A type‐2 casein kinase (YCK‐2), lacking the 25‐kDa autophosphorylatable β 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 β subunit of LCK‐2. The native Mr 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 α2β2 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 Km 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 Km 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 β subunit. These data suggest that the β 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 β subunits. Copyright © 1986, Wiley Blackwell. All rights reserved
Structure and properties of casein kinase-2 from Saccharomyces cerevisiae. A comparison with the liver enzyme.
MEGGIO, FLAVIO;PINNA, LORENZO
1986
Abstract
A type‐2 casein kinase (YCK‐2), lacking the 25‐kDa autophosphorylatable β 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 β subunit of LCK‐2. The native Mr 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 α2β2 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 Km 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 Km 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 β subunit. These data suggest that the β 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 β subunits. Copyright © 1986, Wiley Blackwell. All rights reserved
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