Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

CaSein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant α and β subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7–10 mM for Mg2+, 0.5–0.7 mM for Mn2+ and 1–2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence ofMg2+. The apparent K m values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 μM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and α subunit. Inhibition of the holoenzyme by 400 μM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the α subunit.

Polylysine-containing peptides are found to affect membrane protein kinases, phosphatidylinositol kinases, and adenylate cyclase. Poly(L-lysine), poly(D-lysine), random copolymers of lysine and serine or lysine and alanine, and poly(L-ornithine) produced large increases in the in vitro phosphorylation of some membrane proteins present in Xenopus laevis oocyte membranes. Poly(L-arginine) did not cause a similar stimulation. In these membranes the phosphorylation of polydisperse protein of approximately 25 kDa was also greatly increased by 1 mM spermine and spermidine, by 10 IAM histone H1, or by 200 IAM peptide containing the 14-residue sequence at the carboxyl terminus of the human c-Ki-ras 2 gene product, which has eight lysines. Similar In addition, this work shows a similar effect of polyamines, histone H1, and a synthetic peptide corresponding to the last 14 amino acids in the carboxyl terminus of human c-Ki-ras-2 gene product (6, 7). This peptide has a polylysine cluster that is adjacent to the protein site that binds the membrane (8). MATERIALS AND METHODSThe acetate salt of a polypeptide corresponding to the last 14 amino acid residues of the carboxyl-terminal portion of human c-Ki-ras-2 protein, with the sequence Lys-Lys-LysLys-Lys-Lys-Ser-Lys-Thr-Lys-Cys-Val-Ile-Met (6, 7), was custom synthesized by Peptide Technologies (Washington, DC).Polylysine containing 25 lysine residues (HBr salt, molecular mass 5.3 kDa determined by end group titration) was from Miles. Molecular masses of other polymers (Sigma, determined by viscosity) were as follows: polylysine, 24 kDa; polyornithine, 25 kDa; polyarginine, 40 kDa; poly(lysine75-serine25), 31 kDa; poly(lysine75alanine25) and poly(lysine50-alanine50), 38 kDa; poly(lysine67alanine33), 32 kDa. Histone H1, phosphatidylinositol (PtdIns), pbosphatidylinositol 4-phosphate (PtdIns-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-P2), cAMP, cGMP, phorbol 12-myristate 13-acetate (PMA), and high-performance silica thin-layer plates were obtained from Sigma. 5'-Guanylyl imidodiphosphate (p[NH]ppG) was from Sigma.Membranes from defolliculated full-grown oocytes were prepared as described previously (9), and resedimented in 0.22 M sucrose containing 50 mM Hepes at pH 7.5, 1 mM dithiothreitol, and 1 mM EDTA. Membranes purified from NG-108-15 mouse-rat hybrid nerve cells were prepared as published (10)

The cDNA gene coding for the a subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted elutamic acids for Lys" and LYS~~, changing the charge distribution of a very basic sequence found in the a subunit. Expression of the mutated c6NA in a pT7-7 vector in E. coli yielded an active mut~t r~ombinant protein that was extensively purified. This mutant was not si~ificantly affected in its app. K,,, for casein or a model peptide substrate, nor in its interaction with the activatingfl subunit. Inhibition by quercetin and by 5,6-dichloro-I-J&r+ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII IzE'~E~~ mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:l) and 2,3 diphosphoglycerate.

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