RESUMO

Protein kinase CK2 is a ubiquitous protein that phosphorylates multiple substrates and is composed of catalytic (alpha, alpha') and regulatory (beta) subunits. Abundant evidence relates CK2 to the regulation of cell division. p21(WAF1/CIP1) is a potent inhibitor of cyclin-dependent kinases and of DNA replication and acts as a key inhibitor of cell cycle progression. In this work we examine the relation between these two important proteins. The interaction between the CK2 beta regulatory subunit of CK2 and p21(WAF1/CIP1) has been confirmed. Using a pull-down assay and fusion constructs of glutathione transferase with fragments of CK2 beta and other mutants, it was possible to define that the N-terminal (1-44) portion of CK2 beta contains a p21(WAF1/CIP1) binding site. CK2 reconstituted from recombinant alpha and beta subunits can phosphorylate p21(WAF1/CIP1) in vitro. This phosphorylation is greatly enhanced by histone H1. p21(WAF1/CIP1) can inhibit the phosphorylation of substrate casein by CK2. This inhibition, however, seems to be due to competition by p21(WAF1/CIP1) as an alternate substrate since in order to observe inhibition it is necessary that the concentration of p21 be of the same order of magnitude as the casein substrate concentration. This competition is not related to the binding of p21(WAF1/CIP1) to CK2 beta because it can also be observed when, in the absence of CK beta, CK alpha is used to phosphorylate casein in the presence of the p21.

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Protein kinase CK2 is a heteromeric enzyme with catalytic (alpha) and regulatory (beta) subunits which form an alpha2beta2 holoenzyme and utilizes both ATP and GTP as nucleotide substrate. Site-directed mutagenesis of CK2alpha subunit was used to study this capacity to use GTP. Deletion of asparagine 118 (alpha(deltaN118)) or the mutant alphaN118E gives a 5-6-fold increase in apparent Km for GTP with little effect on the affinity for ATP. Mutants alphaN118A and alphaD120N did not alter significantly the Km for either nucleotide. CK2alphaN118 has an apparent Ki for inosine 5' triphosphate 5-fold higher than wild-type and is very heat labile. These studies complement recent crystallographic data indicating a role for CK2alpha asparagine 118 in binding the guanine base.

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RESUMO

Protein kinase CK2 is a ubiquitous eukaryotic ser/thr protein kinase. The active holoenzyme is a heterotetrameric protein composed of catalytic (α and α') and regulatory (ß) subunits that phosphorylates many different protein substrates and appears to be involved in the regulation of cell division. Despite important structural studies, the intimate details of the interactions of the α catalytic subunits with the ß regulatory subunits are unknown. Recent evidence that indicates that both CK2 subunits can interact promiscuously with other proteins in a manner that excludes the binding of their complementary CK2 partners has opened the possibility that the phosphorylating activity of this enzyme may be regulated in a novel way. These alternative interactions could limit the in vivo availability of CK2 subunits to generate fully active holoenzyme CK2 tetramers. Likewise, variations in the ratio of α- and ß-subunits could determine the activity of several phosphorylating and dephosphorylating activities. The promiscuity of the CK2 subunits can be extrapolated to a more widespread phenomenon in which "wild-card" proteins could act as general switches by interacting and regulating several catalytic activities. J. Cell. Biochem. Suppls. 30/31:129-136, 1998. © 1998 Wiley-Liss, Inc.

RESUMO

Protein kinase CK2 is a ubiquitous eukaryotic ser/thr protein kinase. The active holoenzyme is a heterotetrameric protein composed of catalytic (alpha and alpha') and regulatory (beta) subunits that phosphorylates many different protein substrates and appears to be involved in the regulation of cell division. Despite important structural studies, the intimate details of the interactions of the alpha catalytic subunits with the beta regulatory subunits are unknown. Recent evidence that indicates that both CK2 subunits can interact promiscuously with other proteins in a manner that excludes the binding of their complementary CK2 partners has opened the possibility that the phosphorylating activity of this enzyme may be regulated in a novel way. These alternative interactions could limit the in vivo availability of CK2 subunits to generate fully active holoenzyme CK2 tetramers. Likewise, variations in the ratio of alpha- and beta-subunits could determine the activity of several phosphorylating and dephosphorylating activities. The promiscuity of the CK2 subunits can be extrapolated to a more widespread phenomenon in which "wild-card" proteins could act as general switches by interacting and regulating several catalytic activities.

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The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da. The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined. The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20). This inhibition is competitive and shows an approximate K1 of 5 microM. CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.

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The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.

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X laevis ovarian tissue or isolated oocytes contain two major tRNA methyl transferase activities capable of methylating total E. coli tRNA using S-adenosyl methionine as a methyl donor. These enzymes can be resolved by chromatography on DEAE-cellulose or by gel filtration on Sephadex G-200 into fractions I and II. The tRNA methyl transferase I which is present mainly in the oocyte nuclei, has a molecular weight of 190,000 and an apparent Km for S-adenosyl methionine of 1.5 microM. The activity of peak II which exists predominantly in the oocyte cytoplasm, has a molecular weight of 125,000 and an apparent Km for S-adenosyl methionine of 17 microM. The most striking difference between these two enzymes, however, resides in their response to spermine or magnesium ions. The nuclear enzyme is activated more that 8 fold by spermine and 4 fold by Mg2+ while the cytoplsmic activity is slightly inhibited by the polyamine and unaffected by Mg2+. The effect of spermine on the nuclear tRNA methyl transferase is highly dependent on the salt concentration since the stimulatory effect of the polyamine decrease at KCI concentrations above 100 mM becoming inhibitory above 200 mM. Spermine increases 4 fold the Vmax of the reaction catalyzed by the nuclear enzyme but does not affect its apparent Km for tRNA which is approximately 2.9 microM. The apparent Km for tRNA of the cytoplasmic enzyme is 3.3 microM.

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The microinjection of transfer RNA into amphibian oocytes permits one to study under in vivo conditions the reactions that affect this important macromolecule. A comparative study has been carried out between the in vivo and in vitro specificity of the aminoacylation reacton. The results obtained show that modifications of the tRNA structure affect aminoacyl-tRNA synthetase recognition in the same fashion in both conditions. The in vivo aminoacylation was not affected by the presence of puromycin (0.5mM) or cycloheximide (0.1 mM) which completely inhibited oocyte protein synthesis. An interesting difference was obtained between the in vivo and in vitro aminoacylation of tRNA with regards to temperature requirements. While the in vivo reaction was optimal at 25 degrees and was totally inhibited at 37 degrees, the in vitro was optimal, at the latter temperature. The inhibition of the in vivo reaction at 37 degrees was not due to inactivation of the enzyme. The transfer of the amino acid moiety to nascent proteins was studied by measuring the transfer of radioactivity from injected (14C) phenylalanyl-tRNA into hot trichloroacetic acid precipitable material. It was found that 30% or more of the amino acid became incorporated into oocyte proteins and that this incorporation was due to direct transfer from the aminoacyl-tRNA and was inhibited by puromycin and cycloheximide.

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The microinjection technique affords us the possibility to introduce purified components into living cells and to answer the question of what effects the change introduced has on cellular metabolism. This technique can therefore be used to test the hypothesis that transfer RNA plays a regulatory role in cellular protein synthesis. Prior to these experiments it is important, however, to test whether transfer RNA microinjected into amphibian oocytes is stable and functional inside this cells. These two questions are answered affirmatively in this report. The stability of tRNA was tested by following the content of TCA precipitable counts inside the oocytes at different times after microinjection of radioactive yeast and E. coli tRNA and by polyacrilamide gel electrophoresis of the material recovered from the cell. The results clearly indicate that tRNAs are resistant to the action of occyte ribonucleases that degrade other RNAs such as 5S RNA. The functionality of the injected tRNA was tested by assaying the intracellular aminoacylation of microinjected yeast tRNA. The aminoacylation of bulk yeast (3H) tRNA introduced into Xenopus laevis oocytes was tested by the capacity of the material recovered 5 hours after injection into the cell to form a ternary complex with wheat protein synthesis elongation factor 1 and GTP. The complex only forms with aminoacyl-tRNA and not with unacylated tRNA. This method showed that at least 80% of the tRNA introduced into the cell was aminoacylated in vivo. A direct assay for internal aminoacylation made use of microinjection of pure tRNAPhe and subsequent determination by phenol extraction of (14C)Phe-tRNA content of oocytes that had been incubated for 2 hours in a medium containing (14C)phenylalanine. The results obtained showed that the oocytes could internally aminoacylate 200-500 times more tRNAPhe that the cell normally contains. Appropiate controls demonstrated that the aminoacylation was aminoacid and tRNA specific and that periodate oxidized tRNAPhe could not be in vivo aminoacylated but tRNAPhe deprived of its Y base could accept the aminoacid. A brief study demonstrated that bulk yeast tRNA and tRNAPhe without its Y base did not inhibit endogenous protein synthesis but a similar amount of tRNAPhe caused 50% inhibition and periodate-oxidized tRNAPhe a 95% inhibition.

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