A gel-electrophoretic analysis for improved sensitivity and specificity ofDNA-dependent protein kinase activity

DNA-dependent protein kinase (DNA-PK) is considered a critical enzyme in th e repair and/or signal transduction of DNA double-strand breaks. DNA-PK act ivity has been mostly measured through "DNA-plus-minus" or "DNA-pull-down" procedures using synthetic peptide as substrate followed by filter-binding analysis, i.e. liquid scintillation counting of acid-insoluble radioactivit y bound to phosphocellulose filter. Considering that non-specific phosphory lation of other cellular proteins in filter-bound acid-insoluble count coul d interfere with the detection of specific phosphorylation of peptide subst rate, we examined the specificity and characteristics of these assay proced ures by SDS gel-electrophoresis of the reaction mixture. The electrophoreti c pattern showed phosphorylation in wide range of non-specific protein band s other than the specific substrate. The very low DNA-PK activity shown by murine L5178Y or FSA1233 cells was unambiguously detectable as the count in substrate band. Even following DNA-pull-down procedure, which would separa te DNA-PK from most of other protein kinases, substantial amount of phospho rylation of other cellular proteins were still contaminated. Thus by select ively counting the particular bands, small amount of specific phosphorylati on of peptide substrate was reliably quantified. These results indicated th at the DNA-PK activity through filter-binding analysis was, as suspected, c ontaminated by non-specific phosphorylation of other cellular proteins and also that the gel-electrophoretic analysis would improve detectability of s pecific phosphorylation by DNA-PK of synthetic peptide substrate and, there fore, would improve the kinase assay in both sensitivity and specificity.