Identification of inhibitory autophosphorylation sites in casein kinase I ε

Casein kinase I epsilon (CKI epsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKI epsilon and the closely related CKI delta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKI epsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal dele deletion mutants was constructed by site-directed mutagenesis, Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants, Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKI epsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKI epsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKI epsilon using I kappa B alpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.