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Originally published In Press as doi:10.1074/jbc.M700517200 on June 8, 2007

J. Biol. Chem., Vol. 282, Issue 31, 22757-22764, August 3, 2007
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Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation*Formula

Lise Bjørkhaug{ddagger}§, Janne Molnes{ddagger}§, Oddmund Søvik{ddagger}, Pål Rasmus Njølstad{ddagger}, and Torgeir Flatmark||1

From the {ddagger}Department of Clinical Medicine, University of Bergen, N-5020 Bergen, §Center for Medical Genetics and Molecular Medicine and Department of Pediatrics, Haukeland University Hospital, N-5021 Bergen, and ||Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway

Received for publication, January 18, 2007 , and in revised form, June 1, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic beta-cells (isoform 1) and hepatocytes (isoforms 2 and 3). Although certain cellular proteins are known to either stimulate or inhibit its activity, little is known about post-translational modifications of this enzyme and their possible regulatory functions. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin-conjugating enzyme system of the rabbit reticulocyte lysate. Both isoforms were polyubiquitinated on at least two lysine residues, and mutation analysis indicated that multiple lysine residues functioned as redundant acceptor sites. Deletion of its C-terminal {alpha}-helix, as part of a ubiquitin-interacting motif, affected the polyubiquitination at one of the sites and resulted in a completely inactive enzyme. Evidence is presented that poly/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, the recombinant hGK was found to interact with and to be allosterically activated up to ~1.4-fold by purified free pentaubiquitin chains at ~100 nM (with an apparent EC50 of 93 nM), and possibly also by unidentified polyubiquitinated proteins assigned to their equilibrium binding to the ubiquitin-interacting motif site. The affinity of pentaubiquitin binding to hGK is regulated by the ligand (D-glucose)-dependent conformational state of the site. Both ubiquitination of hGK and its activation by polyubiquitin chains potentially represent physiological regulatory mechanisms for glucokinase-dependent insulin secretion in pancreatic beta-cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The glucose-phosphorylating enzyme glucokinase (GK)2 (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.2 [EC] ) plays a pivotal role in the regulation of glycolytic flux in hepatocytes and pancreatic beta-cells at physiological millimolar concentrations of glucose (Glc). This 50-kDa size monomeric hexokinase catalyzes the phosphorylation of D-glucose to form glucose 6-phosphate with MgATP2- as the phosphoryl donor and is characterized by a low affinity for Glc ([S]0.5 ~ 8.0 mM), a positive kinetic cooperativity of Glc binding (nH ~ 1.7), and no feedback inhibition by its product.

Besides its expression in hepatocytes (isoforms 2 and 3) as a cytoplasmic and nuclear enzyme, where its translocation and activity is regulated by the glucokinase regulatory protein and the metabolic state of the cell (1), human GK is also expressed in pancreatic beta-cells (isoform 1, the neuroendocrine isoform) mainly as a soluble cytoplasmic enzyme. In pancreatic beta-cells GK has been found to be partitioned between the cytoplasm and the insulin secretory granules as a peripheral membrane protein (2-4) and in a regulated manner, i.e. mediated by Glc/insulin. In pancreatic beta-cells, GK acts as the glucose sensor (5), a concept supported by the finding that complete GK deficiency leads to neonatal diabetes (6, 7). More than 200 different mutations have been identified in the hGK gene (8), and most of them lead to reduced enzyme activity and are associated with mild diabetes, maturity-onset diabetes of the young type 2 (MODY2). Others are characterized by an in vitro thermal instability when expressed as recombinant glutathione S-transferase (GST) fusion proteins (9). A few activating mutations have also been identified, leading to a hypoglycemic hyperinsulinism of infancy (8).

In both hepatocytes and pancreatic beta-cells GK is activated by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (10). Little is known about covalent post-translational modifications of GK and their possible regulatory functions, and the molecular and cellular mechanisms involved in its degradation/turnover are also poorly understood. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin (Ub)-conjugating enzyme system of the rabbit reticulocyte lysate. The functional implications of this posttranslational modification have been studied in vitro with reference to the major roles ubiquitination plays in regulating a broad array of basic cellular processes (11) and in particular in relation to the described regulatory function on the Glc-induced insulin synthesis and secretion in pancreatic beta-cells (12-15).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-GK (H-88, rabbit polyclonal), peroxidase-conjugated anti-rabbit IgG (SC-2313, donkey) and peroxidase-conjugated anti-mouse IgG (SC-2031, goat) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Ub (13-1600, mouse) that recognizes conjugated mono- and poly-Ub and free Ub was from Zymed Laboratories Inc. (San Francisco, CA). The QuikChange site-directed XL mutagenesis kit was obtained from Stratagene (La Jolla, CA). MagicMarkTM XP Western Protein Standard and expression vector pcDNA3.1/HisC were purchased from Invitrogen. TNT T7 Quick-coupled Transcription/Translation System and MagZ Protein Purification System were from Promega (Madison, WI). L-[35S]Met (code AG 1094) and 14C-methylated protein standards were purchased from GE Healthcare. Rabbit reticulocyte Fraction II, human recombinant Ub aldehyde and pentaubiquitin (Ub5, Lys-48-linked), ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme UbcH1 (E2-25K), and the proteasome inhibitor MG-132 were from Boston Biochemicals (Cambridge, MA). Polyubiquitin chains (Ub2-7-Lys-48) were from Affiniti Research Products (Mamhead, UK). Hexokinase was from Sigma-Aldrich. Creatine phosphokinase was from Sigma-Aldrich, factor Xa was from Protein Engineering Technology ApS, and the Detergent Surfact-AmpsTM 20 was from Pierce. Chaps, IPG ampholyte solution, pH 3-10 NL buffer, and glutathione-Sepharose 4B were from Amersham Biosciences. Other two-dimensional electrophoresis components were ZOOM IPGRunner cassette, ZOOM Strip, electrode wicks, and MOPS buffer from Invitrogen. The protease inhibitors phenylmethylsulfonyl fluoride, phenantrolin, aprotinin, benzamidin, pepstatin, and leupeptin were from Sigma-Aldrich. All reagents for cell culture were obtained from Invitrogen. Complete Protease Inhibitor Mixture was from Roche Diagnostic, and Protein A-Sepharose CL-4B was from Amersham Biosciences.

Plasmid Constructs, Expression, and Purification of Recombinant Forms of hGK—Plasmid constructs, protein expression, and purification are described in supplemental methods.

In Vitro Expression of hGK—hGK in the pcDNA3.1+ or pcDNA3.1/HisC vector was expressed in a coupled in vitro transcription/translation system (TNT® T7 Quick-coupled Transcription/Translation System; Promega) in the presence of [35S]Met. The reaction contained in a final volume of 50 µl: 1 µl of [35S]Met (10 µCi), 2 µg of plasmid DNA, 20 mM dithiothreitol, and 10 µM Ub (in addition to the endogenous content of Ub) and 40 µl of rabbit reticulocyte lysate. The standard incubation time was 90 min at 30 °C, and the reaction was quenched by adding 1 mM cold Met. His6-tagged hGK was alternatively isolated by affinity purification using the MagZ Protein Purification System (Promega).

In Vitro Ubiquitination of hGK—In vitro ubiquitination of His6-hGK, hGK, and its mutant forms was performed in a reconstituted rabbit reticulocyte lysate (RRL) system from Promega at 30 °C for 90 min in a final volume of 50 µl containing 15 µl of purified [35S]Met-labeled His6-hGK or hGK protein, 12.5 µM Ub (in addition to the endogenous content of the lysate), 2 µM Ub aldehyde, and 0-50% (v/v) RRL. Subsequently, the high molecular mass reaction products were separated on SDS-PAGE (10% gel) and analyzed by autoradiography and densitometry. The generation of high molecular mass forms of unlabeled purified GST-hGK was alternatively studied in the same reconstituted RRL system after cleavage (by factor Xa) of the GST fusion partner. The reaction contained in a final volume of 50 µl: 1.7 µg of hGK protein, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM dithiothreitol, 2 mM ATP, 2 µM Ub aldehyde, 12.5 µM Ub, and 0-50% (v/v) RRL. The reaction products were separated on SDS-PAGE (10% gel) and subjected to immunoblot analysis.

Degradation of Newly Synthesized hGK in a Proteasome- and ATP-enriched Reticulocyte Lysate System—Degradation of newly synthesized and partially ubiquitinated [35S]Met-labeled His6-hGK was assayed at 30 °C, with a reaction time of 30 min, in an incubation mixture containing in a final volume of 25 µl: 1 µg of plasmid DNA, 0.5 µl[35S]Met (10 µCi), 5 mM ATP, 20 mM dithiothreitol, 10 µM Ub (in addition to the endogenous content of Ub in the RRL lysate), 5 mM MgCl2, E1 (200 nM) and UbcH1-E2 (1 µM), 10 mM phosphocreatine, 0.2 mg/ml creatine phosphokinase, and 20 µl of RRL preincubated in the absence or presence of 100 µM proteasome inhibitor MG132 and 2 µM Ub aldehyde.

One- and Two-dimensional Polyacrylamide Gel Electrophoresis—hGK and its Ub conjugates generated in the in vitro assays were precipitated with precooled acetone (sample/acetone 1:4, by volume at -20 °C for 1-2 h prior to centrifugation at 14 000 rpm, 30 min). Samples prepared for one-dimensional electrophoresis containing [35S]Met-labeled hGK were dissolved in SDS sample buffer and denatured for 15 min at 56 °C before SDS-PAGE (10% gel). The 14C-methylated protein standard contained myosin (220 kDa), phosphorylase-b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), and lysozyme (14.3 kDa). Samples prepared for two-dimensional electrophoresis were dissolved in a sample buffer containing 8 M urea, 2% (w/v) Chaps, 1% (v/v) IPG ampholyte solution (pH 3-10 NL), and 50 mM dithiothreitol. The first dimension was performed at 200 v for 20 min, 450 v for 20 min, 750 v for 20 min, and 2000 v for 1.10 h. The second dimension was run in a gradient of 4-12% (w/v) precast acrylamide gel (1.0 mm). The gel was subjected to immunoblot analysis with a GK-specific Ab. Gels were analyzed on a Fuji bas-2500 and band intensities calculated by Image Gauge v4.0 (Fujifilm).

Immunoblot Analysis—High molecular mass forms of unlabeled recombinant hGK proteins (isoforms 1 and 2) generated in the in vitro ubiquitination assay were separated by SDS-PAGE (10% gel) and immunoblotted by standard procedures. The primary Ab (anti-GK) and the secondary Ab (peroxidase-conjugated anti-rabbit IgG) were diluted 1:2000. Ub-conjugated proteins were detected with an anti-Ub primary Ab that recognizes both unconjugated and conjugated mono- and polyubiquitin and peroxidase-conjugated anti-mouse IgG as the secondary Ab, both diluted 1:1500. The ladder of reference proteins was MagicMarkTM XP Western Protein Standard (Invitrogen) with nine recombinant proteins, range 20-220 kDa. The enhanced chemiluminescence detection method was employed to develop the immunoblots.

INS-1 and HepG2 cells were lysed, GK immunoisolated from the cytosolic fractions by Protein A-Sepharose, electrophoresed, and immunoblotted with anti-ubiquitin or anti-GK Ab. For details, see supplemental methods.

Assay of hGK Activity—In the standard assay the catalytic activity of purified recombinant hGK was measured in a reaction volume of 1 ml containing: 25 mM sodium Hepes, pH 7.4, 25 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.1% (w/v) bovine serum albumin, 2.5 mM ATP, 1 mM NAD+, 0.35 units of glucose-6-phosphatase, 0.5 µg of recombinant hGK, and 50 mM D-glucose. The time course of the NADH formation was followed at 340 nm in a thermostated cuvette (37 °C) of the Hewlett Packard photodiode array spectrophotometer (Agilent 8453), and the activity was calculated from the linear slope.

Intrinsic Tryptophan Fluorescence—Intrinsic tryptophan fluorescence was performed on a PerkinElmer LS-50B instrument at 25 °C in a buffer containing 20 mM Hepes, 100 mM NaCl, and 1 mM dithiothreitol, pH 7.0, and a protein concentration of 0.03 mg/ml. The excitation and emission wavelengths were 295 and 340 nm, respectively, with slit widths of 4 and 7 nm.

Equilibrium Binding of Polyubiquitin Chains to hGK and hGK{Delta}C24—The chromatographic holdup assay was performed essentially as described by Charbonnier et al. (16). For details, see supplemental methods.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Structural Analysis—An analysis of the three-dimensional structure of hGK revealed the sequence EEGSGRGAALVSAVA at positions 442-456 at the C-terminal {alpha}-helix (Fig. 1A). This sequence is homologous to the core ubiquitin-interacting motif (UIM) sequence eeeX{phi}XXAXXXSXXe, where e is a negatively charged residue, {phi} is most often the hydrophobic residues Leu, Ile, or Ala, and X is any amino acid (17, 18). Interestingly, the helix length, and thus the conformation of this site, changes upon binding of Glc and an allosteric activator (Fig. 1B), and its orientation relative to the specifically interacting helix 6 (residues 204-217) changes as well (Fig. 1C).3 In general, UIM sites promote ubiquitination as well as binding of polyubiquitin chains (17, 18), and both possibilities were addressed in the present study.

In Vitro Synthesis and Posttranslational Ubiquitination of hGK—As seen from Fig. 2, lane 1, [35S]methionine (Met)-labeled His6-hGK synthesized in the coupled in vitro transcription-translation system of the RRL is a substrate for the Ub-conjugating enzyme system of the lysate. Several high molecular mass bands were observed in addition to the full-length His6-hGK (56 kDa). Some minor low molecular mass bands were regularly observed, presumably representing incomplete chains (19) because their presence was not affected by protease or proteasome inhibitors. An identical electrophoretic pattern was observed with a construct of WT-hGK (Fig. 2, lane 2), but of slightly lower molecular mass. The apparent molecular masses of the individual radioactive bands were 56, 67.5, 74, and 79 kDa for His6-hGK and 53, 64.5, 71, and 76 kDa for WT-hGK, respectively, and the high molecular mass bands amounted to ~17% of the total. The [35S]Met-labeled His6-hGK was purified by affinity binding to and gradient elution from Ni2+-chelated magnetic beads. The 56-kDa His6-hGK was preferentially eluted at 1 M imidazole (Fig. 2, lane 3). Using the purified His6-hGK as the substrate in the in vitro RRL system the level of Ub conjugates was found to increase in proportion to the concentration of the RRL lysate used in the incubation (supplemental Fig. S1A, lanes 1-6, and S1B).

To further characterize the Ub conjugates of hGK, purified recombinant hGK (isoforms 1 and 2) was used as substrate for the Ub-conjugating enzyme system of the RRL. When analyzed by two-dimensional electrophoresis in a high-resolution polyacrylamide gradient gel, immunodetection with anti-GK revealed that the high molecular mass forms were positioned in a diagonal pattern at a progressively higher pI than the unmodified hGK, as expected for a polyubiquitinated protein (Fig. 3, A and B). Immunodetection with anti-ubiquitin Ab revealed a similar pattern (Fig. 3A, inset). The conjugates in these experiments represented ~50% of the total immunoreactive protein in the gels for both isoforms. Interestingly, the molecular mass position of hGK·Ub1 and hGK·Ub2 revealed a double spot. The respective spots demonstrated the same pI but different mobilities as SDS denatured proteins, presumably due to different conformations, thus indicating two putative ubiquitin acceptor sites in hGK.

To possibly identify the target residue(s) for ubiquitination, the 22 lysine residues common to the isoforms were individually mutated to arginine (Arg) (supplemental Table S1) and the mutant forms were expressed in vitro as His6-tagged and [35S]Met-labeled proteins in the RRL system. For all mutant forms, identical electrophoretic patterns of Ub conjugates were observed, as shown for WT-hGK in Fig. 2. The lysine residues are distributed rather uniformly along the protein, with some residues closely spaced in the linear sequence of flexible loop structures or closely spaced in the three-dimensional structure (20). Hence, multiple lysine residues of hGK presumably function as redundant acceptor sites. When the hGK{Delta}C24-truncated form, devoid of the putative UIM site (Fig. 1), was expressed in the RRL system the double bands observed for His6-hGK·Ub1 and His6-hGK·Ub2 of the WT-hGK were replaced by single bands (Fig. 4, A and B). This finding indicates that the ubiquitination of one of the target sites is coupled to the UIM site. Interestingly, the C-terminal-truncated form revealed a total loss of catalytic activity (data not shown).

Proteasomal Degradation of Newly Synthesized and Ubiquitinated hGK—Proteasome-dependent degradation of hGK was studied co-translationally by expressing [35S]Met-labeled His6-hGK for only 30 min in the standard RRL system. Then its stability was followed in a proteasome- and ATP-enriched RRL system, in the absence and presence of the proteasome inhibitor MG-132. As seen from Fig. 5, the total intensity of the hGK signal (radioactivity), including His6-hGK and its ubiquitinated forms, increased ~1.8-fold (n = 5 and p = 0.008) in the presence of MG-132. Thus, inhibition of the proteasomal activity resulted in a stabilization of the newly synthesized enzyme. The degree of inhibition observed may to some extent have been affected by the relatively high concentration of nonspecific Ub conjugates in the RRL representing competitive substrates in terms of proteasomal degradation (21).


Figure 1
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  		FIGURE 1.
The C-terminal domain of hGK with the putative UIM site and the conformational changes induced upon binding of D-glucose and allosteric activator (Compound A). A, close-up view into the putative UIM site in the three-dimensional structure of the nonliganded (super-open) conformation (Protein Data Bank 1v4t) of hGK with the key interacting residues indicated. B, schematic representation of the change in the backbone dihedral torsion angles ({Delta}{varphi} +{Delta}{Psi}) for residues 442-464 upon binding of D-glucose and allosteric activator as calculated from the structures of the unliganded (super-open) form (PDB 1v4t) and the liganded (closed) form (PDB 1v4s). The boxes represent the difference in the length of the C-terminal {alpha}-helix in the super-open conformation (helix 17, residues 448-459) and the closed conformation (helix 19, residues 444-459). C, the figure demonstrates the change in relative orientation of the interacting C-terminal {alpha}-helix (helix 17/19) and helix 6 (residues 204-217) upon transition from the super-open to the closed conformation. D, schematic representation of the static solvent accessibility of Glu-442, Glu-443, Glu-445, Ala-449, and Ser-453 as calculated by the CUPSAT algorithm (available at cupsat.uni-koeln.de/) in the super-open conformation (open bars) and closed conformation (filled bars) in which the allosteric activator (Compound A) interacts with Val-452 and Val-455. The three-dimensional images were generated with PyMol (www.pymol.org).

 


Figure 2
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  		FIGURE 2.
Expression of hGK cDNA in the in vitro transcription-translation system. His6-hGK (plasmid pcDNA3.1/HisC) and WT-hGK (plasmid pcDNA3.1+) were translated in a transcription-linked RRL translation system at 30 °C for 90 min as described under "Experimental Procedures." Lanes 1 and 2 represent total [35S]Met-labeled translation products analyzed by SDS-PAGE (10%) with His6-hGK (lane 1) and WT-hGK (lane 2). Lane 3 represents His6-hGK purified by affinity chromatography on a Ni2+-chelate resin (elution with a concentration gradient of imidazole) with partial recovery of the high molecular mass forms (indicated by arrows) at 1 M imidazole; the high molecular mass forms preferentially eluted at lower imidazole concentrations. Lane 4, the recombinant GST-WT-hGK (~76-kDa), and lane 5, the recombinant WT-hGK (~50 kDa) and the GST fusion partner (~26 kDa) after cleavage of the fusion protein with factor Xa.

 


Figure 3
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  		FIGURE 3.
Immunodetection of mono- and poly-/multiubiquitinated recombinant WT-hGK in a reconstituted RRL system. A, hGK pancreatic isoform 1. B, hGK liver isoform 2. Two-dimensional electrophoresis (4-12% (w/v) gradient gel) and Western blot analysis using the anti-GK Ab demonstrated the high molecular mass Ub conjugates as a ladder of diagonal spots characteristic of mono/poly-Ub conjugates of the two isoforms. Inset in panel A, two-dimensional electrophoresis of hGK pancreatic isoform 1 and Western blot analysis using anti-ubiquitin Ab. The arrows indicate hGK and Ub-related spots; asterisks indicate nonspecific ubiquitinated proteins in the reticulocyte lysate.

 


Figure 4
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  		FIGURE 4.
Demonstration of the importance of the UIM site for ubiquitination of hGK and its binding by polyubiquitin chains. A, SDS-PAGE (10%) analysis of total in vitro [35S]Met-labeled translation products of His6-hGK (lane 1) and the hGK{Delta}C24-truncated form (lane 2). B, densitometric analysis of panel A. Arrows indicate the high molecular mass bands of His6-hGK and the hGK{Delta}C24 truncated form to be compared. Note the loss of a high molecular mass band in the hGK{Delta}C24 profile. C, shown are equilibrium binding of Ub5 to glutathione-Sepharose 4B saturated with GST-WT-hGK (lanes 1, 2, 5, and 6) and GST-hGK{Delta}C24 (lanes 3 and 4) as described for the chromatographic holdup assay in the absence (lanes 1, 2, 3, and 4) and presence (lanes 5 and 6) of 50 mM D-glucose, free polyubiquitin (mainly Ub5 Lys-48-linked) in lanes 2, 4, and 6 and bound plus free Ub5 in lanes 1, 3, and 5 as analyzed by SDS-PAGE (10%) and immunoblot detection by the anti-Ub Ab. D, densitometric quantitation of the respective Ub5 band intensities in Fig. 4C for GST-WT-hGK in the absence (columns 1 and 2) and presence of 50 mM Glc (columns 5 and 6), and GST-hGK{Delta}C24 (columns 3 and 4). Each column represents the average of three experiments, and the error bars indicate the resulting S.D. Statistical significance was determined using the Student's t-test; **, p < 0.01.

 


Figure 5
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  		FIGURE 5.
Effect of proteasome inhibitor on the stability of the newly in vitro synthesized and ubiquitinated WT-hGK. A, His6-hGK (plasmid pcDNA3.1/HisC) was translated in a transcription-linked RRL translation system at 30 °C for 30 min at 4 mM MgATP in an ATP-regenerating system in the absence (lane 1) and presence (lane 2) of proteasome inhibitor (MG-132); SDS-PAGE (10%) demonstrates the pattern of newly synthesized [35S]Met-labeled His6-hGK. B, densitometric analysis of SDS-PAGE gels in panel A. C, the total expression of [35S]Met-labeled His6-hGK in the absence (column 1) and presence (column 2) of proteasome inhibitor (MG-132) as shown in panel B; each column represents the mean of five analyses ± S.D. A p value <0.01 (**) was determined by the Student's t-test for the increase in hGK expression in the presence of proteasome inhibitor.

 
Equilibrium Binding of Polyubiquitin Chains and Catalytic Activation of WT-hGK—To further characterize the C-terminal UIM site (Fig. 1), we studied the equilibrium binding of polyubiquitin chains (mainly Ub5-Lys-48 linked) to GST-WT-hGK or GST-hGK{Delta}C24 immobilized on glutathione-Sepharose 4B using a chromatographic holdup assay. The amount of bound and free analyte was measured by SDS-PAGE (Fig. 4C), which revealed that polyubiquitin chains bind to GST-WT-hGK in the absence of Glc (Fig. 4D, columns 1 and 2), but not to GST-hGK{Delta}C24 lacking the UIM site (Fig. 4D, columns 3 and 4). Moreover, in the presence of 50 mM Glc, the binding of the analyte to GST-WT-hGK was markedly reduced (columns 5 and 6) as expected from the lower accessibility of the key interacting residues of the UIM site in the ligand-bound conformation of WT-hGK (Fig. 1D).


Figure 6
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  		FIGURE 6.
Catalytic activation of recombinant hGK by free polyubiquitin chains and the reticulocyte lysate. A, the effect of increasing concentrations of free polyubiquitin, mainly Ub5 Lys-48-linked (Ub5, •) and monoubiquitin (Ub1, {circ}) on the catalytic activity of hGK. Graphic points including error bars represent the mean of four to five measurements ± S.D. The curve was calculated and fit to a rectangular hyperbola by nonlinear regression analysis, giving an apparent EC50 value of 93 nM. B, comparison of the stimulatory effect of 97 nM polyubiquitin (column 2), 0.5% (v/v) RRL lysate (column 3) versus the control with only hGK (column 1). Each column represents the mean of four measurements ± S.D. Statistical significance was determined by the Student's t-test. **, p < 0.01 and ***, p < 0.0001 when compared with hGK alone (column 1).

 
As seen from Fig. 6A and 6B, column 2, free polyubiquitin chains also stimulated the catalytic activity of recombinant hGK in a concentration-dependent manner and in the low nanomolar concentration range. An ~1.4-fold stimulation (n = 4) was observed at 97 nM Ub5-Lys-48, and the [S]0.5 value for Glc was slightly reduced, i.e. from 8.62 ± 0.22 to 8.18 ± 0.27 mM (data not shown). A similar small reduction was observed in the Kd value for the equilibrium binding of Glc at 25 °C, i.e. from 4.8 ± 0.06 to 4.3 ± 0.15 mM, as determined by intrinsic tryptophan fluorescence (data not shown). It is also seen that monoubiquitin was much less efficient in stimulating the activity. In addition, it is seen from Fig. 6B that the reticulocyte lysate, containing a plethora of Ub conjugates (22) and free polyubiquitin chains (supplemental Fig. S2A), has a similar (~1.4-fold) stimulatory effect.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In pancreatic beta-cells the ubiquitin-proteasome pathway has been found to have a regulatory role in the Glc-stimulated insulin release (15), Glc-stimulated (pro)insulin synthesis (13), and in the biogenesis and surface expression of the ATP-sensitive K+ (KATP) channels (12) as well as in maintaining a normal function of the voltage-dependent calcium channel (14). In the present study, we demonstrate that hGK of the pancreatic beta-cell (isoform 1) and the hepatocytes (isoform 2) are posttranslationally modified by multiple moieties of Ub. Two-dimensional electrophoresis (Fig. 3, A and B) demonstrate the formation of hGK·Ub1-3 on at least two putative target lysine residues where the double spot of SDS-denatured hGK·Ub1 and hGK·Ub2 in the second dimension represent two ubiquitinated species with presumably different conformations having the same pI. A similar heterogeneity (double bands) has been reported for in vitro ubiquitinated luciferase (23) as well as for Lys-48-Ub2 and Lys-48-Ub3 synthesized in vitro (24). However, on site-directed mutagenesis, in which 22 of the lysine residues in hGK were individually mutated to Arg and expressed in vitro, no specific acceptor site could be identified. Multiple lysine residues in hGK, therefore, seem to function as redundant acceptor sites as previously exemplified by cyclin B (25) and cyclin A (26). It should be noted that the mutant forms tested were catalytically active with a specific activity in the range of 65-99% of WT-hGK (data not shown). Deletion of the 24 C-terminal amino acids, with the UIM site, resulted in polyubiquitination at an apparently single site (Fig. 4A), indicating that the ubiquitination of one site is promoted by the UIM site as previously shown for UIM-containing proteins (27).

Polyubiquitination often serves as a signal for targeting cytoplasmic and nuclear proteins to the proteasome for subsequent degradation (reviewed in Ref. 11). We found that the 30-min in vitro translated [35S]Met-labeled His6-hGK and its ubiquitinated forms were unstable in a proteasome- and ATP-enriched reticulocyte lysate degradation assay that was sensitive to the proteasome inhibitor MG-132 (Fig. 5). It has recently been proposed (28) that the key targeting step for proteasome-mediated degradation is the conjugation of multiple short ubiquitin chains, independent of linkage type. This finding may explain the proteasomal degradation of newly synthesized hGK in our cell-free system, suggesting that its degradation may function as a regulatory mechanism in the homeostatic control of the cellular GK protein expression as demonstrated for several long-lived proteins (29). In hepatocytes (rat) expressing most of the total GK the sequestration and degradation of cytoplasmic GK occurs by the autophagosomal-lysosomal pathway at a rate of 3.5%/h and a half-life of 12.7 h (30). So far, we have no experimental data to support that ubiquitinated hGK is preferentially targeted to the autophagosomal-lysosomal pathway. An active autophagocytosis has been demonstrated in mouse beta-cells (31, 32) and in NIT insulinoma cells (33), including autophagy of insulin secretory granules, and may in this way be involved in the turnover of the membrane-bound form of GK. In certain diabetes-associated mutations in hGK (MODY 2) a reduced stability of the enzyme has been considered as a possible mechanistic explanation for the hyperglycemia and reduced Glc-stimulated insulin secretion (9). Because ubiquitination of misfolded proteins associated with cytoplasmic chaperones may be degraded predominantly through the ubiquitin-proteasome system (34), further studies are in progress to investigate this possibility for selected loss-of-function mutant forms of hGK.

Ubiquitin-binding domains are found in proteins that function in a vast range of cellular events, including the activation of kinases in the nuclear factor-{kappa}B signaling pathway (35). Here we demonstrate that free poly-Ub chains (Ub5-Lys-48-linked) allosterically activate hGK at low nanomolar concentrations (Fig. 6, A and B), i.e. at ten times lower concentrations than for monoubiquitin. From Fig. 6A it is seen that the catalytic activity of hGK is increasingly stimulated by Ub5 in the concentration range of 20-100 nM, with an apparent EC50 value of 93 nM (Fig. 6A). It is shown that the polyubiquitin chains act by an equilibrium binding to the UIM site (Fig. 1), as demonstrated by the holdup binding assay (Fig. 4, C and D). The UIM site is located in the highly mobile C-terminal part including helix 17/19, which specifically interacts with helix 6 (Fig. 1C) both in the "super-open" (helix 17) and in the closed (helix 19) form of hGK. Interestingly, seven activating mutations of hGK have presently been characterized, all clustered in this defined area, and have been considered to represent an allosteric activator site (36). Two of the mutations (Y214C and Y215A) are located in helix 6 and three (V455M, A456V, and A460R) in helix 17/19. Moreover, several synthetic organic compounds, which activate the enzyme by a Vmax and [S]0.5 effect, have been found to interact with the same site (i.e. Val-452 and Val-455) (20, 37) and thus considered to represent potential antidiabetic drugs (20, 37). The putative physiological endogenous activator interacting at this site has, however, still to be discovered.

Our data support the conclusion that polyubiquitin chains, either free or conjugated to proteins (Fig. 6), may represent such a physiological allosteric activator. Interestingly, the maximum stimulation of catalytic activity by Ub5-Lys-48 (Vmax) was similar to that recently reported for the synthetic allosteric activator RO-28-1675 (~1.5-fold) (9). Within an in vivo context, free polyubiquitin chains or polyubiquitinated proteins in GK-expressing cells (supplemental Fig. S2) may have a similar function. Thus, the level of Ub is reported to be 10-20 µM in a variety of cultured cell lines and in rabbit reticulocytes (38-40), where the Ub conjugates represent ~80% of the total Ub level (38). Furthermore, free polyubiquitin chains represent a substantial portion of the total Ub pool in vivo (41), as in the reticulocyte lysates used in the present study (supplemental Fig. S2A). The bulk concentration of Ub conjugates was estimated by SDS-PAGE and immunoblots to be ~9 µM (Ub2-7 polyubiquitin chains served as a standard of reference), and on two-dimensional electrophoresis polyubiquitin chains represented a major fraction, ~4 µM. Estimation of Ub conjugates in GK-expressing cells, i.e. MIN-6 and HepG2 cell lines, revealed even higher levels of immunoreactive Ub conjugates than in the reticulocyte lysate (supplemental Fig. S2A). Moreover, immunoprecipitated GK from INS-1 and HepG2 cells demonstrated on SDS-PAGE and immunoblotting with anti-ubiquitin Ab several high molecular mass bands (supplemental Fig. S2B, lanes 2 and 4, respectively) distributed over a larger Mr range than that observed in the in vitro RRL system. Several potential mechanisms exist for the homeostatic regulation of the fractional level of Ub conjugates in the cell (39), including the ATP-dependent ligation of Ub (42) and the expression and activity of multiple deubiquitinating enzymes (15, 43). Both the ligation and the degradation system are energy dependent, and the cellular level of conjugated Ub has been found to be regulated by the ATP level (44). In the context of the pancreatic beta-cell the glucose-stimulated increase in ATP level, which triggers insulin secretion by closing the KATP channels, depolarization of the plasma membrane, and Ca2+ influx may also promote the formation of polyubiquitin conjugates and thus amplify the glucose response by activating the glucokinase (positive feedback). However, the Ub-conjugating/-deconjugating enzymes of the beta-cells (12-15) remain to be further characterized.

In general, an unanswered query in the biology of ubiquitin interaction is how ubiquitin/polyubiquitin dissociates from ubiquitin-binding domains. In the case of hGK the affinity of Ub5 binding is higher in the absence of Glc (super-open conformation) than in the presence of Glc (closed conformation) (Fig. 4, C and D). It should be noted that the static solvent accessibility calculated for key interacting residues of the UIM site in the ternary hGK·Glc·GKA complex (Fig. 1D) may not be completely representative for the binary hGK·Glc complex. Thus, it is not clear to what extent the binding of the GK activator (Compound A) perturbs the structure of the binary complex.

Our study supports the conclusion that poly-/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, recombinant hGK interacts with and is allosterically activated up to ~1.4-fold by purified free polyubiquitin chains at low nanomolar concentrations assigned to their equilibrium binding to an UIM site at the C-terminal. Both ubiquitin-mediated processes represent potential physiological regulatory mechanisms for GK as a glucose sensor in pancreatic beta-cells and hepatocytes.


   FOOTNOTES
 
* This work was supported by the Norwegian Research Council, the University of Bergen, Innovest AS, Haukeland University Hospital, Helse Vest, the Norwegian Diabetes Association, the Novo Nordisk Foundation, the Aarskog Foundation, and the Meltzer Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental methods and supplemental Figs. S1 and S2 and Table S1. Back

1 To whom correspondence should be addressed: Dept. of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. Tel.: 47-55586428; Fax: 47-55586360; E-mail: torgeir.flatmark{at}biomed.uib.no.

2 The abbreviations used are: GK, glucokinase; hGK, human GK; Ab, antibody; Glc, D-glucose; GST, glutathione S-transferase; hGK{Delta}C24, C-terminal-truncated form of hGK; RRL, rabbit reticulocyte lysate; Ub, ubiquitin; Ub5, pentaubiquitin; UIM, ubiquitin-interacting motif; WT, wild type; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. Back

3 The helix nomenclature. In this study we used the MolMovDB (available at molmovdb.mbb.yale.edu/molmovdb/) to demonstrate the regions of variable secondary structure in the two determined crystal structures of hGK (20). 17 helices were identified in the ligand-free super-open structure (PDB code 1v4t) versus 19 helices in the Glc and allosteric activator-bound closed structure (PDB code 1v4s); H17 (residues 448-459) -> H19 (residues 444-459) in the closed conformation (see molmovdb.mbb.yale.edu/cgi-bin/morph.cgi?ID=496337-23316). Back


   ACKNOWLEDGMENTS
 
We thank Anita-Merete Nordbø and Gry Sjøholt for technical assistance. HepG2 cells were generously provided by Dr. Jørn V. Sagen at Haukeland University Hospital.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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