Allosteric activation of human glucokinase by free polyubiquitin chains and its ubiquitin-dependent cotranslational proteasomal degradation

Abstract

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic β-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 posttranslational 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 α-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 β-cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.