Regulation of Creatine Kinase by ASB9

Abstract

Creatine kinases (CKs) regulate ATP levels at sites of fluctuating energy demands. Reduced CK activity has been implicated in diseases such as heart failure and multiple sclerosis, however little is known about CK regulation. Ankyrin repeat and SOCS box-containing protein, ASB9, (part of an E3 ubiquitin ligase) has been shown to promote proteasomal degradation of CKs. We hypothesize that ASB9 and its splice variants control the levels and activity of CK at sites where energy is critically needed. Our results show that the ankyrin repeat domain of ASB9 (ASB9-ARD) binds CKB with very high affinity (nM) and 1:1 stoichiometry. Comparison of binding data from several N-terminal truncations of ASB9-ARD suggests that residues 19-35 of ASB9 contribute to the binding affinity. In addition, binding of ASB9-ARD to CK abolishes the enzymatic activity of CK. Hydrogen deuterium exchange mass spectrometry (HDXMS) has revealed that only one region in CKB (residues 182 - 203), was protected upon binding of ASB9 when the deuterium incorporation into CKB peptides alone was compared to those in complex with ASB9-ARDs. This region is right in front of the active site of the enzyme, and was shown previously to undergo a conformational change upon binding of creatine and ADP-NO3 to rabbit CKM. Results from the CK inhibition assay together with the HDXMS data strongly suggest that ASB9 binds directly to CKs and inhibits CK activity by altering regions near the active site of the enzyme. A model of this interaction generated using docking and computational modeling agrees with the binding and HDXMS data. We intend to completely characterize the ASB9-CK interaction and discover inhibitors that would disrupt the interaction, resulting in increased amounts of active CK to treat diseases in which there is a an increased energy demand and decreased CK function.