Expression,purification and characterisation of a novel mutant of the human protein kinase CK2

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant subunit. According to our theoretical predictions, we observed that the -helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.     

Functional conservation between the human, nematode, and yeast CK2 cell cycle genes.

Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms. Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae. We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits. Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits. Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.     

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.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2alpha), which has specificity for both ATP and GTP, shows significant amino acid sequence similarity to the cyclin-dependent kinase 2 (CDK2). We constructed site-directed mutants of CK2alpha and used a three-dimensional model to investigate the basis for the dual specificity. Introduction of Phe and Gly at positions 50 and 51, in order to restore the pattern of the glycine-rich motif, did not seriously affect the specificity for ATP or GTP. We show that the dual specificity probably originates from the loop situated around the position His115 to Asp120 (HVNNTD). The insertion of a residue in this loop in CK2 alpha subunits, compared with CDK2 and other kinases, might orient the backbone to interact with the base A and G; this insertion is conserved in all known CK2alpha. The mutant deltaN118, the design of which was based on the modelling, showed reduced affinity for GTP as predicted from the model. Other mutants were intended to probe the integrity of the catalytic loop, alter the polarity of a buried residue and explore the importance of the carboxy terminus. Introduction of Arg to replace Asn189, which is mapped on the activation loop, results in a mutant with decreased k(cat), possibly as a result of disruption of the interaction between this residue and basic residues in the vicinity. Truncation at position 331 eliminates the last 60 residues of the alpha subunit and this mutant has a reduced catalytic efficiency compared with the wild-type. Catalytic efficiency is restored in the truncation mutant by the replacement of a potentially buried Glu at position 252 by Lys, probably owing to a higher stability resulting from the formation of a salt bridge between Lys252 and Asp208.     

Purification and characterization of the CK2alpha'-based holoenzyme, an isozyme of CK2alpha: a comparative analysis

Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.     

The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme.

The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either alone or in combination with the recombinant non-catalytic beta subunit. Calmodulin is not phosphorylated by the CK2 holoenzyme, in either the native or the reconstituted form, unless polylysine is added. In the presence of polylysine, it becomes a good substrate for CK2 (Km 14.2 microM, Kcat 4.6 mol.min-1.mol CK2-1). The recombinant alpha subunit, however, spontaneously phosphorylates calmodulin, this phosphorylation being actually inhibited rather than stimulated by polylysine. The calmodulin tridecapeptide, RKMKDTDSEEEIR, reproducing the phosphorylation site for CK2, is spontaneously phosphorylated by either CK2 holoenzyme or the recombinant alpha subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant beta subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine. On the contrary, the phosphorylation of the nonapeptide, MSSSEEVSW, reproducing the beta-subunit phosphoacceptor site, is dramatically stimulated by polylysine. Using a variety of small peptide substrates, it was shown that phosphorylation rate is diversely stimulated by polylysine. The observed stimulation, moreover, is variably accounted for by changes in Vmax and/or Km, depending on the structure of the peptide substrate. Maximum stimulation with all protein/peptide substrates tested requires the presence of the beta subunit, since the recombinant alpha subunit is much less responsive than CK2 holoenzyme, either native or reconstituted. While the phosphorylation of the peptide RRRDDDSDDD by CK2 is stimulated 2.8-fold, with 15 nM polylysine being required for half-maximal stimulation, a stimulation of only 1.9-fold, with 80 nM polylysine required for half-maximal stimulation, is attained with recombinant alpha subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.     

The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme.

The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.     

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.     

Direct regulation of microtubule dynamics by protein kinase CK2

Microtubule dynamics is essential for many vital cellular processes such as morphogenesis and motility. Protein kinase CK2 is a ubiquitous protein kinase that is involved in diverse cellular functions. CK2 holoenzyme is composed of two catalytic alpha or alpha' subunits and two regulatory beta subunits. We show that the alpha subunit of CK2 binds directly to both microtubules and tubulin heterodimers. CK2 holoenzyme but neither of its individual subunits exhibited a potent effect of inducing microtubule assembly and bundling. Moreover, the polymerized microtubules were strongly stabilized by CK2 against cold-induced depolymerization. Interestingly, the kinase activity of CK2 is not required for its microtubule-assembling and stabilizing function because a kinase-inactive mutant of CK2 displayed the same microtubule-assembling activity as the wild-type protein. Knockdown of CK2alpha/alpha' in cultured cells by RNA interference dramatically destabilized their microtubule networks, and the destabilized microtubules were readily destructed by colchicine at a very low concentration. Further, over-expression of chicken CK2alpha or its kinaseinactive mutant in the endogenous CK2alpha/alpha'-depleted cells fully restored the microtubule resistance to the low dose of colchicine. Taken together, CK2 is a microtubule-associated protein that confers microtubule stability in a phosphorylation-independent manner.     

The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds.

The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar ratios of 4 : 1. The presence of dithiothreitol markedly reduced the anti-aggregation effects of grp94-CT on CK2alpha without altering the solubility of the chaperone. It is concluded that the chaperone activity of the C-terminal domain of human grp94 requires the maintenance of its quaternary structure (dimers and oligomers), which seems to be stabilised by disulphide bonds.     

Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme.

Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the catalytic alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit, counteracts the stimulatory effect of the beta subunit on the alpha subunit activity and partially substitutes the beta subunit in conferring thermal stability to the alpha subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the beta subunit including the autophosphorylation site. It is suggested that the acidic domain of the beta subunit, encompassing residues 55-71, plays a role in the interactions between the beta and alpha subunits.     

Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.     

Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica

The locations have been determined, with respect to the plasma membrane, of lysine alpha 380 and lysine gamma 486 in the alpha subunit and the gamma subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the alpha subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the gamma subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine alpha 380 and lysine gamma 486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine alpha 380 in the native acetylcholine receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine gamma 486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine alpha 380 is on the inside surface of a vesicle and lysine gamma 486 is on the outside surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducible expression of protein kinase CK2 in mammalian cells. Evidence for functional specialization of CK2 isoforms.

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2alpha or CK2alpha') together with the regulatory CK2beta subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2alpha or CK2alpha' (i. e. GK2alpha (K68M) or CK2alpha'(K69M)) together with CK2beta. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2alpha or CK2alpha' resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2alpha differed significantly from the effects of induced expression of kinase-inactive CK2alpha'. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2alpha'(K69M), but not following induction of CK2alpha(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions.     

Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2

The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.     

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.     

Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.