Purification and characterization of a type II casein kinase from Drosophila melanogaster

A cyclic nucleotide-independent protein kinase has been isolated from Drosophila melanogaster by chromatography on phosphocellulose and hydroxylapatite followed by gel filtration and glycerol gradient sedimentation. As determined by sodium dodecyl sulfate gel electrophoresis, the purified enzyme is greater than 95% homogeneous and is composed of two distinct subunits, alpha and beta, having Mr = 36,700 and 28,200, respectively. The native form of the enzyme is an alpha 2 beta 2 tetramer having a Stokes radius of 48 A, a sedimentation coefficient of 6.4 S, and Mr approximately 130,000. The purified kinase undergoes an autocatalytic reaction resulting in the specific phosphorylation of the beta subunit, exhibits a low apparent Km for both ATP and GTP as nucleoside triphosphate donor (17 and 66 microM, respectively), phosphorylates both casein and phosvitin but neither histones nor protamine, modifies both serine and threonine residues in casein, and is strongly inhibited by heparin (I50 = 21 ng/ml). These properties are remarkably similar to those of casein kinase II, an enzyme previously described in several mammalian and avian species. The strong similarities among the insect, avian, and mammalian enzymes suggest that casein kinase II has been highly conserved during evolution.     

Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.     

Phosphorylation of casein kinase II

Casein kinase II from rabbit reticulocytes is a tetramer with an alpha,alpha' beta 2 or alpha 2 beta 2 structure; the alpha subunits contain the catalytic activity, and the beta subunits are regulatory in nature [Traugh, J.A., Lin, W. J., Takada-Axelrod, F., & Tuazon, P. T. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 224-229]. When casein kinase II is isolated from rabbit reticulocytes by a rapid two-step purification of the enzyme, both the alpha and beta subunits are phosphorylated to a significant extent. In vitro, purified casein kinase II undergoes autophosphorylation on the beta subunit. In the presence of polylysine and polyarginine, phosphorylation of the beta subunits is inhibited, and the alpha subunits (alpha and alpha') become autophosphorylated. The effectiveness of polylysine coincides with the molecular weight. With basic proteins, including a number of histones and protamine, autophosphorylation of both subunits is observed. With histones, autophosphorylation of each subunit can be greater than that observed with the autophosphorylated enzyme alone or with a basic polypeptide. Thus, the potential exists for modulatory proteins to alter the autophosphorylation state of casein kinase II. Taken together, the data suggest that phosphorylation of the alpha subunit of casein kinase II in vivo may be due to an unidentified protein kinase or due to autophosphorylation. In the latter instance, casein kinase II could be transiently associated with specific intracellular compounds, such as basic proteins, with a resultant stimulation of autophosphorylation.     

Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.     

Further studies on the quaternary structure of yeast casein kinase II

Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.     

Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.     

Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.     

Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the alpha subunit of animal casein kinase-2.

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase-2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the alpha/alpha' subunits of animal CK2 and devoid of the autophosphorylatable 25-kDa alpha subunit of animal CK2, which display an heterotetrameric alpha 2 beta 2/alpha alpha' beta 2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39,000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38,000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical beta subunits; (3) the 39-kDa band immunoreacts with antisera that recognize the alpha subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the beta subunit could be detected in CKIIB by Western-blot analyses with antisera that recognize animal beta subunits; (5) the recombinant beta subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation. While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non-catalytic subunits. Its properties, compared to those of animal CK2, suggest that the beta subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.     

Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.     

Immunocytochemical localization of casein kinase II during interphase and mitosis

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization

S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.     

Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.

The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.     

Properties of protein kinases in brain coated vesicles

Coated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48,000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 microM. With phosvitin as a substrate, the value of its apparent Km was 20 microM. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.