Casein Kinase I Subfamily.

doi:10.1074/jbc.270.21.12717

Casein Kinase I Subfamily.

MOLECULAR CLONING, EXPRESSION, AND CHARACTERIZATION OF THREE MAMMALIAN ISOFORMS AND COMPLEMENTATION OF DEFECTS IN THE SACCHAROMYCES CEREVISIAE YCK GENES (*)

From the (1) Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, the Department of Biochemistry and Molecular Biology, Louisiana State University, School of Medicine, Shreveport, Louisiana 71130-3932, the
(2) Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, and the
(3) Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

Abstract

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK11, CK12, and CK13, have predicted molecular masses of 43,000, 45,500, and 49,700. CK13 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK11 (2 kilobases (kb)), CK12 (1.5 and 2.4 kb), and CK13 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK13 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK11 and CK12 messages were restricted to testis. All three CK1 isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1s although less effectively than the CK1α or CK1 isoforms. All three CK1s underwent autophosphorylation when incubated with ATP and Mg. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK11 or CK13 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.

Footnotes

↵* This work was supported by National Institutes of Health Grants DK27221 (to P. J. R.), GM26217 (to M. R. C), and GM44140 (to M. H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U22296, U22297, and U22321.
↵¹ The abbreviations used are:

CK1

casein kinase I

kb

kilobase(s)

PCR

polymerase chain reaction

bp

base pair(s)

cpm

counts/min.
↵²For these experiments, the chosen start codon was based on that proposed in the original partial CK1 sequence analysis (12); further inspection of the predicted sequences for the three CK1 forms, though, shows an upstream, in-frame ATG present in all three isoforms, and this is most likely the true start site. Thus, the proteins that we first expressed in E. coli started at Met-18, Met-18, and Met15 for CK11, CK12, and CK13, respectively, in relation to the protein sequences reported in this paper. Subsequently, we have also expressed full-length CK13 and found no major differences in properties as compared with the shorter form described herein (L. Zhai and P. J. Roach, unpublished results).
↵³L. C. Robinson, unpublished results.
↵⁴D. Virshup, unpublished data.