BUDDING AND FISSION YEAST CASEIN KINASE-I ISOFORMS HAVE DUAL-SPECIFICITY PROTEIN-KINASE ACTIVITY

We have examined the activity and substrate specificity of the Sacchar omyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotyp es of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, eac h of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Ph osphoamino acid analysis of P-32-labeled proteins showed phosphorylati on on serine, threonine, and tyrosine residues. The E. coli produced f orms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protei n kinase synthetic peptide substrate polymer poly-E(4)Y(1). Immune com plex protein kinases assays from S. pombe cells showed that Hhp1-conta ining precipitates were associated with a protein-tyrosine kinase acti vity, and the Hhp1 present in these immunoprecipitates was phosphoryla ted on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyr osine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold incr ease in the Km for poly-E(4)Y(1) and casein. These data demonstrate th at four different CKI isoforms from two different yeasts are capable o f protein-tyrosine kinase activity and encode dual-specificity protein kinases.