Structure and properties of casein kinase‐2 from Saccharomyces cerevisiae

Abstract

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])

Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro

“…At 200 mM K+ or Na+, activity was inhibited by more than 90%. This property is also shared by CK I1 from yeast (Meggio et al, 1986).…”

Isolation, sequencing, and disruption of the CKA1 gene encoding the alpha subunit of yeast casein kinase II.

“…262: , 1987 Casein kinase II is a cyclic-nucleotide-independent protein kinase which phosphorylates a broad spectrum of both cytoplasmic and nuclear substrates (for a review, see reference 10). The enzyme is widely distributed among eucaryotic organisms and has been purified from a number of mammalian and avian species (10), Drosophila melanogaster (8), and Saccharomyces cerevisiae (19,22 (12,26), and in some systems two related alphalike polypeptides, alpha and alpha', have been observed (4, 10). The beta subunit becomes phosphorylated when the enzyme is allowed to undergo autophosphorylation, but the function of this subunit and the significance of autophosphorylation are poorly understood.…”

Phosphorylation of synthetic random polypeptides by protein kinase P and other protein-serine (threonine) kinases and stimulation or inhibition of kinase activities by microbial toxins.

“…By synthesis of large polypeptides with different amino acid mixtures and different charge distributions, it may be possible to evaluate quantitatively the contributing components. PK-P has some properties in common with yeast casein kinase II (8,9), such as sensitivity to acidic polysaccharides and activation by basic compounds. However, whereas casein kinase II was isolated from a soluble yeast extract and did not require a detergent for stability, we isolated PK-P by extraction of yeast membranes with Triton X-100 and showed that the detergent was required for stability of the enzyme (6).…”

“…10-(3-Aminopropyl)-2-chlorophenothiazine was supplied by Smith Kline & French. Casein kinase II from yeast and skeletal muscle was prepared through the phosphocellulose column step as described (8,9). PK-C was prepared as described (10).…”