Isolation and Characterization of Human Casein Kinase I∊ (CKI), a Novel Member of the CKI Gene Family

Abstract

The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs

“…Analysis of partially puri®ed p53NK (see Materials and methods) using a`blotted protein kinase' assay with p53 as substrate (Ferrell and Martin, 1991) revealed a single species of kinase with a molecular weight of 49 000 (data not shown) consistent with the identity of the p53 kinase as either the delta (d) or epsilon (e) isoform of CK1, but not other CK1 isoforms which are considerably smaller (a, b and g; Christenson et al, 1996). CK1d and CK1e show both striking sequence similarity (98% within the kinase domains (Fish et al, 1995;Graves et al, 1993;DeMaggio AJ, Christenson E and Hoekstra MF, submitted) and functional similarity (DeMaggio AJ, Christenson E and Hoekstra MF, submitted). To determine whether p53 was a substrate for either of these enzymes, the ability of recombinant CK1d or CK1e to phosphorylate a number of GST-p53 fusion proteins was measured.…”

“…In this paper we present evidence that p53 can be phosphorylated in vitro by the delta and epsilon isoforms of CK1 (CK1d and CK1e) respectively which are highly related to each other (Fish et al, 1995;Graves et al, 1993) and show that the identity of the cellular p53NK is entirely consistent with CK1d and CK1e. We also provide compelling evidence that p53 is phosphorylated in vivo by these kinases.…”

“…In addition, the kinase activity and 35 Sradiolabel continued to co-elute during further puri®cation on phosphvitin-sepharose 4B and Mono S columns (data not shown). (The similarity in the elution characteristics of CK1d and CK1e was not surprising since these isoforms show striking similarity to each other at the amino acid level, especially within their kinase domains (Fish et al, 1995;Graves et al, 1993;DeMaggio AJ, Christenson E and Hoekstra MF, submitted). In addition to co-puri®cation with the radiolabelled proteins, partially puri®ed fractions containing p53NK activity could be inhibited by micromolar concentrations of IC261, an inhibitor which is speci®c for CK1d and e (data not shown; DeMaggio AJ, Christenson E and Hoekstra MF, submitted).…”

“…Five members of the CK1 family have now been cloned from mammalian cells (isoforms a, b, g, d, and e; (Fish et al, 1995;Graves et al, 1993;Rowles et al, 1991); reviewed by Christenson et al, 1996). These show striking similarity within their kinase domains but can di er extensively in their C-terminal noncatalytic domains.…”

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…Interestingly, deletion of this C-terminal region of Hrr25 appears to genetically dissociate MMS sensitivity from zymocin resistance, since such mutants display normal vulnerability to MMS but are still resistant to zymocin (86). Although the function of this C-terminal region is still unknown, yeast Hrr25 resembles a subset of CKI family members found in higher organisms that carry an N-terminal protein kinase domain attached to a similar C-terminal extension (94). It may be that for this class of CKI proteins the C-terminal extension harbors another functionality, namely, phosphoCTD binding.…”