The FASEB Journal • Research Communication
Selective gene activation by spatial segregation of insulin receptor B signaling Sabine Uhles, Tilo Moede, Barbara Leibiger, Per-Olof Berggren, and Ingo B Leibiger1 The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden Insulin exerts pleiotropic effects at the cellular level. Signaling via the two isoforms of the insulin receptor (IR) may explain the activation of different signaling cascades, while it remains to be explored how selectivity is achieved when utilizing the same IR isoform. We now demonstrate that insulinstimulated transcription of c-fos and glucokinase genes is activated simultaneously in the insulin-producing -cell via IR-B localized in different cellular compartments. Insulin activates the glucokinase gene from plasma membrane-standing IR-B, while c-fos gene activation is dependent on clathrin-mediated IR-B-endocytosis and signaling from early endosomes. Moreover, glucokinase gene up-regulation requires the integrity of the juxtamembrane IR-B NPEY-motif and signaling via PI3KC2␣-like/PDK1/PKB, while c-fos gene activation requires the intact C-terminal YTHM-motif and signaling via PI3K Ia/Shc/MEK1/ERK. By using IR-B as an example it is thus possible to demonstrate how spatial segregation allows simultaneous and selective signaling via the same receptor isoform in the same cell.— Uhles S., Moede T., Leibiger B., Berggren P.-O., and Leibiger I. B. Selective gene activation by spatial segregation of insulin receptor B signaling. FASEB J. 21, 1609 –1621 (2007) ABSTRACT
Key Words: insulin signaling 䡠 gene expression regulation 䡠 pancreatic beta cell 䡠 endosomes 䡠 life cell imaging 䡠 c-fos gene 䡠 glucokinase gene Understanding selectivity in signal transduction is one of the most challenging tasks in current cell biology. Here, insulin signaling has served as one of the model examples in hormone-induced signal transduction. Insulin exhibits pleiotropic effects involving mitogenic and/or metabolic events that are tissue- as well as development-dependent. Selectivity in insulin signaling is currently discussed as the result of the activation of selective signal transduction pathways. Data observed from experiments performed on different cell types suggest that activation of specific adaptor proteins, such as insulin receptor substrate (IRS) proteins 1– 6, Gab-1, APS, or Shc, “channels” the insulin signal in more defined ways by engaging specific downstream located effector proteins (1–3). However, the molecular mechanisms by which these different signaling cascades are activated remain unclear. More importantly, although 0892-6638/07/0021-1609 © FASEB
often taken for granted, the coexistence of different, selective insulin signaling cascades within the very same cell is poorly studied and thus not proven. We have recently shown that insulin, secreted by pancreatic -cells on glucose stimulation, activates its own gene via insulin receptor-A/IRS2/PI3K Ia/ mTOR/p70s6k, while it simultaneously stimulates transcription of the -cell glucokinase gene (GK) via insulin receptor-B/PI3K class II like activity/PDK1/PKB (4 – 6). Our data show that the two insulin receptor isoforms reside in different microdomains of the -cell plasma membrane. This may allow the access to different pools of adaptor proteins and thus enable the utilization of different signaling cascades within the “metabolic” branch in insulin signal transduction (5, 6). Hence, we have shown that coexistence of different insulin signaling cascades at the single cell level can be gained by utilizing signal transduction through the two isoforms of the insulin receptor (IR), i.e., A-type (IR-A), and B-type (IR-B). However, it remains unclear whether different insulin signaling cascades can be activated by a given, single IR isoform in the same cell. To address this problem, we investigated the molecular mechanisms underlying the insulin-dependent activation of c-fos gene transcription in the pancreatic -cell at the single cell level. We did chose c-fos as a candidate gene because it is activated by insulin through the “mitogenic” branch, i.e., involving mitogen-activated protein kinases (MAPKs) ERK1/2, p38SAPK2a, or JNK/SAPK1 in several tissues and cell types (7). This guaranteed that the signaling cascade activating c-fos transcription will be different from the already identified cascades leading to the up-regulation of GK and insulin genes in the -cell. We now show that in the same -cell both c-fos and GK genes are activated simultaneously but selectively via IR-B by spatial segregation of the signaling events. While GK gene transcription is up-regulated from plasma membrane-standing IR-B, IR-B endocytosis, and signaling from the early endosome pool are needed to activate the c-fos gene. Moreover, GK gene up-regulation requires the integrity of the juxtamembrane IR-B 1
Correspondence: The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, S-171 76 Stockholm, Sweden. E-mail:
[email protected] doi: 10.1096/fj.06-7589com 1609
NPEY-motif and signaling via PI3K-C2␣-like/PDK1/ PKB, while c-fos gene activation requires the intact C-terminal YTHM-motif and signaling via PI3K Ia/Shc/ MEK1/ERK.
MATERIALS AND METHODS Materials Protein kinase inhibitors wortmannin, PD98059 and SB203580 were from Calbiochem (San Diego, CA, USA), SP600125 was from A.G. Scientific, Inc. (San Diego, CA, USA). Blocking rabbit antiinsulin receptor [␣IR(AB)] and rabbit anti-Insulin receptor B [␣IR(B)] antibodies were from Biodesign (Saco, ME, USA), and mouse monoclonal insulin-like growth factor (IGF)-IR␣ was from Pharmingen (San Diego, CA, USA). Inhibitors or antibodies were added to the culture medium 30 min prior to start of stimulation and were kept throughout stimulation. Cell culture INS1 cells were obtained from Dr. C.B. Wollheim (Centre Me´dical Universitaire, Geneva, Switzerland) and MIN6m9 cells from Dr. S. Seino (Cellular and Molecular Medicine, Kobe University, Kobe, Japan). INS1 cells were cultured in RPMI 1640 culture medium (i.e., 11.1 mM glucose) supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, 10% fetal calf serum (FCS), 1 mM pyruvate, 10 mM HEPES, and 50 M -mercaptoethanol at 5% CO2 and 37°C. MIN6m9 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) culture medium containing 11.1 mM glucose and supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, 10% FCS, and 75 M -mercaptoethanol at 5% CO2 and 37°C. Prior to experiments, cells were starved for 6 h in fully supplemented culture medium containing 2 mM glucose. Cells were stimulated, if not indicated otherwise, for 10 min with 5 mU/ml insulin in fully supplemented culture medium.
dextrin-experiments). Excitation light was obtained from a SPEX fluorolog-2 MM1T11I spectrofluorometer (Spex Industries, Edison, NJ, USA). The following settings were used: for GFP: excitation at 485 nm, a 505 nm dicroic mirror, and a 505–535 nm band-pass emission filter; for DsRed2: excitation at 558 nm, a 565 nm dicroic mirror, and a 580 – 620 nm band-pass emission filter. Fluorescence was imaged using a cooled CCD-camera (CH250 with KAF 1400; Photometrics, Tucson, AZ, USA) connected to an imaging system (Inovision, Durham, NC, USA). Online monitoring was initiated 60 min following start of stimulation, and cells to be monitored were chosen randomly in 6 to 12 fields of view containing at least 9 cells. For calculation, the fluorescence intensity of an individual cell at the beginning of the experiment (t⫽60 min after start of stimulation) was set as 1. The fluorescence intensity of each monitored cell was followed over time and calculated relative to its intensity at t ⫽ 60 min. Fluorescence intensities were calculated by using the Isee-sofware for UNIX (Inovision, Durham, NC, USA). RNA analysis c-fos mRNA was analyzed by comparative RT-polymerase chain reaction (RT-PCR) as described in (8) using primers 5⬘-AGCGCAGAGCATCGGCAGAA-3⬘ (localized in exon 2) and 5⬘-ATCTTGCAGGCAGGTCGGTG -3⬘ (localized in exon 4) (Raytest, Straubenhardt, Germany). -actin mRNA was analyzed by RT-PCR using primers 5⬘-CGTGGGCCGCCCTAGGCACCA-3⬘ and 5⬘-TTGGCCTTAGGGTTCAGAGGGG-3⬘. PCR conditions were chosen, which guaranteed the amplification of c-fos and actin fragments within the linear range, as verified by testing various numbers of amplification cycles (cycles 32 to 42). 32P-labeled PCR products were separated on a 6% polyacrylamide sequencing gel and analyzed by phosphoimaging. Quantification was performed with TINA-software 2.07d (Raytest, Straubenhardt, Germany). Values of c-fos mRNA were normalized by -actin values. Expression constructs c-fos promoter variants
Mouse islet cells Islets were prepared from 10 month-old normo-glycemic ob/ob mice. Isolation of pancreatic islets, culture of islets, and islet cells as well as their transfection was performed as described previously in (4, 8). Online monitoring of GFP and DsRed2 expression Expression of DsRed2 and GFP were detected using digital imaging fluorescence microscopy as reported in (4 – 6, 8). For online monitoring, cells were grown on 24-mm glass coverslips and transiently transfected overnight using the lipofectamine technique. After transfection, cells were cultured for at least 36 h in fully supplemented RPMI 1640 culture medium. After starvation for 6 h at 2 mM glucose, cells were stimulated with insulin at 2 mM glucose in fully supplemented medium. Expression of DsRed2 and GFP was detected using digital imaging fluorescence microscopy as follows. Glass coverslips with transfected cells were placed in a perifusion chamber and mounted on an inverted microscope (Zeiss Axiovert 133TV, Carl Zeiss, New York, NY, USA) equipped with a Zeiss plan NEOFLUAR x25/0.8 Imm Korr lens (Carl Zeiss). During the experiment, cells were kept at 37°C and perifused with fully supplemented culture medium at 2 mM glucose containing 10% FCS or 0.5% BSA (cyclo1610
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Vectors c-fos.GFP/rIns1.DsRed, c-fos.DsRed/GK.GFP, and c-fosSREKO.DsRed/GK.GFP were generated as follows. The GK cassette in pd2.rIns1.DsRed2.bGHpA/rGK.GFP. SV40pA (6) was exchanged vs. the c-fos cassette from pcfos.GFP (4), generating pd2.rIns1.DsRed/c-fos.GFP. Plasmid pd2.c-fos.DsRed/rGK.GFP was obtained by replacing the rIns1.DsRed cassette in pd2.rIns1.DsRed/rGK.GFP vs. the c-fos.DsRed cassette from pc-fos.DsRed. The SRE-mutated c-fos promoter construct was generated by site-directed mutagenesis of the SRE element in pd2.c-fos.DsRed/rGK.GFP replacing the SRE-motif 5⬘-CAGGATGTTCATATTAGGACAT-3⬘ by 5⬘-CAGAGTGTCAACGTTAGGACAT-3⬘ (SRE-KO). Insulin receptor variants Construction of pRcCMVi.hIR(A) and pRcCMVi.hIR(B) is described in (5). To obtain FLAG-tagged IR-B variants, a ClaI-site was introduced by exchanging nucleotides TAA CAT to ATC GAT, thereby removing the stop-codon. Nucleotides encoding the FLAG-tag were inserted inframe via the ClaIsite, thus creating pRcCMVi.hIR(B)-FLAG. Using site-directed mutagenesis, NPEF-, FTHM-, ITAA-, RDAA and APLAmutants were generated by exchanging nucleotides encoding the respective residues in NPEY, YTHM, ITLL, RDII, and in GPLY, thus obtaining pRcCMVi.hIR(B)NPEF-FLAG, pRcCMVi.
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hIR(B)FTHM-FLAG, pRcCMVi.hIR(B)ITAA-FLAG, pRcCMVi. hIR(B)RDAA-FLAG, pRcCMVi.hIR(B)APLA-FLAG, and pRcCMVi. hIR(B)APLA⫹ITAA-FLAG. p52-Shc-variants Mouse p52-Shc was obtained by RT-PCR cloning from MIN6 mRNA using primer combinations (5⬘-CGGTGACTTAAGCAGACAGT-3⬘ and 5⬘-CGGTGGATTCCTGAGATACT-3⬘) and (5⬘-GTGAATCAGAGAGCCTGCCA-3⬘ and 5⬘-GCATCCTGTTGGAGAAGCTG-3⬘) that resulted in the generation of two PCR products encoding the C- and the N-terminal sequence of p52-Shc. The full-length p52-Shc was obtained by fusing N- and C-terminal Shc-sequences via a common StuI-site in both cDNA fragments. pRcCMVi.Shc-myc/His was generated by creating a ClaI-site Val (474), which allowed inframe insertion of the Shc-fragment into pRcCMVi.hIR(A)⌬380myc/His thereby replacing the hIR(A)-fragment. To obtain pCMVi.DN-Shc-myc/His, nucleotides encoding amino acids Tyr (239), Tyr (240) and Tyr (313) were changed by sitedirected mutagenesis in order to encode Phe instead. pRcCMVi.Shc-S154P-myc/His was obtained by mutating Ser-154 to Pro in pRcCMVi.Shc-myc/His and pRcCMVi.Shc-S154P, R397L-myc/His by exchanging Arg (397) to Leu in pRcCMVi.Shc-S154P-myc/His. Rab variants Rat Rab5 and Rab7 cDNAs were obtained by RT-PCR cloning from INS1 mRNA using the following primer combinations. Rab5a (5⬘-CATGGCTAATCGAGGAGCAA-3⬘ and 5⬘-ACCCCCTCAGTTGCTACAACAC-3⬘), Rab5b (5⬘-GGGAGGAGCATATGACTAGCA-3⬘ and 5⬘-ACCCCCTCAGTTGCTACAACAC-3⬘), Rab7 (5⬘-CTTCAAGGATGACCTCTAGG-3⬘ and 5⬘GTGTGTGTTCTTGGTCTGTG-3⬘) into pCRII (Invitrogen). Myc-tagged and mCFP-tagged variants were generated by subcloning the respective cDNAs inframe into pCMV-Myc (Clontech, Palo Alto, CA, USA) or pRcCMVi.mCFP0, respectively. Dominant-interfering mutants were generated by exchanging the nucleotides by site-directed mutagenesis encoding the following amino acids in Rab5a and Rab5b: Ser-34 to Asn (dominant-negative) and Gln (79) to Leu (dominantpositive), in Rab7: Thr (22) to Asn and Asn (125) to Ile (dominant-negative) and Gln (67) to Leu (dominant-positive). Expression constructs for caveolin-1␣, -1, and -2 were described in (6). Dominant-negative adaptor protein (AP)-2 2 was generated by exchanging nucleotides encoding Thr (156) for Ala in pcDNA3.hemagglutinin (HA)-2 (gift from Dr. A Sorkin, Dept. Pharmacology, University of Colorado Health Science Center, USA). All mutations were performed by employing the QuikChange Mutagenesis kit (Stratagene, La Jolla, CA, USA) and respective oligonucleotides purchased from Proligo (Paris, France). All constructions were verified by DNA sequence analysis. Plasmid dynamin-2K44A was previously described (6). pcDNA3Zeo.⌬p85 was a gift from Dr. C.P. Downes (Dept. Biochemistry, University of Dundee, UK). siRNA Small interfering RNAs, against mouse p85 Pik3r1 (siRNAID #151108) and mouse Shc1 (siRNAID #151658) and nontargeting control (#4613) were purchased from Ambion. MIN6 cells were first transfected using Lipofectamine 2000 with the siRNA and transfected 48 h later with pd2.c-fos.DsRed/ GK.GFP. Expression of GFP and DsRed was monitored online after further incubation for 48 h. The expression of all constructs was verified by Western blot analysis. SELECTIVE IR-B-MEDIATED SIGNALING
Preparation of plasma membranes and early endosomes Transfected INS1 cells were lysed and plasma membranes and early endosomes were fractionated on a flotation gradient as described in (9). For the preparation of each gradient, two 10 cm dishes (5⫻106 cells) were used. After transfection with calcium phosphate overnight, cells were grown for an additional 24 h, starved for 6 h in fully supplemented culture medium containing 2 mM glucose, and stimulated with insulin. Cells were washed two times with PBS and lysed in 300 l cold homogenization buffer (HB⫹) containing 250 mM sucrose, 3 mM imidazole (pH 7.4), 10 g/ml cycloheximide and phosphatase and protease inhibitors (see in Immunoprecipitation and Western blot analysis). The cells were gently homogenized by slowly passing them 10 times through a 23G needle and 20 times through a 27G needle. The homogenates were subjected to brief centrifugation (5 min, 600 g, at 4°C) and the postnuclear supernatants were collected while the pellets were resuspended in 200 l HB⫹ buffer, homogenized again, centrifuged (5 min, 600 g, at 4°C) and combined with previous supernatants. Protein determination was performed using the Bradford method, and equal amounts of protein (1 mg) were subjected to preparative fractionation. The postnuclear supernatants were adjusted to 40.6% sucrose by adding a solution containing 62% sucrose (w/w); 3 mM imidazole (pH 7.4); 1 mM EDTA; and 10 g/ml cycloheximide, phosphatase, and protease inhibitors. This was loaded at the bottom of a 5 ml Beckman Ultra-Clear centrifuge tube, to which were added sequentially 1.5 ml of a 35% sucrose (w/w) solution containing 3 mM imidazole (pH 7.4); 1 mM EDTA; 10 g/ml cycloheximide, phosphatase and protease inhibitors; 1 ml of a 25% (w/w) sucrose solution containing 3 mM imidazole (pH 7.4); 1 mM EDTA; 10 g/ml cycloheximide, phosphatase, and protease inhibitors; and finally 400 l HB⫹ buffer. The samples were centrifuged (1 h, 35,000 rpm, at 4°C) with a Beckman SW 55 Ti rotor in an OptimaTM L-100 XP Ultracentrifuge (Beckman, Fullerton, CA, USA). Early endosomes accumulated between 25% and 35% sucrose interface, whereas plasma membrane and cytosolic proteins remained between 35% and 40.6% interface. The early endosome and plasma membrane fractions were carefully collected, diluted in HB⫹ buffer up to 4.5 ml, and centrifuged for an additional 30 min (50,000 rpm, at 4°C). The supernatants were discarded and the pellets resuspended in 100 l HB⫹ buffer and 4⫻ SDS-sample buffer and separated over a 7–12% SDS-polyacrylamide gel and analyzed by Western blot analysis employing antibodies against phosphorylated IR -subunit [rabbit polyclonal to insulin receptor (phospho Y972) Abcam, Cambridge, UK], FLAG-tag (Monoclonal ANTI-FLAG M2; Sigma, St. Louis, MO, USA) and the IR -subunit [insulin R (C-19), rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA]. Cell-surface biotinylation and IR-B internalization analysis Transfected INS1 cells were biotinylated with Sulfo-NHS-LCbiotin (Pierce, Rockford, IL, USA) (10, 11) and IR-B internalization was studied using Pronase digestion as described in (10). INS1 cells (5⫻106 cells/10 cm dish) were transfected with FLAG-tagged insulin receptor constructs by the calcium phosphate method. Forty-eight hours after transfection cells were starved for 6 h in fully supplemented culture medium containing 2 mM glucose. The cells were put on ice and washed three times with ice-cold PBS-Ca-Mg buffer (PBS pH 7.4 containing 0.1 mM CaCl2 and 1 mM MgCl2). Cell-surface proteins were biotinylated by incubation with a solution containing 0.375 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS-Ca-Mg for 50 min at 4°C. The reaction was quenched by 1611
washing the dishes three times with ice-cold PBS-Ca-Mg containing 15 mM glycine. Cells were then stimulated with insulin in fully supplemented culture medium containing 2 mM glucose for 20 min at 37°C. After being washed three times with ice-cold PBS, cell surface proteins were subjected to Pronase digestion by incubating the cells for 50 min at 4°C in RPMI containing Pronase (0.1 mg/ml, Roche Diagnostics, Germany), 20 mM HEPES (pH 7.6), and 2 mM glucose. The Pronase activity was quenched by adding ice-cold PBS containing 10 mg/ml BSA. The cells were washed off the dishes, pelleted, and washed three times with ice-cold PBS. The cells were solubilized in 500 l lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.4, 1% Triton X-100, 4 mM sodium pyrophosphate, 2 mM PMSF, 100 mM NaF, 2 mM Na3VO4, 1 g/ml aprotinin, and 0.1 mg/ml leupeptin). Insulin receptors were immunoprecipitated from equal amounts of total protein with polyclonal anti-FLAG-antibodies (Sigma). Immune complexes were precipitated with Protein-G-Plus Agarose, and the pellets were washed three times with lysis buffer at 4°C. The immunoprecipitates were analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes, and the membranes were probed with HRP-conjugated streptavidin (1:2000 in PBST, GE Healthcare, Buckinghamshire, UK). The membranes were reprobed with anti-insulin receptor ␣-subunit antibodies N-20 (1:200 in TBST; Santa Cruz Biotechnology). Immunoprecipitation and Western blot analysis INS1 cells were grown in 10 cm dishes (5⫻106 cells/dish) and transiently transfected overnight with FLAG-tagged IR-B variants according to the calcium phosphate transfection protocol and cultured for additional 24 h. After starving for 6 h, cells were stimulated with insulin for the indicated periods of time. Cells were washed with PBS, lysed with lysis buffer [137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 20 mM Tris (pH 8.0), 1% Triton X-100, phosphatase and protease inhibitors (4 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 1 g/ml aprotinin), and 10% glycerol] homogenized, and the amount of protein was measured in the supernatants by the Bradford method. Up to 1 mg lysate was incubated with 4 g rabbit Anti-FLAG (Sigma) antibody on a rotator for 16 h at 4°C. Protein-G Plus Agarose (50 l) was added and incubated for an additional 3 h. Immunoprecipitates were washed twice with lysis buffer (see above), twice with buffer #2 [137 mM NaCl, 100 mM TrisHCl (pH 8.0)], once with buffer #3 [150 mM NaCl, 10 mM Tris HCl (pH 7.6), 1 mM EDTA], and once with buffer #4 [20 mM HEPES (pH 7.6), 1 mM DTT, 5 mM MgCl2]. All buffers contained phosphatase and protease inhibitors (see above). All working steps were performed on ice. Immunoprecipitates or lysates, respectively, were separated over a 7.5–15% SDS-polyacrylamide gel (buffering system according to Laemmli), and proteins were electrotransferred to PVDF membrane. In case of immunoprecipitates the membrane was probed with mouse p-Tyr (PY99), rabbit anti IR phosphoY972 (Abcam) and rabbit insulin R (C-19) (Santa Cruz Biotechnology, Inc.), rabbit Anti-SHC (Upstate Biotechnology, Lake Placid, NY, USA), mouse SHC (Transduction Laboratories, Lexington, KY, USA) and rabbit Anti-PI3 Kinase p85 (Upstate). For Shc detection in cell lysates, the membrane was probed with rabbit Anti-Shc (Upstate), for GAPDH detection with mouse anti-GAPDH (Ambion, Austin, TX, USA). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibodies using the ECL system (Amersham, Piscataway, NJ, USA). Immunofluorescence and colocalization analysis of IR-B and EEA1 INS1 cells were cultured on 24 mm glass coverslips and transfected using Lipofectamine. Seventy-two hours after 1612
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transfection cells were preincubated in medium containing 2 mM glucose for 6 h and stimulated with insulin. Ten minutes after start of stimulation, cells were fixed and permeabilized by treatment with PBS containing 3% paraformaldehyde for 15 min and with PBS containing 0.05% saponin for 15 min. Immunofluorescence staining was performed utilizing a FITC-labeled mouse monoclonal anti-FLAG antibody (Monoclonal ANTI-FLAG M2-FITC, Sigma) and a rabbit polyclonal antibody against the early endosomal marker EEA1 (EEA1 antibody-early endosome marker, Abcam) combined with an Alexa546-labeled goat anti-rabbit antibody (Molecular Probes, Eugene, OR, USA). After the staining procedure cells were embedded using the ProLong Antifade Kit (Molecular Probes). FITC- and Alexa546-fluorescence were visualized utilizing a Leica TCS-SP2 confocal microscope with the following settings: excitation: 488 nm for FITC and 546 nm for Alexa546, a 488/543/633 triple dichroic mirror and emission detection at 505–540 nm for FITC and 560 – 600 nm for Alexa546. Fifteen FITC-fluorescence-positive cells were randomly selected for each experimental condition and an image stack covering the central portion of the cell was recorded. To estimate the colocalization between FLAGtagged IR-B and EEA1 staining, a maximum projection of the image stack was generated utilizing the Leica LCS-software. Visible vesicle-like structures that stained positive either for EEA1 or for both EEA1 and FLAG-tagged IR-B were counted in these images.
RESULTS Insulin activates c-fos gene transcription in the pancreatic -cell via MEK1/ERK It has been shown that c-fos gene activation by insulin can involve signaling through various MAPK, i.e., ERK1/2, p38/SAPK2a, or JNK/SAPK1 (7). To test which MAPK are involved in insulin-mediated activation of the c-fos gene in pancreatic -cells, we performed comparative RT-PCR analysis of c-fos mRNA and reporter gene assays with human c-fos promoterdriven (–710/⫹1) fluorescent proteins. Insulin stimulation led to a more than 2-fold increase in c-fos mRNA levels and to a significant increase in c-fos promoter activation in INS1 cells and primary mouse -cells after 30 min (Fig. 1A, B). The involved MAPK were studied by the effect of the pharmacological inhibitors of ERK kinase MEK1 (PD98059), p38 (SB203580), and JNK (SP600125). INS1 cells were transfected with a vector encoding both c-fos promoter-driven DsRed and GK promoter-driven GFP (c-fos.DsRed/GK.GFP). This allowed us to directly compare the influence of the inhibitors on the activation of two different promoters in the same cell. In agreement with our earlier data (5), GK promoter activation was not sensitive to inhibition of MEK1, p38, and JNK (data not shown). In contrast, treatment of INS1 cells and primary -cells with the MEK1-inhibitor PD98059 led to an almost complete loss of insulin-stimulated c-fos promoter-driven reporter gene expression (Fig. 1C) as well as an abolished increase in c-fos mRNA levels (Fig. 1D). In agreement with this, mutation of the serum response element of the c-fos promoter (SRE-KO), i.e., of the binding motif for the ternary transcription factor complex activated
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by ERK pathways (12, 13), abolished the insulin-dependent up-regulation seen with the wild-type c-fos promoter (Fig. 1E). Taken together, these data demonstrate that insulin stimulates c-fos gene transcription in the -cell by signaling via MEK1/ERK and activation of transcription through the SRE of the c-fos promoter. Signaling via IR-B activates c-fos gene transcription
Figure 1. Insulin activates c-fos gene transcription in the pancreatic -cell by signaling via the ERK1/2 pathway. A) c-fos mRNA levels in response to insulin in INS1 cells. B) Online monitoring of insulin-stimulated c-fos promoter-driven GFP expression in transfected INS1 cells and mouse -cells. Pancreatic -cells were identified by insulin promoter-driven DsRed expression conferred by a second expression cassette in the same plasmid, i.e., c-fos.GFP/rIns1.DsRed. C) Effect of various MAP kinase inhibitors on insulin-stimulated c-fos promoter activation in INS1 cells. D) Effect of MEK1 inhibitor on c-fos mRNA levels in INS1 cells. E) Effect of mutation of the serum response element (SRE-KO) on insulin-stimulated c-fos promoter activation in transfected INS1 cells. A, D) The amount of c-fos mRNA was measured by comparative RT-PCR and presented as the percentage of mRNA levels of nonstimulated control (given as 100%). Data are shown as mean values ⫾ sem (n⫽3). B, C, E) Changes in promoter activity were measured as ratios of fluorescence obtained at 240 min vs. 60 min after stimulation with insulin and are represented SELECTIVE IR-B-MEDIATED SIGNALING
Next we wanted to identify the IR isoform that is involved in insulin-dependent activation of the c-fos promoter in -cells. Mitogenic signaling via MAPK is described to involve mainly IGF-I receptors (IGF1R) or, in case of insulin receptors, IR-A rather than IR-B (14, 15). -cells express the IGF1R (16) and both IR-A and IR-B (4, 17). We assessed the roles of IGF1R and IR isoforms in c-fos promoter activation by utilizing receptor-specific antibodies that block signal transduction. INS1 cells were treated with antibodies that block signaling through either IGF1R (␣IGF1R), both IR-A and IR-B [␣IR(AB)], or through only IR-B [␣IR(B)]. Treatment with ␣IR(AB) abolished the insulin-stimulated increase in both c-fos mRNA levels (Fig. 2A) as well as c-fos promoter-driven GFP expression in transfected INS1 cells (Fig. 2B). In contrast, application of ␣IGF1R, which thereby blocked signal transduction through IGF1R, did not affect either c-fos mRNA levels or c-fos promoter activation. However, application of the IR-B-specific antibody completely abolished upregulation of c-fos mRNA levels (Fig. 2A) as well as inhibited c-fos promoter activation in transfected INS1 cells (Fig. 2B) and -cells (Fig. 2C), suggesting that insulin activates the c-fos promoter through IR-B. Western blot analysis of immunoprecipitated FLAG-tagged IR-A and IR-B showed that treatment of INS1 cells with ␣IR(B) led to a significant decrease in tyrosine phosphorylation of IR-B but not IR-A (Fig. 2D), while the application of ␣IGF1R, as expected, had no effect. Furthermore, we investigated the effect of transient overexpression of IR-A and IR-B on promoter activation in INS1 cells. While overexpression of IR-B led to an enhanced stimulatory effect on insulin-stimulated c-fos promoter activity, overexpression of IR-A had no effect (Fig. 2E). Collectively, these data demonstrate that in -cells insulin-stimulated c-fos promoter activation is regulated by signaling via IR-B. c-fos promoter activation involves Shc After having established that insulin activates c-fos gene transcription in pancreatic -cells by signaling via IR-B, i.e., the same IR isoform that activates GK gene transcription (5), we wanted to compare the mechanism(s) that allow the simultaneous but selective acti-
as mean values ⫾ sem (n⫽8). Cells were incubated with the pharmacological inhibitors for MEK1 (20 M PD98059), p38 (20 M SB203580), or JNK (25 M SP600125) 30 min prior to and throughout stimulation. 1613
Figure 2. Insulin activates the c-fos promoter via IR-B. A) Effect of blocking antibodies of IGF1R (␣IGF1R), of both IR-isoforms [␣IR(AB)], and of IR-B [␣IR(B)] on c-fos transcription in INS1 cells. c-fos mRNA levels were measured by comparative RT-PCR. Data are shown as mean values ⫾ sem (n⫽3). B) Effect of blocking antibodies on insulin-stimulated c-fos promoter-driven GFP expression in INS1 cells. C) Effect of ␣IR(B) on insulin-stimulated c-fos promoterdriven GFP expression in mouse islet cells. D) Effect of blocking antibodies on tyrosine phosphorylation of expressed IR-A-FLAG and IR-BFLAG in INS1 cells. Tyrosine phosphorylation of IR isoforms was analyzed by Western blotting of FLAG immunoprecipitates. A representative blot is shown. Data are shown as mean values ⫾ sem (n⫽3). E) Effect of overexpressed wild-type IR-A or IR-B on insulin-stimulated c-fos promoter activation. INS1 cells were cotransfected with c-fos promoter-driven GFP and either empty vector (mock), IR-A or IR-B. B,C,E) Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 after stimulation and represent mean values ⫾ sem (n⫽8).
vation of the two genes. Hence, we next wanted to know which adapter protein is involved in IR-B-mediated activation of c-fos gene transcription. A number of studies implicate the role of Shc in the activation of the MAPK cascade in response to insulin. Shc is expressed as three isoforms of ⬃46, 52, and 66 kDa (18). Western blot analysis revealed the expression of all three isoforms in -cells (Fig. 3A). IR preferentially phosphorylate p52-Shc (18). Three tyrosine residues in the CH1 region of Shc function as docking sites in signal transduction following phosphorylation, i.e., in binding of Grb2-Sos, resulting in the activation of Ras, Raf-1, and ERK (18, 19). To analyze whether signal transduction through Shc is required for up-regulation of c-fos promoter activity, we employed a dominantinterfering variant of p52-Shc where all three tyrosine residues within the CH1 region were replaced by phenylalanine (DN-Shc) as well as siRNAs against Shc to knock-down endogenous Shc expression. As shown in Fig. 3B, C, expression of DN-Shc or Shc-siRNA inhibited c-fos promoter activation by insulin. The latter approach resulted in a knockdown of expression of all Shc isoforms, including p52-Shc, by 50% (Fig. 3D). Although we cannot exclude the involvement of p66and p46-Shc in insulin-induced activation of c-fos gene transcription, we favor p52-Shc because it coimmunoprecipitates with IR-B (see Fig. 4B). It has been shown that interaction between Shc and the IR is dependent on receptor tyrosine autophosphorylation of the NPEY-motif in the juxtamembrane 1614
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region, and that the interaction is mediated via the phosphotyrosine binding (PTB) domain of Shc or via the involvement of IRS1 (20 –22). We next analyzed whether the NPEY-motif of IR-B is involved in the Shc/MEK/ERK-pathway leading to up-regulation of c-fos promoter activity. Therefore, INS1 cells were cotransfected with c-fos.DsRed/GK.GFP and with either nontagged or FLAG-tagged wild-type IR-B, or with a FLAG-tagged IR-B variant bearing a mutation in the NPEY-motif, i.e., IR-BNPEF-FLAG. As shown in Fig. 4A, expression of FLAG-tagged IR-B led to the same enhanced increase in c-fos and GK promoter activation as observed with nontagged IR-B, demonstrating that the tag does not interfere with receptor function. Surprisingly, expression of the NPEF-mutant had no inhibitory effect on insulin-stimulated c-fos promoter activation, while it inhibited further activation of the GK promoter in the same cell. In the latter process, the NPEY-motif might be important for recruitment of PI3K-C2␣ to IR-B, which is essential to confer signaling via PDK1 and PKB. Mutation of NPEY to NPEA showed the same inhibitory effect as NPEF in insulin-stimulated GK promoter up-regulation. The effect of this mutation on c-fos promoter is less clear since the differences between NPEA and “mock” on the one hand and between NPEA and wild-type or NPEF on the other were not statistically significant (Fig. 4A). Since earlier studies showed that binding of Shc to the IR via its PTB-domain requires phosphorylation of the tyrosine residue in NPEY (22), the data obtained from the
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YTHM-motif, i.e., IR-BFTHM-FLAG, led to a significant decrease in c-fos promoter activation, while the pronounced activation of the GK promoter was not affected (Fig. 4A). To test a possible direct interaction of the YTHM-motif with Shc, we expressed FLAGtagged wild-type IR-B or FTHM-mutant in INS1 cells and stimulated with insulin for different periods of time. Western blot analysis of FLAG-immunoprecipitates showed an increase in p52-Shc-binding to wildtype IR-B after insulin stimulation, whereas no binding of p52-Shc to the FTHM-mutant could be detected (Fig. 4B). These results suggest both the involvement of Shc in insulin-stimulated c-fos gene activation and its binding to the C-terminal pYTHM-motif of IR-B. c-fos promoter activation requires a PI3K class Ia activity
Figure 3. The role of Shc in c-fos promoter activation. A) Identification of Shc isoforms in insulin-producing cell lines by Western blot analysis. Lysates obtained from HIT-T15, INS1, MIN6, and RINm5F cells and mouse islets were probed with an antibody against Shc. B) Effect of overexpressed wild-type (WT-Shc) and dominant-interfering mutant (DNShc) on insulin-stimulated c-fos promoter activation. INS1 cells were cotransfected with c-fos.GFP and either empty vector (mock), WT-Shc or DN-Shc and GFP expression was monitored online. C) Effect of siRNA against Shc (Shc-siRNA) on insulin-stimulated c-fos promoter activation. MIN6 cells were cotransfected with c-fos.GFP and either control siRNA (mock) or Shc-siRNA and GFP expression was monitored online. B, C) Data are presented as the ratio of fluorescence obtained at minutes 240 and 60 after stimulation and represent mean values ⫾ sem (n⫽9). D) Effect of Shc-siRNA treatment on Shc protein levels. MIN6 cells were transfected with either control siRNA (mock) or Shc-siRNA and protein levels were analyzed by Western blotting 96 h after transfection. Shc protein levels were shown in relation to GAPDH protein levels. The average effect in three independent experiments was a reduction in p52-Shc levels to 53 ⫾ 4.7% (mean ⫾ sem) compared to control siRNA treated cells.
mutation analysis in Fig. 4A suggest that the NPEYmotif is not involved in Shc-mediated activation of the c-fos promoter. However, since IR-B-NPEY becomes tyrosine phosphorylated in response to insulin (see Fig. 6B), we cannot completely rule out that this motif contributes, at least in part, to Shc binding. An alternative, but so far excluded (19), possibility for Shc/IR interaction is binding of Shc via its SH2domain to the YTHM-motif in the C-terminus of IR-B (23). Interestingly, expression of IR-B with a mutated SELECTIVE IR-B-MEDIATED SIGNALING
The mutation of the YTHM-motif also abrogates direct binding of PI3K Ia to the phosphorylated pYTHM-motif and PI3K Ia activation (24, 25). Besides the ability of the PTB-domain of Shc to bind to NPEY-motifs of receptor tyrosine kinases, it can also function as a PH-domain and thus can interact with the PI3K Ia lipid product PI(3,4,5)P3 (26). Therefore, a PI3K activity might be necessary to recruit Shc to the plasma membrane in close proximity to the IR where it then becomes activated by the IR. Data by Ugi et al. (27) showed that insulin-stimulated tyrosine phosphorylation of Shc in 3T3-L1 adipocytes is sensitive to the PI3K inhibitor wortmannin and that expression of a dominant-negative PI3K mutant inhibited insulin-induced Shc phosphorylation. Moreover, expression of a Shc mutant with a disrupted PTB-domain, i.e., Shc-S154P, did not localize to the plasma membrane and was not tyrosine-phosphorylated in response to insulin stimulation (27). To analyze whether the activation of the Shc/MEK1/ERK-pathway, leading to up-regulation of c-fos gene transcription in the -cell, requires a PI3K activity, we transfected INS1 cells and mouse islet cells with the vector c-fos.DsRed/GK.GFP. Treatment with 100 nM wortmannin abolished insulin-stimulated c-fos promoter activity in both INS1 (Fig. 5A) and mouse islet cells (Fig. 5B), indicating the involvement of a class Ia PI3K. However, this was not sufficient to inhibit activation of the GK promoter in the same cell via the less wortmannin-sensitive class II PI3K (5). Western blot analysis showed the presence of the PI3K Ia adaptor protein p85 in wild-type IR-B FLAG-immunoprecipitates following insulin stimulation, whereas binding of p85 to the FTHM-mutant was drastically reduced (Fig. 5C). Moreover, expression of either the dominantnegative form of PI3K Ia adaptor protein p85 (⌬p85) or a siRNA against p85 reduced insulin-stimulated c-fos promoter activity (Fig. 5D–F). Finally, expression of the Shc-S154P PTB-mutant did not allow the pronounced elevation in c-fos promoter activity seen in cells overexpressing p52-Shc, but, surprisingly, reduced c-fos promoter activity in a dominant-negative way to almost nonstimulated levels (Fig. 5G). The most reasonable 1615
Figure 4. The role of IR-B NPEY- and YTHMmotifs in IR-B-mediated GK and c-fos transcription. A) Effect of overexpressed wild-type and mutant IR-B variants on insulin-stimulated c-fos promoter-driven DsRed and GK promoter-driven GFP expression in INS1 cells. Changes in promoter activity were measured as ratios of fluorescence obtained at minutes 240 and 60 after stimulation and are presented as mean values ⫾ sem (n⫽10). B) Western blot analysis of Shc coimmunoprecipitated with either wild-type IR-B or FTHM-mutant. INS1 cells were transfected with FLAG-tagged IR-B variants and stimulated with insulin for indicated periods of time. Immunoprecipitates from 1 mg lysate, obtained with anti-FLAG antibody, and 150 g lysate were probed with antibodies against phospho-tyrosine (antipTyr), IR -subunit (anti-IR) and Shc (anti-Shc). The p52Shc isoform is indicated by an arrow; *shows unspecific binding. A representative Western blot out of two independent experiments is shown. In compiled data, binding to IR-B-FLAG at 10 min is set as 100%.
explanation for the observed effect would be that Shc-S154P still binds to the IR via its SH2-domain but, because of the mutation of its PTB-domain and the resulting missing interaction with PI(3,4,5)P3 in the plasma membrane and/or the NPEpY-motif, does not become activated by the IR. Thus, it interferes with signaling by competing for IR interaction with intact endogenous Shc molecules. If this is to be true, then mutation of the SH2-domain, i.e., R397L (21), in Shc-S154P should abolish its interaction with IR and thus eliminate its dominant-negative effect. Indeed, expression of the double-mutant Shc-S154P, R397L, where both the PTB- and the SH2-domain were disrupted, resulted in a c-fos promoter activation similar to that observed in mock-transfected cells (Fig. 5G). These data suggest that, in contrast to IR-B-mediated activation of GK transcription, PI3K Ia activity is needed to recruit Shc to IR-B and to allow further signal transduction to activate the c-fos promoter. 1616
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c-fos promoter activation requires clathrin-dependent endocytosis of IR-B To investigate whether the signaling pathways leading to up-regulation of the GK and the c-fos promoter via IR-B originate from receptor complexes located in the same or different plasma membrane compartments, we used -cyclodextrin as a tool to differentially deplete cholesterol from plasma membrane domains and thereby to interfere with IR-B signaling. However, no differences were found in cholesterol dependency with regard to IR-B-mediated activation of GK vs. c-fos promoters in response to insulin (data not shown). Another possibility to explain how IR-B can activate different signaling cascades is that activation occurs in different cellular compartments, i.e., plasma membrane-standing vs. internalized receptor complexes. Ceresa and co-workers demonstrated that the dominant-interfering dynamin mutant dynamin-K44A abolishes clathrin-dependent endocytosis of IRs, which
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Figure 5. Involvement of PI3K Ia in insulin-dependent c-fos promoter activation. A, B) Effect of wortmannin on insulinstimulated c-fos promoter-driven DsRed and GK promoter-driven GFP expression in (A) INS1 cells and (B) in mouse islet cells. C) Western blot analysis of PI3K coimmunoprecipitated with wild-type IR-B or FTHM-mutant. INS1 cells were transfected with FLAG-tagged IR-B variants and stimulated with insulin for indicated periods of time. Immunoprecipitates obtained from 800 g lysate, obtained with anti-FLAG antibody, and 100 g lysate were probed with antibodies against phospho-tyrosine (antipTyr), IR -subunit (anti-IR), and PI3K Ia adaptor protein p85 (antip85). A representative Western blot out of two independent experiments is shown. In compiled data, binding to IR-B-FLAG at 10 min is set as 100%. (D-G) Effects of (D) dominant-negative p85 (⌬p85), (E,F) siRNA against p85 (p85-siRNA) and (G) Shc-mutants (Shc-S154P and Shc-S154P,R397L) on insulin-stimulated c-fos promoter-driven DsRed and GK promoter-driven GFP expression. D, G) INS1 cells were cotransfected with c-fos.DsRed/ GK.GFP, and either empty vector (mock), ⌬p85 or Shc-variants. E, F) MIN6 cells were cotransfected with c-fos.DsRed/GK.GFP and either control siRNA (mock) or p85-siRNA. F) Effect of p85-siRNA treatment on p85 protein levels. MIN6 cells were transfected with either control siRNA (mock) or p85-siRNA and protein levels were analyzed by Western blotting 96 h after transfection. p85 protein levels were shown in relation to GAPDH protein levels. The average effect in three independent experiments was a reduction in p85 levels to 41 ⫾ 4.6% (mean⫾sem) compared to control siRNA treated cells. A, B, D, E, G) Changes in promoter activities were measured as the ratio of fluorescence obtained minutes 240 and 60 after stimulation with insulin and are presented as mean values ⫾ sem (n⫽9).
leads to a reduction in insulin-meditated Shc tyrosine phosphorylation in HII4E cells (28). By using dynamin2K44A as a tool, we have shown that insulin activates GK gene transcription by signaling from plasma membrane-standing IR-B (6). To test whether insulin-dependent activation of the c-fos promoter requires internalization of IR-B, we studied its promoter activation in -cells that were transiently transfected with dynamin2K44A. As shown in Fig. 6A, expression of dynamin2K44A abolished insulin-stimulated c-fos promoter activation, thereby indicating that up-regulation of c-fos is dependent on IR-B internalization. Two major endocytic pathways are facilitated by dynamin-2, i.e., endocytosis via clathrin-coated vesicles and clathrin-indepenSELECTIVE IR-B-MEDIATED SIGNALING
dent endocytosis via caveosomes (29). To test whether insulin-stimulated c-fos gene transcription requires IR-B internalization by caveolar endocytosis, we overexpressed caveolin-1 and -2. -cells express caveolin-1␣, -1, and -2 (6). We found that expression of caveolin-1␣ or -1, respectively, or the combination of either of them with caveolin-2, did not interfere with insulindependent c-fos promoter-driven GFP expression (data not shown). To verify whether c-fos promoter activation requires IR-B internalization via the clathrin-mediated endocytic pathway, we coexpressed a dominant-negative mutant of the adaptor protein-2 (AP-2) subunit 2, i.e., 2T156A (30). Adaptor proteins, such as AP-2, attach 1617
Figure 6. Receptor endocytosis-dependent c-fos promoter activation. A) Effect of dominant-interfering dynamin-2K44A and 2T156A on insulin-stimulated c-fos and GK promoter activation. INS1 cells were transfected with c-fos.DsRed/GK.GFP and with either an empty plasmid (mock), dyn-2K44A or 2T156A. B) Activated IR-B in plasma membrane (PM) and early endosomes (EE). INS1 cells were transfected with either empty plasmid (mock) or IR-BFLAG. Cells were lysed after insulin stimulation, PM and EE were prepared as in Materials and Methods and analyzed by Western blotting. The endogenous pYIR() is indicated by an arrow. A representative Western blot out of three independent experiments is shown. C, D) Effect of mutation of putative internalization motifs on IR-B internalization. INS1 cells were transfected with vectors for FLAG-tagged IR-B, IR-BAPLA, IR-BRDAA, IR-BITAA, or IRBAPLA⫹ITAA. Following biotinylation with sulfo-NHS-LC-biotin, cells were treated or not with insulin for 20 min and, thereafter, treated or not with Pronase, as indicated. Following cell lysis, IR-B variants were immunoprecipitated using anti-FLAG antibodies and immunoprecipitates were analyzed by Western blotting using antiIR␣-antibodies (N-20) and HRP-conjugated streptavidin. One representative series of immunoblots out of three is shown in (C). The calculated data are shown as mean values ⫾ sem (n⫽3) in (D). E) Effect of endocytosis-deficient IR-B mutants on c-fos and GK promoter activation. INS1 cells were cotransfected with c-fos.GFP, GK.GFP and with either empty vector (mock) or vectors for FLAG-tagged IR-B, IR-BAPLA, IR-BRDAA, IR-BITAA or IRBAPLA⫹ITAA. A, E) Changes in promoter activities were measured as the ratio of fluorescence obtained at minutes 240 and 60 after stimulation with insulin and are presented as mean values ⫾ sem (n⫽10).
clathrin to the membrane, select the vesicle cargo, e.g., the receptor, and recruit accessory proteins that regulate clathrin-coated pit formation (31). Phosphorylation of the AP-2 subunit 2 at threonine 156 has been demonstrated to be essential for efficient endocytosis in vitro and in vivo (30). As shown in Fig. 6A, expression of 2T156A led to a complete loss in up-regulation of c-fos promoter activity, indicating that this pathway requires clathrin-dependent endocytosis of IR-B. Both, dynamin2K44A and 2T156A had no effect on IR-B-mediated activation of GK transcription (Fig. 6A). That the endocytosed IR-B is signaling competent is shown by the fact that insulin-stimulation resulted in activated, i.e., autophosphorylated, FLAG-tagged IR-B in both plasma membrane and early endosome preparations of transfected INS1 cells (Fig. 6B). Five motifs within the -subunit of the IR have been reported so far to be involved in IR endocytosis, i.e., GPLY (32, 33), NPEY (32, 33), ITLL (10), RDII (10) and YTHM (34). Our data so far suggest that the C-terminal YTHM-motif and possibly the juxtamembrane NPEY-motif are required for IR-B-mediated up1618
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regulation of c-fos promoter activity. Although we cannot rule out the importance of these motifs for IR-B endocytosis, their involvement in PI3K Ia binding/ activation and subsequent Shc binding makes it unlikely that they are simultaneously involved in AP-2 binding and clathrin-coated pit formation. To test whether GPLY, ITLL, and RDII are required for IR-B endocytosis in insulin-producing -cells, we performed cell-surface biotinylation analysis using FLAG-tagged wild-type IR-B and IR-B variants with individually mutated internalization motifs, i.e., APLA, ITAA, or RDAA. Data in Fig. 6C and D show that mutation of GPLY or ITLL reduces IR-B internalization while mutation of RDII enhanced insulin-stimulated endocytosis of this IR-B mutant. The combined mutation of GPLY and ITLL, i.e., APLA/LLAA, led to maximal inhibition of IR-B endocytosis. Expression of IR-BAPLA/LLAA abolished further up-regulation of c-fos promoter activity by insulin (Fig. 6E). In contrast, mutation of RDII, which positively affected IR-B endocytosis, also allowed the up-regulation of c-fos promoter (Fig. 6E). Expression of these mutants had no effect on insulin-stimulated GK promoter
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activation in the same cell, which served as the control for nonendocytosis-dependent IR-B signaling. Taken together these data suggest that the signaling pathway leading to the up-regulation of c-fos gene transcription requires the sequential binding of PI3K Ia and Shc to IR-B located at the plasma membrane and that internalization of the IR-B/Shc-complex is mediated by clathrin-coated vesicle formation involving the intact GPLY- and/or ITLL-motifs of IR-B and participation of AP-2. c-fos promoter activation requires IR-B signaling from early endosomes Following endocytosis, IR-B, in contrast to IR-A, has been shown to undergo almost no recycling but is targeted to lysosomal degradation (35, 36). The targeting of transport vesicles such as clathrin-coated vesicles to their acceptor compartment is tightly regulated by members of the Rab family, small GTP-binding proteins that are known to be involved in the regulation of membrane trafficking in the endocytic and exocytic pathways (37, 38). We used the expression of dominant-interfering Rab variants as a tool to study the involvement of early vs. late endosomes in IR-B-mediated c-fos gene transcription. Rab7 is important for the regulation of late endocytic trafficking, i.e., between late endosomes and lysosomes. To test, whether late endosomes are involved in IR-B-mediated c-fos gene transcription, we studied the effect of dominant-positive Rab7Q79L and dominant-negative Rab7T22N and Rab7N125I (39). The functionality of these Rab7 variants was tested by confocal microscopy in COS1 cells, which allowed the use of LysoTracker Red as a marker for lysosomes (data not shown). This approach is not possible in -cells because lysotracker here also stains the acidic insulin granules. In agreement with earlier publications (39), expression of mCFP-tagged wild-type and active (Q67L) variants exhibited a distinct pattern with the lysosome marker surrounded by a ring of mCFP-Rab7. COS1 cells and INS1 cells showed increased lysosomal size when wild-type Rab7 or the active mutant were expressed while the dominant-negative variants (N125I and T22N) remained dispersed in the cytosol. However, coexpression of myc-tagged dominant-active and dominant-negative Rab7 variants together with c-fos.DsRed/GK.GFP did not have any effect on insulin-stimulated up-regulation of the c-fos promoter (Fig. 7A). This indicates that the signaling cascade that activates the MAPK pathway does not originate from the late endosome pool. Another possibility is that signaling originates from the early endosome pool. Rab5 is located on the cytoplasmic surface of early endosomes and is a key component of the protein complex responsible for homotypic fusion of early endosomes and cargo sorting in these organelles (40). When we expressed mCFPtagged wild-type and dominant-active variants, i.e., Q79L, of Rab5a and Rab5b in COS1 and INS1 cells, we found an increase in the size of early endosomes, while SELECTIVE IR-B-MEDIATED SIGNALING
Figure 7. The role of Rab7, Rab5a and Rab5b in insulinstimulated c-fos promoter activation. A) Effects of expressed active (Rab7Q67L) and dominant-interfering (Rab7T22N) Rab7 variants on insulin-stimulated c-fos promoter-driven DsRed and GK promoter-driven GFP expression. INS1 cells were transfected with c-fos.DsRed/GK.GFP and with either an empty plasmid (mock), Rab7Q67L or Rab7T22N (n⫽9). B) Effects of expressed dominant-interfering Rab5a and Rab5b on insulin-stimulated c-fos promoter-driven DsRed and GK promoter-driven GFP expression. INS1 cells were transfected with c-fos.DsRed/GK.GFP and with either an empty plasmid (mock), Rab5aS34N or Rab5bS34N and stimulated with insulin. Changes in promoter activity were measured as ratios of fluorescence obtained at 240 min vs. 60 min after stimulation and are presented as mean values ⫾ sem (n⫽9). C) Colocalization of IR-B with EEA1/endosomes. The effects of expressed endocytosis-interfering proteins on the EEA1positive pool and on the colocalization of EEA1/endosomes and IR-B-FLAG were analyzed by laser scanning confocal microscopy. INS1 cells were transfected with expression plasmids encoding FLAG-tagged IR-B (wild-type) in combination with an empty vector (mock), dyn-2K44A, 2T156A, Rab5aS34N, or Rab5bS34N, fixed and treated with antibodies against FLAG and EEA1 (n⫽15). The results are presented as mean values ⫾ sem. Prior to fixation, cells were treated, or not, with insulin.
expression of the dominant-negative acting mutant S34N was dispersed throughout the cytosol (data not shown). This pattern of Rab5 variants is in agreement with earlier observations (41). To test whether the interfering versions of Rab5a and/or Rab5b had any effect on insulin-stimulated c-fos promoter activation, we coexpressed them with c-fos.DsRed/GK.GFP. Interestingly, dominant-negative Rab5b did not lead to a decrease in c-fos promoter activity while, in contrast, expression of dominant-negative Rab5a abolished c-fos promoter activation (Fig. 7B). This is in agreement with the finding that expression of Rab5aS34N impaired colocalization of FLAG-tagged IR-B with EEA1 in coimmunolocalization studies in 1619
INS1 cells, while expression of Rab5bS34N did not affect IR-B/EEA1 colocalization (Fig. 7C). A similar Rab5-isoform dependency was noted in (41), showing that epidermal growth factor (EGF)-induced endocytosis of the EGF receptor requires Rab5a. The underlying molecular mechanisms involved in this isoform dependency remain unclear. We conclude from these data that insulin-stimulated c-fos gene transcription is not only dependent on clathrin-mediated internalization of IR-B, but also that the signal responsible for the activation of the Shc/ MEK1/ERK cascade originates from the endocytosed IR-B located in early endosomes.
DISCUSSION A key to understand the molecular mechanisms that underlie the selective action of insulin is to define the spatio-temporal segregation of insulin signaling at the cellular level (3). We have previously shown that in the pancreatic -cell selectivity in insulin signaling can be gained by signal transduction through the two isoforms of IR, which are situated in different plasma membrane microdomains. While signaling through IR-A and IRS/ PI3K Ia/p70s6k activates transcription of the insulin gene, signaling through IR-B and PI3K-C2␣-like/ PDK1/PKB is required to activate the GK gene (5, 6). Therefore, plasma membrane compartmentalization of receptor isoforms with the subsequent access to a defined pool of adaptor/signaling proteins may represent one mechanistic basis for selective signaling. But how can selectivity be achieved when signal transduction involves the same receptor isoform? In other words, do different, selective signaling cascades originating from the same IR isoform coexist in the same cell? In the present study we show that selectivity in signal transduction through the same IR isoform, here IR-B, can be gained by the involvement of different binding motifs within the receptor and by signal transduction from different cellular compartments. Employing online monitoring of insulin-stimulated promoter activation at the single cell level revealed that the GK gene is activated by plasma membrane-standing IR-B, which trigger signal transduction via PI3K-C2␣-like/ PDK1/PKB. However, the signal that leads to upregulation of c-fos gene transcription originates from the same but now endocytosed IR-B isoform, involving signaling via the PI3K Ia/Shc/MEK1/ERK cascade. The observation that overexpression of IR-B, thus enriching the IR-B receptor pool, leads to an enhanced promoter activation of both GK and c-fos genes simultaneously in the same cell makes signaling from IR-A/ IR-B hybrid receptors unlikely. But is it possible that the same IR-B molecule activates GK and c-fos genes in a sequential way, i.e., first activating the GK gene while still being situated in the plasma membrane and, thereafter, following its internalization, activating the c-fos gene from early endosomes? Since the two signaling pathways do not depend on each other they could 1620
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result from different IR-B molecules. However, the following observations would support the view that signaling may originate from the same receptor molecule. The fact that the activation of both promoters is sensitive to the same degree of cholesterol depletion from the plasma membrane suggests that the B-type receptors activating the two genes are localized in the same plasma membrane microdomain. The activation of the two genes involves different regions of the B-type receptor -subunit. While activation of the GK gene requires the integrity of the juxtamembrane NPEYmotif, c-fos gene activation requires the sequential binding of first PI3K Ia and then Shc to the C-terminal YTHM-motif of IR-B. This is in accordance with the finding that organization of the signaling complex that is involved in the activation of the c-fos gene already starts at the plasma membrane. The two signaling pathways involve different PI3K activities, which would allow selectivity in the initial events at the plasma membrane. While IR-B-mediated activation of GK gene transcription requires a class II PI3K-C2␣-like activity that preferentially generates PI(3,4)P2 and allows the subsequent activation of PDK1/PKB, up-regulation of the c-fos promoter involves the activity of PI3K Ia, which preferentially generates PI(3,4,5)P3 and in our model would allow recruitment of Shc to the receptor. However, recruitment of Shc to IR-B at the plasma membrane is not sufficient to activate c-fos gene transcription; this is achieved only after clathrin-mediated internalization of the receptor complex and by signal transduction from the early endosome pool. Although these observations would allow signaling via the same receptor molecule, we cannot rule out the possibility that signaling occurs through different IR-B molecules. The potential requirement of the NPEYmotif in both signaling cascades would argue for the latter scenario. Future research allowing single receptor resolution in live-cell imaging will be needed to provide further evidence to clarify this matter. Taken together, we now demonstrate that selectivity in insulin signaling via the same receptor isoform can be gained by signal transduction from different cellular compartments, i.e., plasma membrane-standing vs. internalized receptors. Thus, our data allow a mechanistic understanding of how selective signaling via the same receptor isoform in the same cell simultaneously activates different signaling pathways in response to the same stimulus, here exemplified by insulin. This work was supported by funds from Karolinska Institutet and by grants from the Swedish Diabetes Association, the Swedish Research Council, the Novo Nordisk Foundation, Berth von Kantzow’s Foundation, Juvenile Diabetes Research Foundation (JDRF), EuroDia (FP6 –518153), and the Family Stefan Persson Foundation.
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