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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^*

Lise Bjørkhaug, Janne Molnes, Oddmund Søvik, Pål Rasmus Njølstad^¶, and Torgeir Flatmark^||1

From the 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

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic -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 -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 -cells.

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

^* 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.

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.

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.

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