin D failed to block changes in two-thirds of thespots, supporting their post-transcriptional regulation. Morespots changed in response to IBMX than to glucose aloneconceivably because of phosphorylation. Fourteen mRNA-binding proteins responded to stimulation, thus representingthe most prominent class of rapidly regulated proteins. Bioin-formatics analysis indicated that the mRNA 5"- and 3"-un-translated regions of 22 regulated proteins contain potentialbinding sites for polypyrimidine tract-binding protein 1,which promotes mRNA stability and translation in stimu-lated !-cells. Overall our findings support the idea thatmRNA-binding proteins play a major role in rapid adaptivechanges in insulin-producing cells following their stimula-tion with glucose and cAMP-elevating agents. Molecular& Cellular Proteomics 8:393–408, 2009.

Pancreatic !-cells store insulin within secretory granules(SGs).1 Hyperglycemia triggers the fusion of SGs with the

plasma membrane and the extracellular release of insulin,which in turn lowers glycemia by promoting glucose uptakeinto cells. In addition to insulin secretion, glucose promotesthe biogenesis of SGs by enhancing the synthesis of theircomponents, including preproinsulin, prohormone converta-ses 1/3 (PC1/3) (1, 2) and 2 (PC2) (2), chromogranin A (3), andICA512 (4). This rapid up-regulation of SG biogenesis islargely attributed to post-transcriptional mechanisms be-cause it is insensitive to the transcription blocker actinomycinD (AmD) (5–9). These post-transcriptional mechanisms in-clude reduced degradation of mRNAs encoding SG compo-nents (10–13), recruitment of these mRNAs from the cytosolto the endoplasmic reticulum (14), and increased translation(15–18). Elevation of cAMP levels also increases stability andtranslation of mRNAs encoding insulin SG proteins (12, 17). Akey factor for glucose/cAMP-mediated up-regulation ofmRNA stability and translation is polypyrimidine tract-bindingprotein 1 (PTBP1), formerly known as PTB1 or heterogeneousnuclear ribonucleoprotein I (hnRNP I) (11, 12, 19, 20). PTBP1was identified in 1989 based on its ability to bind polypyrim-idine tracts of pre-mRNAs and has multiple functions (21). Inthe nucleus it regulates pre-mRNA splicing (22–25) andpoly(A) site cleavage (26). In the cytoplasm, it has been shownto regulate cap-independent translation through the internalribosome entry site (27–29), mRNA localization (30, 31) andthe stability of mRNAs for CD154 (32, 33), inducible nitric-oxide synthase (34), insulin, and other SG proteins (11, 12, 19,35). Alternative splicing of the Ptbp1 transcript generatesdifferent isoforms (36), the largest of which measures 59 kDa(20) and contains four RNA recognition domains.

In this study we examined the proteomic changes occurringshortly after stimulation of INS-1 cells, an in vitro model of!-cells, with glucose and/or the cAMP-elevating agent

From the ‡Experimental Diabetology, Departments of §Proteomics,!Bioinformatics, and ‡‡Medicine III, ¶Biotechnology Center, **Centerfor Regenerative Therapies, Dresden University of Technology, and§§Max Planck Institute of Molecular Cell Biology and Genetics,Dresden 01307, Germany

Received, April 9, 2008, and in revised form, September 25, 2008Published, MCP Papers in Press, October 14, 2008, DOI 10.1074/

mcp.M800157-MCP2001 The abbreviations used are: SG, secretory granule; IBMX,

3-isobutyl-1-methylxanthine; UTR, untranslated region; PTBP1, poly-pyrimidine tract-binding protein 1; PC, prohormone convertase; AmD,

actinomycin D; hnRNP, heterogeneous nuclear ribonucleoprotein;2-D, two-dimensional; RNAi, RNA interference; 2-DE, 2-D gel elec-trophoresis; PANTHER, Protein Analysis through Evolutionary Rela-tionships; KH, K homology; BVA, Biological Variation Analysis; PAI,plasminogen activator inhibitor; CPE, carboxypeptidase E; CGA,chromogranin A; CBF-A, CArG binding factor A.

Research

© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 8.3 393This paper is available on line at http://www.mcponline.org

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/DC2http://www.mcponline.org/cgi/content/full/M800157-MCP200Supplemental Material can be found at:

http://www.mcponline.org/cgi/content/full/M800157-MCP200/DC2

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3-isobutyl-1-methylxanthine (IBMX). To this aim, protein sam-ples were analyzed by mass spectrometry following their sep-aration by fluorescent two-dimensional (2-D) DIGE (37–39).This method facilitates quantitative comparisons of samplesby labeling proteins prior to 2-D electrophoresis with dyes thatdiffer in fluorescence spectra, such as Cy3 and Cy5.

2-D DIGE routinely allows the separation of !2,000 and!1,600 spots in the range of pH 4–7 and pH 6–9, respec-tively, for a total number of !3,000 distinct spots. For com-parison, 800–2,500 spots over the wider range of pH 3–10 aretypically resolved in proteomics studies on isolated islets thatrely on non-fluorescent dyes for protein staining (40–42). 2-DDIGE is also preferable to other procedures such as silverstaining because of the greater linearity, sensitivity (0.025 ng),and wide dynamic range of the fluorescence signal (39). Usingthis approach, we identified mRNA-binding proteins as a ma-jor class of molecules whose expression pattern rapidlychanges in response to glucose and IBMX stimulation.

MATERIALS AND METHODS

Cell Culture and Stimulation of INS-1 Cells

Rat insulinoma INS-1 cells were grown as described previously(43). Cells in 75-cm2 flasks were preincubated in resting medium (15mM HEPES, pH 7.4, 5 mM KCl, 120 mM NaCl, 24 mM NaHCO3, 1 mM

MgCl2, 2 mM CaCl2, 0 mM glucose, 1 mg/ml ovalbumin) for 1 h beforestimulation for 2 h by addition of fresh medium containing 25 mM

glucose and/or 1 mM IBMX (Sigma). Transcription was blocked using5 "g/ml actinomycin D (AppliChem, Darmstadt, Germany), which wasadded to both the resting and stimulating media as indicated.

Transfection of INS-1 Cells

The cDNA of rat PTBP1 in INS-1 cells was cloned into pcDNA3.1(Invitrogen) as described previously (12). INS-1 cells were transientlytransfected with cDNA vectors using a Laboratory PulseAgile Elec-troporation System (Model PA-3000, Cyto Pulse Sciences, Inc., GlenBurnie, MD). Cells were harvested by trypsinization of a 175-cm2

confluent flask (sufficient for "4 transfections) followed by centrifu-gation (500 # g, 5 min, room temperature). The cell pellet wasresuspended in 250 "l of Cytoporation Medium (Cyto Pulse Sciences,Inc.). For overexpression of PTBP1, 4 "g of pcDNA3.1-PTBP1(PTBP1-V5) and pcDNA3.1 (empty control vector) were added beforecells were transferred into an electroporation cuvette (Eppendorf AG,Hamburg, Germany; 4-mm gap). The electroporation program wasperformed twice in an interval of 1.5 min (560 V; 0.2-"s pulse width;0.2-s pulse interval four times). After electroporation, 600 "l of INS-1cell medium (43) was added, and cells were seeded in two to threewells of a 6-well plate covered with sterile coverslips. Seventy-twohours after transfection, 10 mM sodium butyrate (Sigma) was addedto fresh medium, and cells were harvested 24 h later.

RNA Interference (RNAi)

Knockdown of rat Ptbp1 mRNA in INS-1 cells by RNAi was per-formed using the pGENEClip U1 Hairpin vector (Promega, Madison,WI). The hairpin 5$-TCTCGTCATGGAAGAGTGTAAATTTCAAGA-GAATTTACACTCTTCCATGACCT-3$ was designed as describedpreviously (12). Four micrograms of the pGENEClip-PTBP1 or thescrambled pGENEClip control (5$-TCTCGTGAAATAGAGTGTAG-GAATTCAAGAGATTCCTACACTCTATTTCACCT-3$) vectors were in-dependently electroporated in INS-1 cells as described above.

Cell Extract Preparation

For 2-D gel electrophoresis (2-DE), cells were washed poststimu-lation three times with ice-cold PBS (Dulbecco’s PBS, 1# withoutCa2% and Mg2%) (PAA Laboratories GmbH, Pasching, Austria) andtwice with ice-cold sucrose buffer (250 mM sucrose, 10 mM Tris-HCl,pH 7.5). Cells were scraped in sucrose buffer, pelleted by centrifuga-tion (500 # g, 5 min, 4 °C), and then lysed in 7 M urea, 2 M thiourea,4% (w/v) CHAPS, 30 mM Tris-HCl, pH 9.3, 1# protease inhibitormixture (GE Healthcare). Cell lysates were further passed throughneedles of decreasing sizes (18, 22, and 25 gauge). Interfering DNAwas broken by sonication (5 # 10 s, 2-min break). The lysates werethen centrifuged at 15,800 # g for 10 min at 4 °C to remove insolublematerial. Protein concentration was measured using the RC DC pro-tein assay (Bio-Rad). Proteins were used for 2-DE or shock frozen inliquid nitrogen and stored at &80 °C.

For other applications, cells were washed with ice-cold Dulbecco’sPBS, scraped in PBS, pelleted by centrifugation (500 # g, 5 min, 4 °C)and then lysed in 20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA,1% (v/v) Triton X-100, 1# protease inhibitor mixture P8340 (Sigma),1# phosphatase inhibitor mixture set II (Merck). Cell lysates wereincubated for 10–30 min on ice and then centrifuged at 23,800 # g for10 min at 4 °C to remove insoluble material. Protein concentrationwas measured using the BCA protein assay kit (Pierce). Proteins werefrozen and stored at &80 °C or directly used for SDS gel electro-phoresis after adding 6# SDS sample buffer (350 mM Tris-HCl, pH6.8, 36% (v/v) glycerol, 12% (w/v) SDS, 600 mM DTT, 0.12% (w/v)bromphenol blue).

2-D DIGE

The minimal labeling of protein samples with CyDye DIGE FluorsCy2, Cy3, and Cy5 (EttanTM DIGE, GE Healthcare) was performedaccording to the manufacturer’s instructions. Briefly, 50 "g of proteinextracts from resting and stimulated cells was labeled with 200 pmolof Cy3 and Cy5, respectively. All experiments were independentlyrepeated six times using different samples. To ensure the samelabeling efficiency, three of six experiments were labeled in the op-posite manner. Fifty micrograms of protein resulting from the mixtureof resting and stimulated cell extracts in an equal ratio was labeled inparallel with 200 pmol of Cy2 as an internal control for normalization(38, 39, 44). After labeling, the proteins were reduced with 50 mM DTT,and the differentially labeled samples were mixed and supplementedwith 0.5% (v/v) ampholytes (Bio-Lyte 3/10 ampholyte, 40% (Bio-Rad)for pH 4–7; IPG Buffer pH 6–11 (GE Healthcare) for pH 6–9). To betterresolve protein spots and minimize their chance of co-migration, weused the longest available IPG strips (24 cm) with overlapping narrowrange pH (pH 4–7 and 6–9) (45). For separation on ImmobilineTM

DryStrip Gels pH 4–7 (IPG strips; 24 cm, GE Healthcare) labeledsamples were brought to a final volume of 450 "l with rehydrationbuffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 50 mM DTT) andpassively loaded during rehydration of the strips. For separation onIPG strips pH 6–9, labeled samples were instead brought to a finalvolume of 80 "l with rehydration buffer and “cup-loaded” on stripsalready rehydrated in 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 5%(v/v) glycerol, 0.5% (v/v) IPG Buffer pH 6–11, 12 "l/ml DeStreak (GEHealthcare)). First dimension IEF was performed using the Ettan IPG-phor IEF IITM unit (GE Healthcare). Focusing conditions were asfollows: pH 4–7: (i) 0.5 h, linear 0–150 V; (ii) 1.5 h, 150 V; (iii) 1 h, 250V; (iv) 4 h, linear 250–1,000 V; (v) 1.5 h, linear 1,000–5,000 V; (vi) 2 h,linear 5,000–10,000 V; and (vii) 8 h, 10,000 V; pH 6–9: (i) 4 h, 150 V;(ii) 3 h, 300 V; (iii) 6 h, linear 300–10,000 V; and (iv) 7 h, 10,000 V at20 °C. According to the pH, a maximum of 50 "A (pH 4–7) or 30 "A(pH 6–9) was applied to the IPG strip. After IEF, IPG strips were firstsoaked with equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea,

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30% (v/v) glycerol, 2% (w/v) SDS) supplemented with 20 mg/ml DTTfor 20 min and then in new equilibration buffer supplemented with 25mg/ml iodoacetamide for additional 20 min. The equilibrated IPGstrips were transferred on the top of 10% polyacrylamide gels for thesecond dimension, which was run at 1 watt/gel at 20 °C using anEttan DALTsix electrophoresis unit (GE Healthcare).

Image Analysis

DIGE-labeled gels were scanned with a Typhoon 9410 VariableMode Imager (GE Healthcare) using excitation/emission wavelengthsspecific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670nm). For statistical analysis, DeCyder Differential Analysis Softwarewith the Biological Variation Analysis (BVA) module version 5.0 (GEHealthcare) was used. Spots were automatically detected, matched,and normalized to the internal standard labeled with Cy2. Afterwardsspots were manually checked to guarantee correct matching acrossthe gels. The 18 images from the corresponding six gels for eachcondition and pH range were used to calculate the average ratio. Onlyspots that were detected in at least four of six gels and showed athreshold limit of 1.5-fold difference and a Student’s t test of 99%(p # 0.01) were regarded to differ significantly. For each comparisonof condition sets a separate BVA setup was used.

Protein Identification by MS

For MS, 1–1.5 mg of protein extracts was separated by 2-DE, andproteins were stained with colloidal Coomassie Brilliant Blue G-250(Bio-Rad) as described by Kang et al. (46). Protein spots were excisedfrom gels, processed, and digested with 50–100 ng of trypsin (Pro-mega), and peptides were extracted as described previously (47).MALDI-TOF spectra were obtained using an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in thereflectron mode and $-cyano-4-hydroxycinnamic acid as matrix.MALDI-TOF/TOF measurements were carried out in the LIFT mode.Peptide mass fingerprint spectra were internally calibrated with tryp-sin autolysis peaks. All spectra were processed, and peak lists weregenerated using flexAnalysis software (version 2.2) and the followingparameters: signal-to-noise threshold of 5 and exclusion of contam-inant ion masses as given in supplemental Table 1. Peptide massmapping and fragment ion analysis were performed using the on-lineavailable Mascot version 2.2 (Matrix Sciences Ltd., London, UK) (48).The following search criteria were used: (i) taxonomy, Rattus norve-gicus; (ii) enzyme specificity, trypsin; (iii) mass accuracy, 50 ppm and0.5 Da for peptide mass fingerprinting and fragment ion analysis,respectively; (iv) fixed and variable modifications, cysteine carbam-idomethylation and methionine oxidation, respectively; (v) maximumof one missed cleavage site; and (vi) databases, National Center forBiotechnology Information (NCBI) version 20080221 (6,122,577 se-quences; 2,096,230,148 residues; Rattus: 68,243 sequences; Feb-ruary 27–29, 2008) and NCBI version 20080229 (6,251,073 se-quences; 2,135,462,495 residues; Rattus: 68246 sequences; March3–4, 2008) (as indicated). Proteins were considered as identified ifthe peptide mass fingerprint exhibited a significant Mascot score(score ! 61; p ' 0.05).

Western Blot

Protein extracts separated by 2-DE or one-dimensional SDS-PAGEwere transferred onto nitrocellulose membrane. Proteins were immuno-blotted with the following antibodies: mouse monoclonal anti-PTBP1(Zymed Laboratories Inc., South San Francisco, CA); rabbit polyclonalanti-phospho-PTBP1 (12); rabbit polyclonal anti-hnRNP K (H-300) andrabbit polyclonal lamin A/C (H-110) (Santa Cruz Biotechnology Inc.,Santa Cruz, CA); mouse monoclonal anti-hnRNP A1 (ab50949) and

rabbit polyclonal anti-hnRNP A3 (ab10685) (Abcam, Cambridge, UK);mouse monoclonal anti-chromogranin A (CGA) (BD Biosciences); rabbitpolyclonal anti-PC1/3, anti-PC2, and anti-carboxypeptidase E (CPE)(Millipore, Billerica, MA); mouse monoclonal anti-PAI-RBP1 and mousemonoclonal anti-PCBP2 (M07) (Abnova Corp., Taipei, Taiwan); mousemonoclonal anti-ICA512 (49); mouse monoclonal anti-%-tubulin (Sigma);polyclonal rabbit anti-N-CBF-A (CArG binding factor-A) (a gift from T.Leanderson); and polyclonal rabbit anti-Staufen2 (a gift from M. Kiebler).Blots were incubated with horseradish peroxidase-conjugated goat an-ti-mouse or goat anti-rabbit IgG (Bio-Rad), developed with SuperSignalWest Pico Chemiluminescent Substrate or SuperSignal West FemtoMaximum Sensitivity Substrate (Pierce) according to the manufacturer’sinstructions, and visualized using a LAS-3000 imaging system (Fuji FilmCo. Ltd., Tokyo, Japan). Alternatively the ECL Plex Western BlottingSystem (GE Healthcare) containing secondary antibodies conjugatedwith CyDyes was used for protein detection. The secondary antibodysignal was detected by scanning the membrane with a Typhoon 9410Variable Mode Imager.

Computational Prediction of PTBP1 Binding Sites

Collection of Sequences—Regulated proteins of rat INS-1 cellswere mapped to their NCBI Entrez Gene identifiers. Human andmouse orthologs were obtained using the InParanoid database (50).NCBI Entrez Nucleotide was then queried for available mRNA se-quences associated with the rat, mouse, and human genes. 5$- and3$-untranslated regions (UTRs) as defined in Entrez Nucleotide wereseparated, and the 3$-UTR poly(A) tail was removed. On average,seven 5$-UTR and eight 3$-UTR sequences were retrieved per proteinand species. The average length of the 5$- and 3$-UTR (withoutpoly(A) tail) was 144 and 654 nucleotides, respectively.

Conservation—To identify conserved regions, a multiple sequencealignment of rat, mouse, and human sequences was constructed foreach UTR of a regulated protein using the multiple alignment programMAFFT with the L-INS-i iterative refinement parameter (51). A con-sensus sequence was calculated from residues that were at least50% conserved in the multiple sequence alignment. For each con-served position of the alignment, the number of species showing theconserved amino acid was recorded. Positions conserved in only onespecies were excluded from further analyses.

Search for PTBP1 Binding Sites—Each UTR was screened for thepresence of the motif CYYYYCYYYYYG, corresponding to the con-sensus for PTBP1 binding according to Tillmar et al. (19). Hits werecounted whenever a match was found considering zero, one, or twomismatching nucleotides. Overlapping hits were counted separately,allowing a continuous binding region to give rise to several hits.

Significance of Detected Binding Sites—To assess the significance ofhits matching the PTBP1 binding motif, we calculated the p value foreach binding site found in a UTR. To this end, each UTR sequence wasshuffled 100,000 times and subsequently searched for the bindingmotif. Shuffling ensured that the nucleotide composition was preserved.The frequency of observing at least the same number of hits in therandom sequences as in the original sequence was recorded. The pvalue was calculated as this frequency divided by 100,000. It resemblesthe probability that the same number of hits observed occurs bychance. Hits with a p value '0.01 were considered to be significant.

Secondary Structure Assessment—Because structural studiesshow that PTBP1 RNA recognition motif domains bind single-strandedRNA (52), we predicted the secondary structure of consensus UTRsequences using RNAfold (53), which calculates the minimum freeenergy structure of a given RNA sequence. We mapped binding sitehits onto the predicted secondary structure. A binding site wascounted as single-stranded if more than half of its nucleotides werenon-paired in the minimum free energy structure. Significant hits weremanually checked for their secondary structure.


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RESULTS

Analysis of Spots That Rapidly Change Expression uponStimulation—To identify the potentially large set of proteinsregulated shortly after stimulation of INS-1 cells with glucoseand/or IBMX, we separated and analyzed protein samples by2-D DIGE. Among the four different conditions that weretested (25 mM glucose, 1 mM IBMX, 25 mM glucose % 1 mM

IBMX, and 25 mM glucose % 1 mM IBMX % 5 "g/ml AmD), wedetected 2,224 ( 182 and 1,694 ( 99 spots within the rangesof pH 4–7 and 6–9, respectively (supplemental Table 2). Onlyspots that were detected in at least four of six gels andshowed an average increase or decrease &1.5-fold and a#0.01 t test were considered to differ significantly.

First we compared cell extracts of INS-1 cells incubated for2 h in either resting (0 mM glucose) or stimulating (25 mM

glucose % 1 mM IBMX) buffer. Upon stimulation, 33 spotsincreased, and 42 decreased (Fig. 1, A and B). Next wecompared the profiles of cells kept at rest or stimulated witheither 25 mM glucose or 1 mM IBMX. In cells stimulated with 25mM glucose, eight spots increased, and five decreased (sup-plemental Fig. 1, A and D), whereas upon stimulation with 1mM IBMX, 40 spots increased, and 37 decreased (supplemen-tal Fig. 1, B and E). Four (two up-regulated and two down-regulated) of the 13 glucose-responsive spots did not changein response to IBMX. Of the remaining nine spots, six wereup-regulated and three were down-regulated in IBMX-treatedcells. Thus, the number of regulated spots (33 % 42 ) 75) inINS-1 cells co-stimulated with glucose and IBMX was lower

than the sum of the spots independently regulated by glucoseand IBMX (13 % 78 & 9 ) 82). This discrepancy is evengreater considering that only 39 of these 82 spots (47.6%)also changed upon co-stimulation with glucose and IBMX,thus indicating that synergism of glucose and cAMP is onlypartial.

To detect changes among proteins with low molecularweight, samples were also separated on 15% polyacrylamidegels. Only four spots with low molecular weight increased incells stimulated with 25 mM glucose % 1 mM IBMX, whereasfive spots decreased (data not shown).

To determine whether the rapid proteomic changes ob-served in response to stimulation with 25 mM glucose % 1 mM

IBMX depend on post-transcriptional mechanisms, we com-pared the DIGE profiles of cells co-stimulated in the presenceor absence of 5 "g/ml AmD (supplemental Fig. 1, C and F). Inthe presence of AmD, 20 of 33 (61%) spots still increased, and28 of 42 spots (67%) decreased (Fig. 2A, overlapped area ofgreen and red rectangles), indicating that their regulation oc-curs because of post-transcriptional mechanisms. NotablyAmD treatment of co-stimulated cells correlated with an in-crease in 18 spots and a decrease in 21 spots that wereunresponsive to glucose and/or IBMX stimulation alone (Fig.2A, non-overlapped area of red rectangle).

Identification of Rapidly Regulated Proteins by Mass Spec-trometry—MALDI-TOF-MS analysis was performed to identifythe spots regulated upon stimulation with 25 mM glucose % 1mM IBMX in the presence or absence of AmD (Fig. 1, C and D,

FIG. 1. 2-DE proteomic profile ofINS-1 cells after stimulation with 25mM glucose and 1 mM IBMX for 2 h.The 2-DE proteomic profiles of INS-1were compared using the DIGE technol-ogy (A and B). Shown are representativegray images of stimulated cell extracts ofsix independent experiments. Onlyspots detected in &4 gels and having anaverage &1.5-fold increase or decreaseand a #0.01 t test were regarded todiffer significantly. Using this threshold,18 (pH 4–7) and 15 (pH 6–9) spots weresignificantly increased (red spots) uponstimulation with 25 mM glucose % 1 mM

IBMX, whereas 24 (pH 4–7) and 18 (pH6–9) spots were significantly decreased(green spots). Representative images ofpreparative gels containing "1.5 mg ofloaded protein extracts stained with col-loidal Coomassie Brilliant Blue G-250are shown (C and D). IEF was performedon pH 4–7 (A and C) and 6–9 (B and D)IPG strips. The second dimension wasseparated by 10% SDS-PAGE.



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FIG. 2. Functional annotation of identified proteins. A, number of rapidly up- (1) or down-regulated (2) spots by stimulation with 25 mM

glucose (Glc) % 1 mM IBMX (green rectangle), 25 mM glucose (blue rectangle), 1 mM IBMX (yellow rectangle), and 25 mM glucose % 1 mM IBMX %5 "g/ml AmD (red rectangle). The number of spots regulated by multiple conditions is represented in overlapped areas. B and C, number ofidentified genes that were differentially expressed in one or more of the four stimulation conditions separated according to their molecular function (B)or biological process (C) annotation in the PANTHER database. Fold enrichments were calculated by comparing the number of occurrences of a termin proteins found in this study with the number of occurrences of that term in the PANTHER database. To assess significance of the enrichment,p valueswere calculated using the hypergeometric distribution. The enrichment and its significance are shown below the diagram. The significance isindicated as *** for p # 0.001, ** for 0.001 ' p # 0.01, and * for 0.01 ' p # 0.05 (for more detailed information see supplemental Table 7).



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supplemental Fig. 2, and supplemental Table 3). We identified63 (84%) of the 75 spots whose levels changed in the ab-sence of AmD (see Table I) and 70 (72%) of the 97 spotswhose expression changed in the presence of AmD. Many ofthe unidentified spots had a basic isoelectric point, makingdifficult their separation and isolation when preparative pro-tein samples were loaded on the IPG strips.

Proteins recognized by MS belonged to 22 molecular func-tion categories in the PANTHER database (54) (see Fig. 2, Band C, and supplemental Tables 4–7 for detailed information).Interestingly the largest class of regulated proteins, corre-sponding to 26.9%, was represented by nucleic acid-bindingproteins (Fig. 2B), including the following 14 mRNA-bindingproteins: CUGBP1, hnRNP A1, hnRNP A3, hnRNP A/B (CBF-A), hnRNP E2 (PCBP2), hnRNP K, hnRNP L, hnRNP H1,KH-type splicing regulatory protein, PAI-RBP1, PRP19/PSO4pre-mRNA processing factor 19, RNA binding motif protein8A, Srp20 (splicing factor arginine/serine-rich 3), andStaufen2. According to their biological process annotation inthe PANTHER database, 27 (34.6%) of the regulated proteinsare involved in nucleoside, nucleotide, and nucleic acid me-tabolism, whereas 19 (24.4%) are implicated in protein me-tabolism and modification (Fig. 2C). A drawback of 2-DE is thelack of detection of low abundance proteins and the poorresolution of membrane and large hydrophobic proteins (55).Thus, we cannot rule out that the identification of nucleicacid-binding proteins as a major class of regulated factors isbecause of their preferential detection by the selected methodrelative to other protein classes. Changes in 10 mRNA-binding proteins (CUGBP1, hnRNP A/B (CBF-A), hnRNP K,hnRNP L, hnRNP H1, KH-type splicing regulatory protein,PAI-RBP1, RNA binding motif protein 8A, Srp20, andStaufen2) were not inhibited by AmD. The changed expres-sion patterns of hnRNP K, PAI-RBP1, PCBP2, and lamin A,another protein identified in our screen, were verified by im-munoblotting of 2-D gels (Fig. 3). The immunoreactive patternof hnRNP K and PAI-RBP1 correlated well with the one de-tected by 2-D DIGE (equivalent spots are indicated by thesame numbers in Figs. 1 and 3). In the case of PCBP2 andlamin A, the immunoblotting pattern showed a shift similar tothe one visualized by 2-D DIGE, but an accurate match of thespots detected with the two procedures was not possible.Additionally analysis by one-dimensional gel electrophoresisshowed increased levels or an upward shift suggestive ofphosphorylation in the case of hnRNP A1, hnRNP A3, the52-kDa isoform of Staufen2, and lamin A (Fig. 4).

We have shown previously that stimulation of INS-1 cellswith IBMX induces the cAMP-dependent protein kinase A-de-pendent phosphorylation of PTBP1 on serine 16 (12). Havinga predicted pI of 9.17, PTBP1 migrates beyond the range ofproper separation by the pH 6–9 IPG strips. Thus, its absencein the list of regulated spots identified by 2-D DIGE/MS wasnot unexpected. Its change upon stimulation, however, wasvisualized by 2-DE immunoblotting with an antibody that spe-

cifically binds to PTBP1 phosphorylated on serine 16 (Fig. 3,I and J).

Insulin, ICA512, PC1/3, PC2, and chromogranin A are SGcomponents whose increased expression in cells stimulatedwith glucose/IBMX is regulated by PTBP1 (11, 12) but thatwere not identified in our screen. These proteins, however, areeither too small to be detected (insulin) or too hydrophobic tobe properly separated (all other cases) by conventional 2-DE.Nonetheless 2-DE immunoblotting confirmed that in cellsstimulated with glucose % IBMX, the levels of chromogranin A(Fig. 5, A and B), PC1 (Fig. 5, C and D), and PC2 (Fig. 5, E andF) increased, whereas the transmembrane fragment ofICA512 (ICA512-TMF) (Fig. 5, G and H) and carboxypeptidaseE (CPE) (Fig. 5, I and J) decreased either because of proteo-lytic cleavage (4, 56) or secretion (3), respectively.

Bioinformatics Prediction of PTBP1 Binding Motifs in Reg-ulated Proteins—Next we investigated whether mRNAs en-coding proteins regulated by glucose and IBMX contain po-tential PTBP1 binding sites in their 5$- and 3$-UTRs.Conservation of these putative binding sites among rat,mouse, and human (Table II) or just between rat and eithermouse or human (Table III) was considered a necessary cri-terion for significance. Thirteen of the identified proteins in-cluded fully conserved potential PTBP1 sites in mRNA UTRsfrom all three species. By lowering the conservation criterionto include regions conserved in at least two species, we foundputative PTBP1 binding sites in mRNA UTRs of 22 proteins inour list (Table III), including PAI-RBP1, PCBP2, and Staufen2.The changed expression of PAI-RBP1 and Staufen2, but notPCBP2, was most likely because of post-translational mech-anisms because of its insensitivity to AmD. Eighteen of theseregulated proteins, including Staufen2 but not PAI-RBP1,were identified as a single spot by 2-D DIGE, suggesting thattheir changed level was not the result of phosphorylation. Inmost cases, at least one of the putative PTBP1 binding sitesin each of these mRNA UTRs was predicted to be locatedwithin single-stranded regions by secondary structure analy-ses (supplemental Fig. 3). This is interesting in view of theprevailing data suggesting that PTBP1 preferentially bindssingle-stranded RNA; albeit this opinion has recently beenchallenged (57). For insulin, ICA512, PC1/3, PC2, and chro-mogranin A, which are regulated by PTBP1 through its bind-ing to their 3$- and/or 5$-UTRs (11, 19),2 our method confirmspotential PTBP1 binding sites fully conserved among rat,mouse, and human with at least one potential binding site ina single-stranded region (data not shown).

Validation of PTBP1 Binding Predictions—To test whetherPAI-RBP1, Staufen2, and PCBP2 are regulated by PTBP1, weanalyzed their expression following Ptbp1 knockdown byRNAi or overexpression of PTBP1 tagged at its C terminuswith a V5 epitope (PTBP1-V5). After Ptbp1 knockdown, only

2 K.-P. Knoch, H. Schneider, and M. Solimena, unpublishedobservation.



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TABLE ISummary of proteins with increased and decreased levels after stimulation with 25 mM glucose and 1 mM IBMX

Proteins were identified by MALDI-TOF-MS. Glc, glucose; ND, spots were not detected in these conditions; —, spots were not significantlychanged in these conditions; FUSE, far upstream element.

Spot no.a Accessionno. (gi) Protein

Expression changeb

GenesymbolGlc %

IBMX Glc IBMX Glc %IBMX % AmD

pH 4–7, increased1 16923998 Heterogeneous nuclear ribonucleoprotein K 3.57 — 3.22 3.07 Hnrpk

51592098 Staufen, RNA-binding protein, homolog 2isoform LS

Stau2

11560133 Tubulin, $1 Tuba12 16758782 Lamin B1 3.29 — 3.48 1.98 Lmnb13 56799436 Chromobox homolog 3 3.22 — — 2.12 Cbx34 8393696 Stathmin 1 2.94 — — — Stmn15 16923998 Heterogeneous nuclear ribonucleoprotein K 2.60 — — 2.28 Hnrpk

149040328 Internexin, $ Inexa6 16758782 Lamin B1 2.56 — 3.44 5.81 Lmnb18 149040328 Internexin, $ 2.25 1.73 2.10 1.81 Inexa9 62078893 Ubiquitin-activating enzyme E1 2.01 1.69 1.66 1.91 Ube110 157816973 Protein phosphatase 1, regulatory (inhibitor)

subunit 81.90 1.50 3.88 2.00 Ppp1r8

11 16923998 Heterogeneous nuclear ribonucleoprotein K 1.82 — — 1.58 Hnrpk12 13592133 !-Actin 1.71 1.72 1.88 — Actb

14010837 NSFL1 (p97) cofactor (p47) Nsfl1c13 56971386 Gars protein (glycyl-tRNA synthetase) 1.67 — — 1.76 Gars

1346413 Lamin A Lmna14 16923998 Heterogeneous nuclear ribonucleoprotein K 1.63 — — — Hnrpk15 13929082 Pyridoxal (pyridoxine, vitamin B6) kinase 1.61 — — 2.19 Pdxk16 1346413 Lamin A 1.57 — — 1.65 Lmna17 16923998 Heterogeneous nuclear ribonucleoprotein K 1.56 — — — Hnrpk18 149053241 Rabaptin, RAB GTPase binding effector

protein 11.50 — — 1.63 Rabep1

pH 4–7, decreased19 157786744 Dihydropyrimidinase-related protein 2 &1.51 — — &1.93 Dpysl220 71361625 Adaptin ear-binding clathrin-associated protein &1.52 &1.55 &2.22 — Necap121 16923998 Heterogeneous nuclear ribonucleoprotein K &1.54 — — &1.94 Hnrpk22 149040328 Internexin, $ &1.58 — — &1.52 Inexa24 74095899 PRP19/PSO4 pre-mRNA processing factor 19

homolog&1.60 — — — Prpf19

25 16923998 Heterogeneous nuclear ribonucleoprotein K &1.65 — — &2.15 Hnrpk26 16923998 Heterogeneous nuclear ribonucleoprotein K &1.66 — — &2.47 Hnrpk27 58865398 Leucine aminopeptidase 3 &1.66 &1.52 — &1.80 Lap328 8393855 Nucleoporin 54 kDa &1.75 — &1.97 &1.74 Nup5429 149040328 Internexin, $ &1.78 — — — Inexa30 58865550 N-myc downstream regulated gene 1 &1.79 — — — Ndrg131 1346413 Lamin A &1.80 — &1.97 &2.57 Lmna32 20302113 Stress-induced-phosphoprotein 1 &1.83 — &1.95 &1.76 Stip133 11693176 Acidic ribosomal phosphoprotein P0 &1.92 — &2.52 &2.75 Arbp34 203941 Vitamin D-binding protein precursor &2.08 — — — Gc

27465535 Tubulin, !5 Tubb2b35 8393696 Stathmin 1 &2.23 — — — Stmn136 13592133 !-Actin &2.31 &1.97 &2.93 &3.02 Actb

14010837 NSFL1 (p97) cofactor (p47) Nsfl1c38 56799436 Chromobox homolog 3 &2.72 — &2.62 &2.62 Cbx340 56799436 Chromobox homolog 3 &3.38 — &3.91 &3.23 Cbx3

pH 6–9, increased43 206205 M2 pyruvate kinase 2.90 — — 1.93 Pkm244 40538742 ATP synthase, H%-transporting, mitochondrial

F1 complex, $ subunit, isoform 12.57 1.99 — 2.09 Atp5a1

45 62825891 Phosphofructokinase, muscle 2.42 — 3.44 2.94 Pfkm46 13592065 Ribosomal protein S6 kinase polypeptide 1 2.38 — 1.94 — Rps6ka147 13592065 Ribosomal protein S6 kinase polypeptide 1 2.26 — 1.89 1.62 Rps6ka148 112984344 Adaptor-related protein complex AP-1,

" subunit 12.09 2.78 — — Ap1m1

49 140971918 Heterogeneous nuclear ribonucleoprotein A/B 2.02 — 2.33 1.73 Hnrpab157816973 Protein phosphatase 1, regulatory (inhibitor)

subunit 8Ppp1r8



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the levels of the 52-kDa isoform of Staufen2 were modestlyincreased, whereas they did not change upon PTBP1-V5overexpression. The levels of the other Staufen2 isoforms,PAI-RBP1, and PCBP2 did not significantly differ in eithercondition (Fig. 6). In view of these findings, the possibility thatPAI-RBP1, Staufen2, and PCBP2 are regulated by PTBP1seems unlikely.

DISCUSSION

Previous studies have shown that stimulation of !-cells andinsulinoma cells rapidly increases the expression of many SGgenes primarily by activating post-transcriptional mecha-nisms (11, 15–17). However, a comprehensive and unbiasedproteomics analysis of these changes has not yet been re-ported (58). For this reason we have compared the proteomic

TABLE I—continued

Spot no.a Accessionno. (gi) Protein

Expression changeb

GenesymbolGlc %

IBMX Glc IBMX Glc %IBMX % AmD

50 34327779 Heterogeneous nuclear ribonucleoprotein A3isoform c

1.93 — 3.48 — Hnrpa3

51 52783155 Plasminogen activator inhibitor 1 RNA-bindingprotein

1.82 — — — Serbp1

52 40538860 Aconitase 2, mitochondrial 1.78 — — 2.44 Aco253 6981602 Syntaxin-binding protein 1 1.70 — — — Stxbp154 8394162 Aconitase 1 1.65 — 1.57 — Aco155 162287306 ATP-citrate lyase isoform 1 1.56 — 1.98 — Acly56 8393418 Glyceraldehyde-3-phosphate dehydrogenase 1.55 — 2.16 — Gapdh

34327779 Heterogeneous nuclear ribonucleoprotein A3isoform c

Hnrpa3

57 13592065 Ribosomal protein S6 kinase polypeptide 1 1.55 — — — Rps6ka1pH 6–9, decreased

59 66911068 Pcbp2 protein &1.51 — — — Pcbp261 1346413 Lamin A &1.56 — — — Lmna62 1346413 Lamin A &1.60 — — &2.63 Lmna

76253725 Chaperonin subunit 6a (') Cct6a63 1346413 Lamin A &1.71 — &1.72 &1.70 Lmna64 52783155 Plasminogen activator inhibitor 1 RNA-binding

protein&1.79 — — &2.35 Serbp1

65 13592065 Ribosomal protein S6 kinase polypeptide 1 &1.81 — &1.94 &2.40 Rps6ka166 52783155 Plasminogen activator inhibitor 1 RNA-binding

protein&1.91 — — &2.30 Serbp1

68 157816973 Protein phosphatase 1, regulatory (inhibitor)subunit 8

&1.97 — &2.18 — Ppp1r8

6978487 Aldolase A Aldoa81294202 Psmc6 protein Psmc6

69 52783155 Plasminogen activator inhibitor 1 RNA-bindingprotein

&2.00 — — &2.20 Serbp1

71 162287306 ATP-citrate lyase isoform 1 &2.14 — &2.12 &2.54 Acly73 19424312 KH-type splicing regulatory protein

(far upstream element (FUSE)-binding protein 2)&2.39 &1.86 &2.50 &4.28 Khsrp

74 1346413 Lamin A &4.85 — &2.87 &3.08 Lmna75 1346413 Lamin A &5.15 — &5.82 &5.44 Lmna

pH 4–7, increased,15% SDS-PAGE

76c 8393696 Stathmin 1 10.42 ND ND ND Stmn180c 8393696 Stathmin 1 4.28 ND ND ND Stmn182c 112983968 Sorting nexin 3 2.87 ND ND ND Snx3

pH 4–7, decreased,15% SDS-PAGE

77c 40018580 Hypothetical protein LOC308869 &1.50 ND ND ND C11orf5979c 228542 Myosin: subunit ) regulatory light chain &4.46 ND ND ND Mylc2b81c 8393696 Stathmin 1 &1.77 ND ND ND Stmn1

pH 6–9, increased,15% SDS-PAGE

83c 149037907 DnaJ (Hsp40) homolog, subfamily B, member 1(predicted), isoform CRA_b

1.92 ND ND ND Dnajb1

83c 38328245 Hnrpa1 protein Hnrpa1a Numbering of spots is according to the 2-D gels (10% SDS-PAGE) shown in Fig. 1. Spots 7, 23, 37, 39, 41, 42, 58, 60, 67, 70, 72, 78c could

not be identified.b Average ratio of changed expression between resting and stimulated (25 mM glucose % 1 mM IBMX) INS-1 cells calculated using the BVA

module version 5.0 (p # 0.01).c Proteins identified from regulated spots as detected by 2-D DIGE using 15% SDS-PAGE (data not shown).



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profile of resting and stimulated INS-1 cells by 2-D DIGEfollowed by MS, which is a reliable platform for proteomicsstudies (37, 59). In total, we identified 165 spots whose levelssignificantly changed in response to stimulation for 2 h with 25mM glucose and 1 mM IBMX, either alone or together, and inthe presence or absence of AmD. We identified 117 (70.9%)of these spots, corresponding to 78 different proteins. This yieldis comparable to that achieved in other proteomics studies in!-cells (40, 60, 61). The remaining 30% of the regulated spots

could not be identified either because of their insufficientamount or because their spectrum could not be assigned to anyentry in the queried databases. Thirteen of the identified spots(11.1%) included multiple proteins that co-migrated at the sameposition in the gel. In these cases it was not possible to deter-mine the identity of the regulated protein(s).

Except insulin and other components of the secretory gran-ules, none of the proteins identified in this study have beenreported previously to exhibit rapid level changes in !-cells.

FIG. 3. Validation of 2-D DIGE results by Western blotting on 2-D gels. Selected rapidly regulated proteins were identified from extractsof cells kept in resting (0 mM glucose) or stimulating (25 mM glucose % 1 mM IBMX) buffer for 2 h. Immunoprobing was carried out usinganti-hnRNP K (pH 4–7; A and B), anti-PAI-RBP1 (pH 6–9; C and D), anti-PCBP2 (pH 6–9; E and F), and anti-lamin A/C (pH 4–7 and 6–9; G andH) antibodies. PTBP1 in extracts from resting (I) or stimulated (J) cells was detected using the ECL Plex system in combination with a mousemonoclonal antibody directed against PTBP1 (green channel) and an affinity-purified rabbit antibody against PTBP1 phosphorylated on serine16 (red channel). The numbering of spots detected in A, B, C, and D is according to the 2-D gels (10% SDS-PAGE) shown in Fig. 1. Changesin spot pattern are indicated with arrows if no direct match to the 2-D DIGE was possible.



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Earlier studies investigated the proteome of mouse islets imme-diately after isolation (42, 62, 63) and following 24-h exposure to11 mM glucose (40). More recent studies have focused onproteomic differences between glucose-responsive and non-responsive MIN-6 cells (64), INS-1 832/13 cells exposed to 16.7mM glucose versus 2.8 mM glucose for 48 h (61), and INS-1Ecells treated with different cytokines (60). Despite differences inexperimental design, 27 (34.6%) of the 78 regulated proteinsidentified here were also found in other studies (see supplemen-tal Table 8). According to their molecular functional classifica-tion in the PANTHER database, these 27 regulated proteins

belong to the following groups: nucleic acid binding (five), cy-toskeletal (four), chaperone (three), lyase/transferase (three),membrane trafficking (two), oxidoreductase (two), kinase (two),ion channel (two), protease (one), transfer/carrier (one), miscel-laneous function (one), and molecular function unclassified(one). The inclusion in this list of enzymes involved in glucosemetabolism and mitochondrial function, such as glyceralde-hyde-3-phosphate dehydrogenase, pyruvate kinase L, aconi-tase, and glutamate dehydrogenase is not surprising given theirabundance and relevant regulatory role in !-cells. Many of these27 proteins were also shown to change levels after treatment of

FIG. 4. mRNA-binding proteins are regulated upon stimulation. Western blots for several mRNA-binding proteins in extracts of cells keptin resting (0 mM glucose (Glc)) or stimulating (25 mM glucose % 1 mM IBMX) buffer for 2 h are shown. Immunoprobing was carried out withanti-Staufen2 (Stau2) (A), anti-hnRNP A1 (B), anti-hnRNP A3 (C), anti-PCBP2 (D), anti-CBF-A (E), or anti-lamin A/C (F) antibodies. Forquantification, three lanes were probed for each protein under each condition. Equal loading was monitored by immunoblotting for %-tubulin.G, quantification of the immunoblots shown in A–F. The level of each detected protein in resting cells was equaled to 100%. Each bar showsquantification from three independent experiments normalized to %-tubulin (*, p # 0.05; **, p # 0.01). Error bars represent standard devia-tion of three independent experiments.



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INS-1E cells with interleukin-1! and interferon-% for 24 h (60),although these changes were often antithetical to those de-tected in our screen and in some cases below the threshold of1.5-fold applied here. A remarkable example for this oppositeregulation is hnRNP K. D’Hertog et al. (60) resolved hnRNP K insix spots, all of which were down-regulated in INS-1E cells

exposed to cytokines. Here hnRNP K was separated in ninespots, five of which were up-regulated and four of which weredown-regulated.

Notably our 2-D DIGE screen did not detect preproinsulin,pro-PC1/3, pro-PC2, prochromogranin A, or pro-ICA512among the regulated proteins. The up-regulation of these SG

FIG. 5. SG proteins are regulated upon stimulation. Western blotting on 2-D gels (pH 4–7) for some SG proteins in extracts of cells keptin resting (0 mM glucose) or stimulating (25 mM glucose % 1 mM IBMX) buffer for 2 h is shown. Immunoprobing was carried out usinganti-chromogranin A (CGA) (A and B), anti-PC1 (C and D), anti-PC2 (E and F), anti-ICA512 (G and H), and anti-carboxypeptidase E (CPE) (I andJ) antibodies. Changes in spot pattern are indicated with arrows or numbers.



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components following stimulation was nevertheless con-firmed using antibody-based approaches, such as radioim-munoassay/ELISA for insulin (not shown) or Western blottingfor the other SG proteins. The proteomic composition offractions enriched in insulin SGs of INS-1E cells was recentlyanalyzed by one-dimensional gel electrophoresis and nano-LC-ESI-MS/MS (65). Of the 130 identified proteins, 110 hadnot been associated previously with SGs. Yet not all knownSG components, such as PC1/3 and ICA512, were detected.On the other hand, five of the proteins identified, namelyglyceraldehyde-3-phosphate dehydrogenase; actin; vitaminD-binding protein precursor; ATPase, H%-transporting, V1subunit A, isoform 1; and heat shock protein 40 homolog(DnaJ), were in our list of rapidly regulated proteins.

The changes in the proteomic pattern that we observed incells stimulated with glucose alone were less extensive thanthose produced by IBMX stimulation. Indeed only 13 spotswere glucose-responsive, whereas 77 were IBMX-respon-sive. Thus, most of the proteins identified in our study aretargets of cAMP regulation. Glucose-induced proteomicchanges are most likely underestimated because of thereduced glucose responsiveness of INS-1 cells comparedwith primary !-cells, a limit shared with other insulinoma celllines such as mouse MIN-6 cells (66). Co-stimulation ofINS-1 cells with glucose and IBMX changed the levels of 75spots of which 61% were still increased and 67% weredecreased in the presence of the transcription inhibitorAmD, indicating the post-transcriptional nature of thischange. These data add to the evidence that change in theexpression profile of !-cells shortly after stimulation islargely driven by post-transcriptional mechanisms (5–8).

Phosphorylation is the most common post-translationalmodification, and many of the spots that changed in responseto IBMX are either already known or likely targets of cAMP-dependent protein kinase A or other Ca2%-stimulated kinases,such as protein kinase C. Many proteins regulated in re-sponse to co-stimulation with glucose and IBMX, with orwithout AmD, migrated as multiple spots that characteristi-cally shifted to a more acidic pI upon stimulation as expectedin the case of phosphorylation. A prominent example of thisbehavior was again hnRNP K, whose overall pattern shiftedtoward a more acidic pH range. A similar shift towards lowerpI was observed in the case of PAI-RBP1 and lamin A. Phos-phorylation of hnRNP K and lamin A was further validated bytreatment with alkaline phosphatase, which resulted in adownward shift of the protein doublet observed by SDS-PAGE (data not shown).

The most novel finding of our study is the identificationamong regulated proteins of seven heterogeneous nuclearribonucleoproteins and seven additional mRNA-bindingproteins, which account for 17.9% of the total rapidly reg-ulated proteome. Some of them, namely hnRNP K, hnRNPH1, and splicing factor arginine/serine-rich 3 have alreadybeen shown to be affected following the long exposure ofINS-1 832/13 cells to glucose (61) or INS-1E cells to cyto-kines (60). However, this is the first demonstration thatmRNA binding factors represent a major class of rapidlyregulated proteins in a !-cell model. Regulation of mRNAstability and translation by mRNA-binding proteins isemerging as a relevant post-transcriptional mechanism torapidly increase insulin granule biosynthesis following !-cellstimulation (67, 68). In particular, stimulation of INS-1 and

TABLE IIPredicted conserved (human, rat, and mouse) PTBP1 binding sites in the mRNA UTRs from regulated proteins

Hits are sorted by p value, p ' 0.01. Stm1 is listed twice in the table because of a different number of mismatches detected in the PTBP1consensus sequence and consequently different p values. NECAP, adaptin ear-binding clathrin-associated protein.

EntrezGene

symbolProtein

Untranslatedregion

Number ofmismatchesa

Number ofhits

pvalueb

Number ofhits in

single strandc

Aldoa Aldolase A 3$ 2 12 0.0001 4Dpysl2 Dihydropyrimidinase-related protein 2 3$ 2 35 0.0006 8C11orf59 Chromosome 11 open reading frame 59 3$ 2 14 0.0007 0Lmna Lamin A 3$ 2 22 0.0013 7Pkm2 M2 pyruvate kinase 3$ 2 15 0.0017 6Mapk1 Mitogen-activated protein kinase 1 5$ 1 3 0.0026 1Dnm2 Dynamin 2 3$ 1 5 0.0029 1Stmn1 Stathmin 1 3$ 1 3 0.003 1Stub1 STIP1 homology and U-box-containing protein 1 3$ 1 4 0.0033 0Sgta Small glutamine-rich tetratricopeptide repeat

(TPR)-containing, $3$ 1 7 0.0041 2

Stxbp1 Syntaxin-binding protein 1 3$ 2 28 0.0047 15Stmn1 Stathmin 1 3$ 2 8 0.0063 4Ndrg1 N-myc downstream regulated 1 3$ 1 4 0.0098 1Necap1 NECAP endocytosis-associated 1 3$ 1 4 0.0099 2

a Number of mismatches between 0 and 2.b The p value is the probability of obtaining at least the same number of hits in a random sequence of the same composition.c The secondary structure of RNA was predicted using RNAfold (53).



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!-cells with either glucose or IBMX induces the nucleocy-toplasmic translocation of PTBP1, an additional heteroge-neous nuclear ribonucleoprotein (hnRNP I). Binding of cy-tosolic PTBP1 to the 3$- and 5$-UTR of mRNAs encodingSG components in turn promotes their stability and trans-lation (11, 12).2 The ability of IBMX to induce the phospho-rylation of PTBP1 was here confirmed by 2-D immunoblot-ting. As in the case of PTBP1 (12, 69), the rapidphosphorylation of the other hnRNPs identified in this studycould regulate their nucleocytoplasmic transport.

Using bioinformatics tools we found that the mRNAs ofthree regulated mRNA-binding proteins, i.e. PAI-RBP1,

PCBP2, and Staufen2, include potential PTBP1 binding sitesin their 5$- or 3$-UTRs. Neither up-regulation nor down-regu-lation of PTBP1, however, altered their levels except for amodest increase in Staufen2 following the Ptbp1 knockdown.Notably the glucose % IBMX-induced reduction of PCBP2was AmD-sensitive, whereas the changed pI pattern of PAI-RBP1 by 2-DE was compatible with phosphorylation. Basedon these data, the possibility that PTBP1 is involved in thepost-transcriptional regulation of PAI-RBP1, PCBP2, andStaufen2 is unlikely.

In conclusion, our findings demonstrate that rapid modula-tion of mRNA-binding proteins is a major process following

TABLE IIIPredicted conserved (minimum of two species) PTBP1 binding sites in the mRNA UTRs from regulated proteins

Hits are sorted by p value, p ' 0.01. Some genes are listed several times in the table because of a different number of mismatches detectedin the PTBP1 consensus sequence and consequently different p values. NECAP, adaptin ear-binding clathrin-associated protein.

EntrezGene

symbolProtein

Untranslatedregion

Number ofmismatchesa

Number ofhits

pvalueb

Number ofhits in

single strandc

Stau2 Staufen2 3$ 1 9 0 4Lmna Lamin A 3$ 2 33 0 12Dpysl2 Dihydropyrimidinase-related protein 2 3$ 2 42 0 10C11orf59 Chromosome 11 open reading frame 59 3$ 2 23 0 0Aldoa Aldolase A 3$ 2 12 0.0001 4Tubb2b Tubulin ! chain 15 3$ 2 13 0.0001 3Stau2 Staufen2 3$ 2 21 0.0001 5Stxbp1 Syntaxin-binding protein 1 3$ 2 36 0.0001 21Pkm2 M2 pyruvate kinase 3$ 2 18 0.0002 6Stau2 Staufen2 3$ 0 2 0.0005 1Sgta Small glutamine-rich tetratricopeptide repeat

(TPR)-containing, $3$ 1 9 0.0006 4

Serbp1 Plasminogen activator inhibitor 1 RNA-bindingprotein

5$ 1 3 0.0007 3

Snx3 Sorting nexin 3 3$ 1 5 0.0007 2Glul Glutamate-ammonia ligase (glutamine synthase) 3$ 2 19 0.0014 4Dpysl2 Dihydropyrimidinase-related protein 2 5$ 2 9 0.0018 1Inexa $-Internexin 3$ 2 21 0.0026 6Stmn1 Stathmin 1 3$ 1 3 0.003 1Ndrg1 N-myc downstream regulated 1 3$ 2 17 0.003 3Necap1 NECAP endocytosis-associated 1 3$ 1 5 0.0032 1Tubb2b Tubulin ! chain 15 3$ 1 3 0.0037 0Smc3 Structural maintenance of chromosomes

protein 35$ 2 4 0.0038 0

Atp6v1a ATPase, H%-transporting, lysosomal V1subunit A

3$ 2 25 0.0039 6

Pkm2 M2 pyruvate kinase 3$ 1 4 0.0063 1Stmn1 Stathmin 1 3$ 2 8 0.0063 4Pcbp2 Poly(rC)-binding protein 2 3$ 2 9 0.0064 3Dpysl2 Dihydropyrimidinase-related protein 2 3$ 1 7 0.0065 2Ppp1r8 Predicted: similar to protein phosphatase 1,

regulatory (inhibitor) subunit 83$ 2 11 0.0065 4

Cugbp1 CUG triplet repeat, RNA-binding protein 1 3$ 1 4 0.0068 0Cmpk Cytidylate kinase 3$ 2 11 0.0094 5Ppp1cb Protein phosphatase 1, catalytic subunit, !

isoform3$ 2 16 0.0095 1

Ndrg1 N-myc downstream regulated 1 3$ 1 4 0.0098 1a Number of mismatches between 0 and 2.b The p value is the probability of obtaining at least the same number of hits in a random sequence of the same composition.c The secondary structure of RNA was predicted using RNAfold (53).



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FIG. 6. Validation of PTBP1 binding predictions by knockdown and overexpression of PTBP1. Western blots for PTBP1 (A, E, and F) andmRNA-binding proteins with predicted PTBP1 binding sites in their 5$- and 3$-UTRs are shown. In these cells, PTBP1 levels were eitherdown-regulated by RNAi (A and B) or up-regulated by transfection of PTBP1-V5 (E–G). A and E show immunoblots obtained with the anti-PTBP1antibody; F shows the immunoblot obtained with the anti-V5 antibody. For quantification, three lanes were probed for each protein under each condition.Equal loading was monitored by immunoblotting for %-tubulin. C, quantification of PTBP1 knockdown as detected in A. The average level of PTBP1 incells transfected with the control vector for RNAi was equal to 100%. D and I, quantification of immunoblots for the 52-, 59-, and 62-kDa isoforms ofStaufen2 (Stau2) (70, 71), PAI-RBP1, and PCBP2 as detected in B and G. The level of these proteins in control cells was equaled to 100%. H,quantification of endogenous (endog.) and total PTBP1 levels as detected in E. The average PTBP1 level in cells transfected with the control vector wasequaled to 100%. Each bar shows quantification from three independent experiments normalized to %-tubulin (*, p # 0.05; **, p # 0.01; ***, p # 0.001).Error bars represent standard deviation of three independent experiments.



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the stimulation of insulinoma cells with insulin secretagogues.Future studies will be necessary to analyze the significance ofthese changes on !-cell gene expression and function.

Acknowledgments—We thank M. Kiebler and T. Leanderson forproviding antibodies against Staufen2 and CBF-A, respectively; A.Altkruger and C. Wegbrod for cell culture and technical assistance;C. Wollheim for providing INS-1 cells; V. Lange for advice on 2-DDIGE; M. Winzi for bioinformatics programming support, F. Schuitfor discussion; A. Shevchenko for critical reading of the manuscriptand support; L. Rohde for editing the manuscript; and K. Pfriem andR. Liedtke for secretarial assistance.

* This work was supported by grants from the European Foundationfor the Study of Diabetes, Juvenile Diabetes Research FoundationGrant 1-2004-567, German Research Foundation Grant SFB655, andGerman Ministry for Education and Research Grant NBL-3 (to M. So.)and by Deutsche Forschungsgemeinschaft Graduiertenkolleg 864studentship (to C. S.).

!S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

¶¶ To whom correspondence should be addressed: ExperimentalDiabetology, Fetscherstrasse 74, 01307 Dresden, Germany. Tel.: 49-351-4586611; Fax: 49-351-4586330; E-mail: [email protected].

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