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PubMed2006

Mechanism of assembly of G protein betagamma subunits by protein kinase CK2-phosphorylated phosducin-like protein and the cytosolic chaperonin complex.

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit complex (Gbetagamma) that has been recently shown to catalyze the formation of the Gbetagamma dimer from its nascent polypeptides. Phosphorylation of PhLP at one or more of three consecutive serines (Ser-18, Ser-19, and Ser-20) is necessary for Gbetagamma dimer formation and is believed to be mediated by the protein kinase CK2. Moreover, several lines of evidence suggest that the cytosolic chaperonin complex (CCT) may work in concert with PhLP in the Gbetagamma-assembly process. The results reported here delineate a mechanism for Gbetagamma assembly in which a stable ternary complex is formed between PhLP, the nascent Gbeta subunit, and CCT that does not include Ggamma. PhLP phosphorylation permits the release of a PhLP x Gbeta intermediate from CCT, allowing Ggamma to associate with Gbeta in this intermediate complex. Subsequent interaction of Gbetagamma with membranes releases PhLP for another round of assembly.

Carrier Proteins↗
PubMed1999

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2003

Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2.

The three-dimensional crystal structure of the triple-point mutant of the catalytic subunit of human protein kinase CK2alpha has been determined at 2.4 A resolution. Microcrystals of mutant CK2 catalytic subunit were obtained by a protein-crystallization method based on thin-film nanotechnology. These microcrystals (of about 20 micro m in diameter) were used for diffraction data collection by means of the microfocus beamline at the ESRF synchrotron. A comparison between the human protein kinase CK2alpha and the corresponding enzyme from a lower organism (Zea mays) is made.

Amino Acid Sequence↗
PubMed1998

Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays.

The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution. The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.

Casein Kinase II↗
PubMed1999

Crystallization and preliminary x-ray diffraction analysis of the regulatory subunit of human protein kinase CK2.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. A C-terminal truncated form of the beta subunit has been overproduced in Escherichia coli and purified to homogeneity. Two crystal forms of the truncated protein which diffract to at least 2 A resolution have been obtained. Form I belongs to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 92.9, c = 53.7 A, beta = 96.3 degrees, and yields plate-like crystals. Form II belongs to the tetragonal space group P42212, with unit-cell parameters a = 132.19, b = 132.19, c = 63.79 A, and produces rod-shaped crystals. Both crystal forms have a functional dimer in the crystal asymmetric unit.

Casein Kinase II↗
PubMed2001

Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

Adenosine Triphosphate↗
PubMed2005

Regulation of azole drug susceptibility by Candida albicans protein kinase CK2.

Fluconazole resistance of the fungal pathogen Candida albicans can arise through several mechanisms, but the responsible genes and pathways are poorly understood. We report here that mutations in CKA2, identified through an insertional mutagenesis screen, confer fluconazole resistance. CKA2 and its homologue CKA1 specify catalytic subunits of protein kinase CK2. Although cka1 mutations have little effect on fluconazole resistance, CKA1 overexpression suppresses the fluconazole resistance of a cka2 mutant. This observation, along with synthetic cka1-cka2 interactions, argues that Cka1p and Cka2p carry out similar functions. cka2 mutants overexpress CDR1 and CDR2, two fluconazole efflux transporter genes, and a cdr1 mutation decreases resistance of a cka2 mutant, as expected if CDR1 and CDR2 overexpression is responsible for fluconazole resistance of the cka2 mutant. The protein phosphatase calcineurin is required for azole tolerance, and we find that the calcineurin inhibitor cyclosporin reverses fluconazole resistance of cka2 mutants. In addition, a mutation in CRZ1, which specifies a homologue of the Saccharomyces cerevisiae transcription factor that is a major target of calcineurin, suppresses fluconazole resistance of cka2 mutants. Expression analysis of Cka2p-responsive genes argues that Cka2p and Crz1p act through distinct mechanisms. Several clinical fluconazole-resistant isolates overexpress some Cka2p-responsive genes. We suggest that a Cka2p-dependent regulatory pathway is altered by clinically derived azole resistance mutations.

Antifungal Agents↗
PubMed1997

Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit.

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C-terminal, CK2beta-(155-215)-peptide is unaffected or even increased. Moreover, the basal activity of alpha mutants K74-77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more sensitive to inhibition by either beta-subunit or its N-terminal, CK2beta-(1-77)-peptide, while its stimulation by the C-terminal peptide, CK2beta-(155-215), is comparable to that of alpha wild type. These observations suggest an indirect role of Val66 in conferring to the alpha-subunit a conformation less sensitive to down regulation by beta-subunit.

Amino Acid Sequence↗
PubMed2006

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis.

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.

Animals↗
PubMed2003

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality.

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.

Animals↗
PubMed2004

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK.

We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein. Phosphorylation fluctuates during the cell cycle with a moderate peak during G(1) phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un- or underphosphorylated form of DEK.

Amino Acid Sequence↗
PubMed2005

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus.

The heterotetrameric Sec62/63 complex associates with the heterotrimeric Sec61 complex to form the heptameric Sec complex. This complex is necessary and sufficient for post-translational protein translocation across the membrane of the endoplasmic reticulum. We show that Sec63p is phosphorylated at its C-terminal domain by the protein kinase CK2 and that this phosphorylation strengthens the interaction between the cytosolic domains of Sec63p and Sec62p. Exchanging either threonine 652 or threonine 654 against the nonphosphorylatable alanines in Sec63p impairs the binding to Sec62p and interferes with the efficient translocation of proteins across the membrane of the endoplasmic reticulum. These findings show that phosphorylation of Sec63p is required for tightly recruiting the putative signal-sequence-binding subunit Sec62p to the Sec complex.

Casein Kinase II↗
PubMed1999

Intermolecular contact sites in protein kinase CK2.

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55-70 of subunit beta (beta55-70) and 65-80 of subunit alpha (alpha65-80). This has been supported by crosslinking of subunits with peptides alpha65-80 and beta55-70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-alpha65-80; anti-beta55-70). Functionally, beta55-70 is a negative regulatory region for the kinase activity of subunit alpha. The opposite, stimulatory property of subunit beta has been assigned to its C-terminal part. Subdivision of peptide beta155-181, that has stimulatory effect, into overlapping peptides and assaying for alpha binding and binding competition revealed a tight physical contact at beta162-175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit alpha as indicated by crosslinking and peptide competition. The crosslinking data also confirm a beta-beta contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment.

Amino Acid Sequence↗
PubMed2001

A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin.

Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, alpha-subunits, and two regulatory, beta-subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is downregulated by the regulatory subunit. The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme. Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex. The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit. The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex. The interactions with the regulatory subunit and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30 degrees C. The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme. The results obtained suggest that CK2beta subunit and protein substrates share a common, or at least an overlapping, site of interaction on the catalytic subunit. The interaction between both subunits would prevent substrates from binding irreversibly to alpha subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate. The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.

Binding Sites↗