Abstract
We have isolated two genes from Saccharomyces cerevisiae that both encode a calmodulin-dependent protein kinase (CaM kinase). The CMK1 gene has been cloned by hybridization using an oligonucleotide probe synthesized on the basis of the peptide sequence of purified yeast CaM kinase (Londesborough, J. (1989) J. Gen. Microbiol. 135, 3373-3383). The other gene, CMK2, which is homologous to CMK1, has been isolated by screening at low stringency with a CMK1 fragment as a probe. The CMK2 product expressed in bacteria shows Ca(2+)- and CaM-dependent protein kinase activity, indicating that CMK2 also encodes a CaM kinase. The CMK1 and CMK2 products expressed in bacteria were found to have different biochemical properties in terms of autoregulatory activity and preference for yeast CaM or bovine CaM for maximal activity. Antibody raised against a peptide fragment of the CMK1 protein cross-reacts with the CMK2 product. Immunoblotting with this antibody indicated that the CMK1 and CMK2 products have apparent molecular masses of 56 and 50 kDa, respectively, in yeast cells. The predicted amino acid sequences of the two CMK products exhibit highest similarity with mammalian calmodulin-dependent multifunctional protein kinase II (CaM kinase II): the similarity within the N-terminal catalytic domain is about 40%, whereas that within the rest of the sequence is 25%. These data indicate that yeast has two kinds of genes encoding CaM kinase isozymes whose structural and functional properties are closely related to those of mammalian CaM kinase II. Another gene may be substituted for function of the CMK1 and CMK2 kinase in vivo, since elimination of both kinase genes is not lethal.
Highlights
From the $Departmentof Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, the §Department of Molecular Biology, The Tokyo Metropolitan Znstituteof Medical Science, Bunkyo-ku, Tokyo 113, Japan, and the llResearch Laboratories of the Finnish StateAlcohol Company (Alko), Box 350, SF-00101 Helsinki10, Finland
Antibody raised against a peptide fragment phorylates a target proteinspecific forthe kinase, and thereby of the C M K l protein cross-reacts with tChMe K 2 prod- regulates the activity of its own discrete process
Immunoblotting with this antibody indicated that group includes CaM kinase I1 isozymes [12, 13], which phosthe C M K l and C M K 2 products have apparent molec- phorylate more than 10 different target proteins, modulating ular masses of 56 and 50 kDa, respectively, in yeast their functions simultaneously [14]
Summary
Saccharomyces step in theregulatory cascade involved in controlof a diverse cerevisiae that bothencode a calmodulin-dependent range of physiological processes, such as carbon metabolism, protein kinase(CaMkinase). 60 kDa) are distributedwidely in many tissues and organisms malian calmodulin-dependent multifunctional protein [15, 16], suggest that this kinase is responsible for the regukinase I1 (CaM kinase 11): the similarity within thNe terminal catalytic domainis about 40%, whereas that within the rest of the sequence is 25%. These data indicate that yeast has two kinds of genes encoding CaM kinase isozymes whose structural and functional lations of a wide variety of proteins in uiuo and plays central roles in Ca2'/CaM signal transduction pathways. We examined whether yeast contains any gene that is homologous to CMKl by Southern blotting
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