CASEIN KINASE I-GAMMA SUBFAMILY - MOLECULAR-CLONING, EXPRESSION, AND CHARACTERIZATION OF 3 MAMMALIAN ISOFORMS AND COMPLEMENTATION OF DEFECTS IN THE SACCHAROMYCES-CEREVISIAE YCK GENES

Casein kinase I, one of the first protein kinases identified biochemic ally, is known to exist in multiple isoforms in mammals. Using a parti al cDNA fragment corresponding to an isoform termed CK1 gamma, three f ull-length rat testis cDNAs were cloned that defined three separate me mbers of this subfamily. The isoforms, designated CK1 gamma 1, CK1 gam ma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,5 00, and 49,700. CK1 gamma 3 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within t he protein kinase domain but only 51-59% identical to other casein kin ase I isoforms within this region. Messages for CK1 gamma 1 (2 kilobas es (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK1 gamm a 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testi s. All three CK1 gamma isoforms were expressed as active enzymes in Es cherichia coil and partially purified. The enzymes phosphorylated typi cal in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activa ted by heparin whereas phosphorylation of the protein substrates was i nhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms. All three CK1 gamma s underwent autophosphorylation when in cubated with ATP and Mg2+. The YCK1 and YCK2 genes in Saccharomyces ce revisiae encode casein kinase I homologs, defects in which lead to abe rrant morphology and growth arrest. Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast muta nt carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins a nd these CK1 isoforms.