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• W2014380041 abstract "The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSA XXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5′-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion. The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSA XXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5′-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion. glucokinase regulatory protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase glucokinase rapid amplification of cDNA ends nickel-nitrilotriacetic acid enzyme-linked immunosorbent assay polymerase chain reaction activation domain binding domain analysis of variance kilobase(s) digoxigenin long distance synthetic dropout The low affinity glucose-phosphorylating enzyme glucokinase (hexokinase type IV) plays a pivotal role for the coupling of physiological millimolar glucose concentration changes to glycolysis in liver, pancreatic beta cells, and neuroendocrine cells (1Meglasson M.D. Matschinsky F.M. Am. J. Physiol. 1984; 246: E1-E13Crossref PubMed Google Scholar, 2Lenzen S. Panten U. Biochem. Pharmacol. 1988; 37: 371-378Crossref PubMed Scopus (66) Google Scholar, 3Matschinsky F. Liang Y. Kesavan P. Wang L. Froguel P. Velho G. Cohen D. Permutt M.A. Tanizawa Y. Jetton T.L. Niswender K. Magnuson M.A. J. Clin. Invest. 1993; 92: 2092-2098Crossref PubMed Scopus (259) Google Scholar, 4Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 5Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 6Magnuson M.A. Niswender K.D. Pettepher C.C. Draznin B. LeRoith D. Molecular Biology of Diabetes, Part I. Humana Press, Totowa, NJ1994: 155-174Google Scholar, 7Magnuson M.A. Diabetes. 1990; 39: 523-527Crossref PubMed Google Scholar). In pancreatic beta cells glucokinase, acting as a glucose sensor, catalyzes the rate-limiting step for glucose-stimulated insulin secretion (1Meglasson M.D. Matschinsky F.M. Am. J. Physiol. 1984; 246: E1-E13Crossref PubMed Google Scholar, 2Lenzen S. Panten U. Biochem. Pharmacol. 1988; 37: 371-378Crossref PubMed Scopus (66) Google Scholar, 4Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 8Matschinsky F.M. Diabetologia. 1993; 36: 1215-1217Crossref PubMed Scopus (16) Google Scholar). Studies in glucokinase knock-out mice, as well as the metabolic profile of diabetic patients with mutations of the glucokinase gene, provide evidence that the glucose sensor function of this enzyme in pancreatic beta cells can be fulfilled only within a narrow range of enzyme activity (5Matschinsky F.M. Diabetes. 1996; 45: 223-241Crossref PubMed Scopus (0) Google Scholar, 9Bali D. Svetlanov A. Lee H.W. Fuscodemane D. Leiser M. Li B.J. Barzilai N. Surana M. Hou H. Fleischer N. Depinho R. Rossetti L. Efrat S. J. Biol. Chem. 1995; 270: 21464-21467Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Grupe A. Hultgren B. Ryan A. Ma Y.H. Bauer M. Stewart T.A. Cell. 1995; 83: 69-78Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 11Velho G. Blanche H. Vaxillaire M. Bellanne-Chantelot C. Pardini V.C. Timsit J. Passa P. Deschamps I. Robert J.J. Weber I.T. Marotta D. Pilkis S.J. Lipkind G.M. Bell G.I. Froguel P. Diabetologia. 1997; 40: 217-224Crossref PubMed Scopus (221) Google Scholar, 12Permutt M.A. Chiu K.C. Tanizawa Y. Diabetes. 1992; 41: 1367-1372Crossref PubMed Google Scholar). Thus principles of posttranslational regulation of beta cell glucokinase have gained special interest in recent years (13Chen C. Hosokawa H. Bumbalo L.M. Leahy J.L. J. Clin. Invest. 1994; 94: 1616-1620Crossref PubMed Scopus (57) Google Scholar, 14Chen C. Bumbalo L. Leahy J.L. Diabetes. 1994; 43: 684-689Crossref PubMed Scopus (35) Google Scholar, 15Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar, 16Noma Y. Bonner Weir S. Latimer J.B. Davalli A.M. Weir G.C. Endocrinology. 1996; 137: 1485-1491Crossref PubMed Scopus (48) Google Scholar, 17Liang Y. Jetton T.L. Zimmerman E.C. Najafi H. Berner D.K. Matschinsky F.M. Magnuson M.A. Diabetes. 1994; 43: 1138-1145Crossref PubMed Scopus (21) Google Scholar, 18Liang Y. Najafi H. Matschinsky F.M. J. Biol. Chem. 1990; 265: 16863-16866Abstract Full Text PDF PubMed Google Scholar). Liver glucokinase is regulated by insulin and glucagon on the transcriptional and translational level and by the glucokinase regulatory protein (GRP)1 via translocation between cytosol and nucleus (6Magnuson M.A. Niswender K.D. Pettepher C.C. Draznin B. LeRoith D. Molecular Biology of Diabetes, Part I. Humana Press, Totowa, NJ1994: 155-174Google Scholar, 19Iynedjian P.B. Biochem. J. 1993; 293: 1-13Crossref PubMed Scopus (279) Google Scholar, 20Van Schaftingen E. Diabetologia. 1994; 37 (Suppl. 2): S43-S47Crossref PubMed Scopus (62) Google Scholar, 21Van Schaftingen E. Detheux M. Veiga da Cunha M. FASEB J. 1994; 8: 414-419Crossref PubMed Scopus (206) Google Scholar, 22Agius L. Peak M. Biochem. Soc. Trans. 1997; 25: 145-150Crossref PubMed Scopus (19) Google Scholar, 23Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In particular this translocation confers a short term adaptation of glucose metabolism to changes of the glucose concentration in the physiological range (20Van Schaftingen E. Diabetologia. 1994; 37 (Suppl. 2): S43-S47Crossref PubMed Scopus (62) Google Scholar, 21Van Schaftingen E. Detheux M. Veiga da Cunha M. FASEB J. 1994; 8: 414-419Crossref PubMed Scopus (206) Google Scholar, 22Agius L. Peak M. Biochem. Soc. Trans. 1997; 25: 145-150Crossref PubMed Scopus (19) Google Scholar, 23Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 24Shiota C. Coffey J. Grimsby J. Grippo J.F. Magnuson M.A. J. Biol. Chem. 1999; 274: 37125-37130Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 25de la Iglesia N. Veiga-da-Cunha M. Van Schaftingen E. Guinovart J.J. Ferrer J.C. FEBS Lett. 1999; 456: 332-338Crossref PubMed Scopus (82) Google Scholar). In pancreatic beta cells the situation is different from that in liver. The glucokinase activity level in insulin-producing cells is regulated by glucose. However, this modulation is not mediated by the liver type GRP, which was not detected in pancreatic beta cells (13Chen C. Hosokawa H. Bumbalo L.M. Leahy J.L. J. Clin. Invest. 1994; 94: 1616-1620Crossref PubMed Scopus (57) Google Scholar,15Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar, 17Liang Y. Jetton T.L. Zimmerman E.C. Najafi H. Berner D.K. Matschinsky F.M. Magnuson M.A. Diabetes. 1994; 43: 1138-1145Crossref PubMed Scopus (21) Google Scholar, 18Liang Y. Najafi H. Matschinsky F.M. J. Biol. Chem. 1990; 265: 16863-16866Abstract Full Text PDF PubMed Google Scholar). Studies with digitonin-permeabilized insulin-producing RINm5F cells provide evidence that glucokinase interacts with an as yet unidentified protein factor capable of modulating the activity (15Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar). The existence of this protein is supported by morphological data that report changes in the spatial distribution of glucokinase immunostaining in response to the nutritional status (16Noma Y. Bonner Weir S. Latimer J.B. Davalli A.M. Weir G.C. Endocrinology. 1996; 137: 1485-1491Crossref PubMed Scopus (48) Google Scholar, 26Jörns A. Tiedge M. Lenzen S. Virchows Arch. 1999; 434: 75-82Crossref PubMed Scopus (20) Google Scholar). To identify proteins that interact with glucokinase, we used a random peptide phage display library to select epitopes with high binding affinity. Through this strategy two glucokinase-binding peptide consensus sequences were identified that showed a high homology to (i) the GRP of the liver (27Detheux M. Vandekerckhove J. Van Schaftingen E. FEBS Lett. 1993; 321: 111-115Crossref PubMed Scopus (31) Google Scholar) and (ii) to the bifunctional regulatory enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) (28Pilkis S.J. Claus T.H. Kurland I.J. Lange A.J. Annu. Rev. Biochem. 1995; 64: 799-835Crossref PubMed Scopus (226) Google Scholar, 29Okar D.A. Manzano A. Navarro-Sabate A. Riera L. Bartrons R. Lange A.J. Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). In particular, the binding of glucokinase to PFK-2/FBPase-2 may help to explain recent observations of metabolic channeling of glycolysis in liver (30Cascante M. Centelles J.J. Agius L. Biochem. J. 2000; 352: 899-905Crossref PubMed Google Scholar). The interaction of glucokinase with PFK-2/FBPase-2 might as well have a regulatory role in metabolic stimulus-secretion coupling in beta cells because the bifunctional enzyme is also expressed in pancreatic islets. Collagenase P, restriction enzymes, the SP6/T7 transcription kit and the DIG nucleic acid detection kit were obtained from Roche Molecular Biochemicals. Hybond N nylon membranes, the ECL detection system, and the autoradiography films were fromAmersham Pharmacia Biotech. Guanidine thiocyanate was from Fluka (Neu-Ulm, Germany). Restriction enzymes and modifying enzymes for the cloning procedures were from New England Biolabs (Beverly, MA) or Fermentas (Fermentas, St. Leo-Rot, Germany). Custom oligonucleotide synthesis was done by Life Technologies, Inc. or MWG Biotech (Ebersberg, Germany). Media and supplements for culture of yeast were from CLONTECH (Palo Alto, CA). Columns for DNA purification were from Qiagen (Hilden, Germany). All other reagents of analytical grade were from Merck. Human beta cell glucokinase protein was expressed in M15 Escherichia coli bacteria and purified by Ni-NTA metal chelate chromatography as described (31Tiedge M. Krug U. Lenzen S. Biochim. Biophys. Acta. 1997; 1337: 175-190Crossref PubMed Scopus (32) Google Scholar). The recombinant protein was immobilized by His tag on Ni-NTA-HisSorbTM strips (Qiagen). 200 μl of the protein solution (50 μg/ml glucokinase protein in 0.1m NaHCO3) were incubated overnight at 4 °C with gentle agitation in the microplate wells. Thereafter the supernatant was removed, and the wells were blocked with 300 μl of blocking buffer (5 mg/ml bovine serum albumin in 0.1 mNaHCO3) for 1 h at 4 °C with gentle agitation. The selection of peptides able to bind to glucokinase was performed with the Ph.D.-12TM phage display peptide library (New England Biolabs). The combinatorial library of random 12-mer peptides was incubated in glucokinase-coated and blocked wells for 1 h at room temperature. A control experiment using only blocked wells was also performed to exclude unspecific binding. The following biopanning procedure was carried out according to the instructions in the manufacturer's manual. Specifically bound phages were eluted by disruption with glycine buffer or by incubation with 100 μl of the protein solution (200 μg/ml glucokinase protein in 0.1 m NaHCO3). The recovered phages were amplified in ER2537 E. coli bacteria, and the phage titer was expressed as plaque-forming units. The whole procedure was repeated three to four times, and individual phage clones of each panning were selected. Phage DNA of single colonies were prepared using the QIAprep Spin M13 kit (Qiagen) and characterized by dideoxy chain sequencing (32Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar). The programs PeptideSearch (EMBL, Protein & Peptide Group, Heidelberg, Germany; www.mann.embl-heidelberg.de) and Bic2 (EBI, Wellcome Trust Genome Campus, Cambridge, UK; www.ebi.ac.uk) were used for homology searches comparing amino acid sequences of glucokinase interacting peptides with sequences listed in the data bases Swall, Swissprot, and Trembl. The conditions in the ELISA experiments were essentially the same as those used in the panning procedure, and all washing steps were performed as described in the manufacturer's manual. Blocking buffer was prepared with 2.5% skimmed milk in Tris-buffered saline. 2-fold serial dilution of consensus sequence displaying phages starting from 1015 phages/well were analyzed for binding to glucokinase. 200 μl of horseradish peroxidase-conjugated anti-M13 antibody (Amersham Pharmacia Biotech; diluted 1:5000 in blocking solution) was added to each well, and bound phages were visualized using 2′,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) in citrate buffer as substrate for the peroxidase. The A405 values were measured after 20 min of incubation using a htIII microplate reader (Anthos, Köln, Germany). 2 μg of total RNA from freshly isolated islets from Wistar rats were subjected to cDNA synthesis by LD PCR (18 cycles) according to the protocol of the SMART cDNA library construction kit (CLONTECH). 1 or 5 μl from the spin column-purified islet cDNA (corresponding to 50 and 250 ng of cDNA) were used for the specific PFK-2/FBPase-2 RACE PCR reaction (3 min of initial denaturation at 94 °C; 25 cycles of 30 s at 94 °C, 30 s at 55 °C or 58 °C, and 60 s at 72 °C; 5 min of end extension at 72 °C in a total volume of 100 μl) with the SMART 5′ PCR forward primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and the rat PFK-2/FBPase-2 consensus reverse primer (5′-(G/C)AC(A/G)TGGAT(A/G)TTCAT-(G/C)AGGTA(A/G)TA-3′), which contained wobble bases according to the various isoforms. 10 μl of the PFK-2/FBPase-2 RACE PCR products were separated in a 1% Sea-Plaque GTG low melting gel. Specific bands were sliced from the gel (20–30 μl) and used for Taq polymerase-amplified cloning into the pTOPO vector (Invitrogen, Groningen, The Netherlands) according to the manufacturer's protocol. The sequences of the PFK-2/FBPase-2 RACE PCR fragments were obtained by the dideoxy chain termination method (32Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar) using a LiCor 4200 automated sequencer (MWG Biotech). Homology plots were generated by Fasta3 and NCBI-Blast2 searches (EBI) on the basis of GenBankTM cDNA sequences of different PFK-2/FBPase-2 isoforms from the rat. A full-length rat islet PFK-2/FBPase-2 cDNA was amplified from LD islet cDNA with specific primers coding for the brain isoform of rat PFK-2/FBPase-2 (5′-TAGCAGGATCCATGTCTGAGAATAGTACATTTTCCA-3′; 5′-TAGTAGTCGACTCAGGAGAGCAAAGTGAGG-3′) and subcloned with BamHI and SalI restriction sites into the pQE30 vector (Qiagen) followed by sequence analysis. Yeast two-hybrid analysis was performed with the Matchmaker GAL4 system 2 (CLONTECH) as described in the manufacturer's manual. Cloning procedures were performed as described previously (33Sambrook J. Fritsch E.F. Maniatis T. Nolan C. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar,34Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1998Google Scholar). The coding cDNA sequence of the human beta cell glucokinase was amplified by PCR from the pQE30-GK plasmid (31Tiedge M. Krug U. Lenzen S. Biochim. Biophys. Acta. 1997; 1337: 175-190Crossref PubMed Scopus (32) Google Scholar) with primers containing NcoI and XhoI restriction sites and subcloned in frame to the activation domain (AD) into pACT2 (CLONTECH). PFK-2/FBPase-2 coding cDNAs for rat liver (35Darville M.I. Crepin K.M. Vandekerckhove J. Van Damme J. Octave J.N. Rider M.H. Marchand M.J. Hue L. Rousseau G.G. FEBS Lett. 1987; 224: 317-321Crossref PubMed Scopus (60) Google Scholar) (GenBankTM accession number Y00702) and rat brain/islet (36Watanabe F. Sakai A. Furuya E. Uyeda K. Biochem. Biophys. Res. Commun. 1994; 198: 335-340Crossref PubMed Scopus (30) Google Scholar) (GenBankTM accession number S67900) were amplified by PCR from the corresponding pQE30 plasmid with composite rimers containing SmaI and BamHI restriction sites and subcloned in frame to the binding domain (BD) into pAS2-1 (CLONTECH). Additionally the kinase domain (amino acid residues 1–257) and the bisphosphatase domain (amino acid residues 258–470) of the liver PFK-2/FBPase-2 were subcloned separately into the pAS2-1 (BD) vector by the same method. All constructs were verified by sequence analyses of the inserts. The yeast Saccharomyces cerevisiae strain CG1945 (CLONTECH) was simultaneously transformed with pACT2 or pACT2-GK and each of the pAS2-1-GRP wild type and mutant plasmids by using the lithium acetate procedure (37Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2883) Google Scholar). Activation of the HIS3 reporter construct was determined by growth on selection SD agar plates lacking leucine, tryptophan, and histidine after 5 days. For the quantitative measurement of β-galactosidase reporter gene activity, the host strain Y190 (CLONTECH) was transformed in the same way, and yeast was grown on SD agar plates without leucine and tryptophan. The resulting transformed yeast clones were grown in SD medium lacking leucine and tryptophan overnight. The yeast protein was isolated from the cell pellet by vigorous vortexing with glass beads (Roth, Karlsruhe, Germany) in phosphate buffer as described (38Ciriacy M. Breitenbach I. J. Bacteriol. 1979; 139: 152-160Crossref PubMed Google Scholar). Insoluble material was pelleted at 14,000 rpm and 4 °C for a period of 30 s. Protein content of the solution was determined using a Bio-Rad protein assay. For the quantitative chemiluminescent β-galactosidase reporter gene assay, the protein solution was added to Galacto Star reaction buffer (Tropix, Bedford, MA) as described by the manufacturer. The light emission was recorded as a 1-s integral in a microplate using a Victor2luminometer (Wallac, Freiburg, Germany), and the specific activity was calculated in relation to β-galactosidase standard values and protein content. Pancreatic islets from fed male Wistar rats (300–400 g of body weight) were isolated by collagenase digestion. When other tissues were used they were also taken from Wistar rats. Rat pancreatic islets were purified by Ficoll gradient centrifugation and were used immediately for isolation of RNA. Total RNA from rat tissues was isolated by a combined water saturated phenol-chloroform-isoamyl alcohol extraction with an addition of ultrapure glycogen to achieve full precipitation of islet RNA (39Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63087) Google Scholar). 20 μg of total RNA/lane was subjected to electrophoresis on denaturing formamide/formaldehyde 1% agarose gels and transferred to nylon membranes. Hybridization was performed as described before (15Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar) using 11-DIG-UTP-labeled antisense cRNA probes coding for rat liver PFK-2/FBPase-2 and rat islet/brain PFK-2/FBPase-2 (36Watanabe F. Sakai A. Furuya E. Uyeda K. Biochem. Biophys. Res. Commun. 1994; 198: 335-340Crossref PubMed Scopus (30) Google Scholar). The DIG-labeled hybrids were detected by an enzyme-linked immunoassay using an anti-DIG-alkaline-phosphatase antibody conjugate. The hybrids were visualized by chemiluminescence detection on a light-sensitive film for quantification by densitometry with the National Institutes of Health Image 1.58 program. Ribosomal bands were used as control markers for gel loading (not shown). The data are expressed as the mean values ± S.E. Statistical analyses were performed by ANOVA followed by Bonferroni's test for multiple comparison using the Prism analysis program (Graphpad, San Diego, CA). The sequence homologies between the islet and the liver PFK-2/FBPase-2 proteins were calculated by the DNASIS V2.1 program (Amersham Pharmacia Biotech). A 12-mer random peptide M13 phage display library was used to screen immobilized human beta cell glucokinase for potential interaction partners. For this purpose recombinant glucokinase protein was fixed through the N-terminal His tag sequence to Ni-NTA-coated microplates. Aliquots of the phage library were allowed to interact with recombinant protein at room temperature for 1 h followed by wash procedures of increasing stringency (panning). Bound phages were recovered and reamplified for the next round of panning. Sequence analysis of enriched phages from the second to the fourth round of panning showed peptide inserts that matched to the consensus sequences SLKVWT (TableI, part A) and LSA XXVAG (TableI, part B). Of the 150 phage clones sequenced, 102 clones revealed the sequence motif LSAIVAG, indicating a strong interaction with the glucokinase protein (Table I). Through screening of the peptide data bases Swall, Swissprot, and Trembl by the PeptideSearch routine of the EMBL, the LSA XXVAG consensus motif showed a complete homology to the human (swissnew Q14397 GCKR human glucokinase regulatory protein) and rat (swissnew Q07071 GCKR rat glucokinase regulatory protein) GRP of the liver, which is a well characterized interaction partner of the glucokinase. The phage peptide sequence HGMKVWTLPATS, which was found in 10 clones by the panning procedure, could be aligned to the consensus motif SLKVWT (Table I, part A). Data base analysis revealed a complete match to the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, which is also known as phosphofructokinase type 2 (swissprot P0793 F261 rat 6PF-2-K/Fru-2,6-P2ase; swissprot P16118 F26L human 6PF-2K/Fru-2,6-P2ase). This bifunctional enzyme plays an important role for the metabolic and hormonal regulation of glycolysis and gluconeogenesis (or glucose-6-phosphate formation) in various tissues,e.g. liver, muscle, brain, and testis.Table ISequences of GK-binding peptides selected from a M13 phage display libraryThe sequences of peptides displayed by glucokinase-binding phage were isolated in three independent experiments each consisting of four rounds of high stringency panning from a 12-mer random peptide M13 phage display library. Two consensus peptide sequences A and B (shown with black background) were identified which were subjected to homology searches by the Peptide (EMBL) and Bic2 (EBI) program in the databases Swall, Swissprot, and Trembl. Amino acids 298–303 of the bifunctional enzyme PFK-2/FBPase-2 correspond to consensus sequence A, and amino acids 181–188 of the rat liver GRP correspond to consensus sequence B. Open table in a new tab The sequences of peptides displayed by glucokinase-binding phage were isolated in three independent experiments each consisting of four rounds of high stringency panning from a 12-mer random peptide M13 phage display library. Two consensus peptide sequences A and B (shown with black background) were identified which were subjected to homology searches by the Peptide (EMBL) and Bic2 (EBI) program in the databases Swall, Swissprot, and Trembl. Amino acids 298–303 of the bifunctional enzyme PFK-2/FBPase-2 correspond to consensus sequence A, and amino acids 181–188 of the rat liver GRP correspond to consensus sequence B. In the next step the interaction of the PFK-2/FBPase-2 and GRP consensus phage peptides were quantified by an ELISA binding assay (Fig. 1). The titration curves with the preferentially enriched phage clones HGMKVWTLPATS (corresponding to PFK-2/FBPase-2) and EYLSAIVAGPWP (corresponding to GRP) clearly show a significant binding affinity to immobilized glucokinase protein. The GRP motif exhibited a strong interaction with glucokinase that was significantly different from the background level at a concentration of 3.1 × 1013 phages/well (Fig. 1 B), whereas a significant binding of the PFK-2/FBPase-2 consensus motif was detectable at a concentration of 1.2 × 1014phages/well (Fig. 1 A). Control phages displaying 12-mer peptides with a random distribution of amino acids did not show any specific interaction with glucokinase protein (Fig. 1). Furthermore, the specific phages with the PFK-2/FBPase-2 and GRP consensus peptide sequences did not exhibit a specific binding to microplates that were blocked by skimmed milk. Recombinant glucokinase protein, which was eluted from the wells with 300 mm imidazoline, retained full catalytic activity, excluding nonspecific hydrophobic interaction with denatured glucokinase protein during the panning procedures (data not shown). To study the interaction of glucokinase with the novel interaction partner PFK-2/FBPase-2 and, for internal control, with the GRP of liver, the cDNAs were subcloned into the pAS2-1 (fusion to binding domain) or the pACT2 (fusion to activation domain) vector of the GAL4 system. Control experiments excluded nonspecific interactions of glucokinase, PFK-2/FBPase-2, and the GRP of liver with the truncated activation and binding domains of the vectors (data not shown). Furthermore, interactions of the proteins were not affected by the location within the binding domain or the activation domain. Quantitative analysis of the protein interactions by the β-galactosidase reporter enzyme clearly showed a strong binding of the GRP to glucokinase with a 12-fold increase of the reporter activity compared with background by the truncated activation domain and binding domain interaction (Fig. 2). The PFK-2/FBPase-2 protein exhibited a 4-fold increase of β-galactosidase activity above the background level (Fig. 2). The glucokinase protein did not bind to itself as shown by reporter activities that were not significantly different from the background level (Fig. 2). The PFK-2/FBPase-2 enzyme consists of two functional domains that confer the kinase and bisphosphatase activity. From the phage display peptide consensus motif the binding to glucokinase is conferred by the bisphosphatase domain. To verify this epitope for the separate domains of the bifunctional enzyme, yeast two-hybrid interaction studies were conducted with the kinase or the bisphosphatase domain fused to the binding domain of the pACT2 vector (Fig.3). These studies clearly indicated that the kinase domain of the PFK-2/ FBPase-2 enzyme did not interact with the glucokinase" @default.
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• W2014380041 date "2001-11-01" @default.
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• W2014380041 cites W1556483094 @default.
• W2014380041 cites W174714445 @default.
• W2014380041 cites W1788294099 @default.
• W2014380041 cites W1792398103 @default.
• W2014380041 cites W1875175786 @default.
• W2014380041 cites W1967746117 @default.
• W2014380041 cites W1973317343 @default.
• W2014380041 cites W1983011713 @default.
• W2014380041 cites W2000081372 @default.
• W2014380041 cites W2000889838 @default.
• W2014380041 cites W2006750837 @default.
• W2014380041 cites W2007569151 @default.
• W2014380041 cites W2011954709 @default.
• W2014380041 cites W2012400614 @default.
• W2014380041 cites W2012997795 @default.
• W2014380041 cites W2013642513 @default.
• W2014380041 cites W2015373835 @default.
• W2014380041 cites W2016627433 @default.
• W2014380041 cites W2016880720 @default.
• W2014380041 cites W2017019751 @default.
• W2014380041 cites W2017761421 @default.
• W2014380041 cites W2022842991 @default.
• W2014380041 cites W2022945083 @default.
• W2014380041 cites W2023069134 @default.
• W2014380041 cites W2026536182 @default.
• W2014380041 cites W2044980718 @default.
• W2014380041 cites W2045652861 @default.
• W2014380041 cites W2046282293 @default.
• W2014380041 cites W2047341705 @default.
• W2014380041 cites W2047355966 @default.
• W2014380041 cites W2049135773 @default.
• W2014380041 cites W2053864009 @default.
• W2014380041 cites W2070669901 @default.
• W2014380041 cites W2071585472 @default.
• W2014380041 cites W2072088248 @default.
• W2014380041 cites W2076413690 @default.
• W2014380041 cites W2080264337 @default.
• W2014380041 cites W2086660631 @default.
• W2014380041 cites W2092937437 @default.
• W2014380041 cites W2102193304 @default.
• W2014380041 cites W2103809654 @default.
• W2014380041 cites W2103845188 @default.
• W2014380041 cites W2112076791 @default.
• W2014380041 cites W2116828634 @default.
• W2014380041 cites W2132819281 @default.
• W2014380041 cites W2138270253 @default.
• W2014380041 cites W2139132103 @default.
• W2014380041 cites W2141606584 @default.
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• W2014380041 cites W2163627231 @default.
• W2014380041 cites W2166486785 @default.
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• W2014380041 cites W2325743399 @default.
• W2014380041 cites W31037396 @default.
• W2014380041 cites W4294216491 @default.
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