A protein kinase, specific for 60s ribosomal proteins, has been isolated from Saccharomyces cerevisiae cells, purified to almost homogeneity and characterized. The isolated enzyme is not related to other known protein kinases. Enzyme purification comprised three chromatography steps ; DEAEcellulose, phosphocellulose and heparin -Sepharose. SDSjPAGE analysis of the purified enzyme, indicated a molecular mass of around 71 kDa for the stained single protein band. The specific activity of the protein kinase was directed towards the 60s ribosomal proteins L44, L44, L45 and a 38 kDa protein. All the proteins are phosphorylated only at the serine residues. None of the 40s ribosomal proteins were phosphorylated in the presence of the kinase. For that reason we have named the enzyme the 60s kinase. An analysis of the phosphopeptide maps of acidic ribosomal proteins, phosphorylated at either the 60s kinase or casein kinase 11, showed almost identical patterns. Using the immunoblotting technique, the presence of the kinase has been detected in extracts obtained from intensively growing cells. These findings suggest an important role played by the 60s kinase in the regulation of ribosomal activity during protein synthesis. 20 years ago, when Kaltschmidt and Wittmann described the gel electrophoresis procedure for the separation and identification of Escherichia coli ribosomal proteins [l], the two acidic proteins L7 and L12 (12 kDa) of the 50s subunit have become one of the most studied ribosomal proteins with respect to their three-dimensional structure and biological function.The functional and physicochemical equivalents of the procaryotic L7 and L12 are two ribosomal proteins, L44 and L45, from the eucaryotic 60s subunit (for review see [2]). Contrary to in animals, the ribosomes of Saccharomyces cerevisiae cells contain a third low-molecular-mass acidic protein. That protein, named L44', can be separated on DEAEcellulose [3] and by an electrofocusing procedure in acrylamide gel [4]. All three proteins have molecular masses of about 13 kDa, belong to 'split proteins ' [5] The phosphorylation of the crude eucaryotic ribosomes was initially reported by Kabat [lo] and by Loeb et al. [ll]. Later studies by other authors have shown that mammalian ribosomes are associated with cyclic-AMP-independent protein kinases [12-151. One of the enzymes has turned out to be a casein kinase type I1 (CKII) which phosphorylates proteins L44 and L45 [14]. For the yeast ribosomes, it has been found that the majority of phosphoproteins, y-32P labelled in vivo [16 -191 correspond to the proteins phosphorylated in vitro with . Two of them were identified as L44 and L45 [18, 19, 211 and are phosphorylated in vitro by the casein kinase type I1 [21, 221. Studies on the physiological role of the phosphorylation of ribosomal acidic proteins have been in progress for 10 years [23] and involved the yeast system. As shown in Ballesta's laboratory, the phosphorylation of L44, L44 and L45 might control the binding process of those proteins to ribosom...

A type-2 casein kinase (YCK-2), lacking the 25-kDa autophosphorylatable /3 subunit characteristic of animal casein kinases-2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase-2 (LCK-2). A 22-kDa phosphorylatable protein, copurifying with YCK-2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the / 3 subunit of LCK-2. The native M , of YCK-2, deprived of the 22-kDa phosphoprotein, is about 150000. Limited proteolysis experiments show that YCK-2 included 37-kDa catalytic subunits, which can be converted into still active 35-kDa proteolytic derivatives. These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition a2P2 of LCK-2 and other animal casein kinases-2. Although many properties of YCK-2 and LCK-2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturating and dissociating conditions and their response to polybasic peptides are quite different. In particular YCK-2 is more readily denaturated than LCK-2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100 -150 mM NaCl, which conversely stimulates LCK-2 activity 2-3-fold. The K , value of the synthetic peptide substrate Ser-(Glu)5 for YCK-2 is not significantly changed by the addition of polylysine. On the contrary the K , value of the same peptide substrate for LCK-2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its P subunit. These data suggest that the /3 subunit of animal CK-2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK-2 may be at least in part accounted for by its lack of p subunits.The operational terms casein kinase-2 (CK-2), casein kinase-G and casein kinase-TS are used to indicate the same class of ubiquitous cyclic nucleotides and CaZ +-independent protein kinases, which prefer casein and phosvitin over histones as artificial substrates, are drastically inhibited by heparin, capable of using GTP besides ATP as phosphate donor and affect both threonyl and seryl residues of casein fractions (reviewed in [l and 21). These enzymes differentiate from casein kinases-1 (also termed A or S) not only in their distinctive specificity toward both the nucleotide substrate and the peptide phosphorylation sites, but also in their larger molecular mass, denoting an oligomeric structure which includes, in the case of animal CK-2, catalytic a subunits near to 40 kDa and non-catalytic p subunits of around 25 kDa.Several potential targets of CK-2, including among others glycogen synthase, troponin-T, RNA polymerase, translation initiation factors, ornithine decarboxylase (reviewed in [2])

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