Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

ABSTRACT: Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2? subunits and two regulatory CK2? subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2?/CK2? interface, a bioorthogonal click reaction was used to modify the protein kinase ?-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2?. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2?. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate ?S1-casein towards CK2?. This labeling strategy was also applied to CK2? subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

SUBMITTER: Nienberg C 

PROVIDER: S-EPMC5039489 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature