Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II

Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation.     

A Gene Located at 56F1-2 inDrosophila melanogasterEncodes a Novel Metazoan β-like Subunit of Casein Kinase II

Drosophila melanogastercasein kinase II (DmCKII) is composed of catalytic α and regulatory β subunits associated as an α₂β₂heterotetramer. Using the two-hybrid system, we have screened aDrosophilaembryo cDNA library for proteins that interact with DmCKII α. One of the cDNAs encodes a novel β-like polypeptide, which we designate β′.In situhybridization localizes the corresponding gene to 56F1-2, a site distinct from that of both the β gene and theStellatefamily of β-like sequences. The predicted sequence of β′ is more closely related to the β subunit ofDrosophilaand other metazoans than to the Stellate family of proteins, suggesting that it is a second regulatory subunit.In vitroreconstitution studies show that a GST-β′ fusion protein associates with the α subunit to generate a tetrameric complex with regulatory properties similar to those of the native α₂β₂holoenzyme. The data are consistent with the proposed role of the β′ subunit as an integral component of the 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.     

The C-terminal domain of PLD2 participates in degradation of protein kinase CKII β subunit in human colorectal carcinoma cells

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKIIβ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKIIβ were necessary for interaction between the two proteins. PLD2 relocalized CKIIβ to the plasma membrane area. Overexpression of PLD2 reduced CKIIβ protein level, whereas knockdown of PLD2 led to an increase in CKIIβ expression. PLD2-induced CKIIβ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKIIβ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKIIβ degradation in HCT116 cells.     

DNA binding activity of casein kinase II

Casein kinase II, an ubiquitous, oligomeric, messenger-independent protein kinase has previously been shown to concentrate in the nuclear compartment when cells are stimulated to proliferate. The present communication reports that purified mammalian CKII interacts with genomic DNA preparations in vitro. This interaction led to an apparent activation of the kinase, most likely explained by prevention of its aggregation and subsequent denaturation. Binding of CKII was optimum with double stranded DNA preparations; duplex lambda phage DNA exhibited at least two types of binding sites and the high affinity system (Kd approximately equal to 6 x 10(-13) M) represented a binding capacity of about 1 mol CKII per mol DNA. CKII-DNA interaction was stimulated in the presence of a polyamine and inhibited by heparin. Blotting experiments disclosed that DNA binds CKII through its alpha subunit. These observations are in line with the hypothesis that casein kinase II may be examined as a component in the transduction of the mitogenic signal from the cell membrane to the nucleus, in response to growth factors.     

Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expression was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal component. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated 'rpL22-like short') that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like.     

Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock

FREQUENCY (FRQ), a key component of the Neurospora circadian clock, is progressively phosphorylated after its synthesis. Previously, we identified casein kinase II (CKII) as a kinase that phosphorylates FRQ. Disruption of the catalytic subunit of CKII abolishes the clock function; it also causes severe defects in growth and development. To further establish the role of CKII in clock function, one of the CKII regulatory subunit genes, ckb1, was disrupted in Neurospora. In the ckb1 mutant strain, FRQ proteins are hypophosphorylated and more stable than in the wild-type strain, and circadian rhythms of conidiation and FRQ protein oscillation were observed to have long periods but low amplitudes. These data suggest that phosphorylation of FRQ by CKII regulates FRQ stability and the function of the circadian feedback loop. In addition, mutations of several putative CKII phosphorylation sites of FRQ led to hypophosphorylation of FRQ and long-period rhythms. Both CKA and CKB1 proteins are found in the cytoplasm and in the nucleus, but their expressions and localization are not controlled by the clock. Finally, disruption of a Neurospora casein kinase I (CKI) gene, ck-1b, showed that it is not required for clock function despite its important role in growth and developmental processes. Together, these data indicate that CKII is an important component of the Neurospora circadian clock.     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts.     

Stimulation of human DNA topoisomerase II activity by its direct association with the beta subunit of protein kinase CKII

DNA topoisomerase II copurifies with and is phosphorylated by protein kinase CKII. In this study, a yeast two-hybrid system was used to investigate the interaction between human topoisomerase II isozymes and CKII subunits. The two-hybrid test clearly showed that both topoisomerase IIalpha and IIbeta interact with the CKIIbeta, but not the CKIIalpha subunit. The two-hybrid test also demonstrated that topoisomerase IIbeta residues 1099-1263 and topoisomerase IIalpha residues 1078-1182 mediate the interaction with the CKIIbeta subunit, providing evidence that the leucine zipper motif and the major CKII-dependent phosphorylation sites of topoisomerase II are unnecessary for its physical binding to CKIIbeta. Furthermore, a DNA relaxation assay demonstrated that the CKII subunit enhances topoisomerase II activity by physical interaction with topoisomerase II.     

The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity.

We found that a preparation of the 90-kDa heat shock protein, HSP90, purified to apparent homogeneity, contains a serine/threonine kinase which phosphorylates HSP90. The protein kinase was identified as casein kinase II (CKII) according to its properties. The protein kinase was separable from HSP90 by adsorption to heparin-Sepharose or phosphocellulose. CKII was coimmunoprecipitated with HSP90 by anti-HSP90 antibodies from cell extracts. Sucrose density gradient centrifugation analysis revealed that an addition of anti-HSP90 antibodies to cell extracts induces a shift of the sedimentation peak of CKII toward the bottom of a centrifuge tube. These results suggest that CKII is associated with HSP90 in cell lysates at low salt conditions. Furthermore, the CKII.HSP90 complex was reconstituted from purified HSP90-free CKII and CKII-free HSP90. In a buffer at low ionic strength, CKII forms large aggregates, but HSP90 dissociates the aggregates. Finally, we found that HSP90 activates CKII; an addition of HSP90 to CKII dramatically increased phosphorylation of exogenous substrates as well as the CKII beta subunit. Taken altogether, these observations suggest that CKII is structurally and functionally active when it forms a complex with HSP90.     

The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase II

The E7 protein of human papillomavirus type 16 (HPV16) transforms cultured cells and cooperates with the ras or fos oncogenes in the transformation of primary cells. In this study we have investigated the phosphorylation of E7. When we immunoprecipitated E7 from CaSki cells with a rabbit polyclonal antiserum to a bacterial fusion protein (trpE-E7), we found that E7 was phosphorylated at serine residues contained in five characteristic thermolysin peptides. Immunoprecipitated E7, and fusion proteins harboring the E7 protein from various HPV types, could all be specifically phosphorylated in vitro by the ubiquitous, growth factor-activated casein kinase II (CKII). Comparative peptide mapping showed that the sites of in vivo and in vitro phosphorylation are the same. CKII was shown previously to specifically phosphorylate serine or threonine residues within a cluster of acidic amino acids. The E7 protein contains such a sequence between amino acids 30 and 37. When a synthetic peptide corresponding to this region of E7 was phosphorylated by CKII in vitro, its thermolysin digestion products were the same as those in the phosphorylated E7 protein. We conclude that E7 is phosphorylated in vivo only at serines within the predicted CKII site and that CKII, or a CKII-like enzyme, participates in the reaction. Both the E1A and SV40 large T proteins contain similar CKII consensus sites proximal to the regions required for their associations with the retinoblastoma gene product (p105Rb). Thus it is conceivable that CKII phosphorylation can modulate the interaction between the transforming proteins and the retinoblastoma gene product.     

The Drosophila SSL gene is expressed in larvae,pupae, and adults,exhibits sexual dimorphism,and mimics properties of the beta subunit of casein kinase II

Drosophila melanogaster casein kinase II (CKII) is composed of catalytic alpha and regulatory beta subunits that generate the alpha2beta2 holoenzyme. A two-hybrid screen of a Drosophila embryo library using CKIIalpha as bait has resulted in the isolation of multiple cDNAs encoding SSL, a CKIIbeta-like polypeptide. We demonstrate that CKIIbeta, beta', and SSL exhibit robust and comparable interaction with CKIIalpha. Residues in SSL that mediate interaction with CKIIalpha appear similar to those in CKIIbeta, and SSL forms homodimers and heterodimers with CKIIbeta or beta' as well. We have tested all known Drosophila CKIIbeta-like proteins for rescue of the ion-homeostasis defect of yeast lacking beta subunits and find that CKIIbeta and SSL complement, beta' has marginal function, and Stellate appears non-functional. We have used real-time RT-PCR to assess developmental expression, and find that CKIIbeta is robust and ubiquitous, whereas SSL is restricted to males (third-instar-larvae, pupae, and adults), but is nondetectable in females of the corresponding stages. These results indicate that SSL expression encompasses a greater developmental window than that previously suggested and may confer distinct functions to CKII in a sex-specific manner.     

Over-expression of phospholipase D isozymes down-regulates protein kinase CKII activity via proteasome-dependent CKIIβ degradation in NIH3T3 cells

Over-expression of phospholipase D (PLD) 1 or PLD2 down-regulated CKII activity in NIH3T3 cells. The same results were found with catalytically inactive mutants of PLD isozymes, indicating that the catalytic activity of PLD is not required for PLD-mediated CKII inhibition. Consistent with this, 1-butanol did not alter CKII activity. The reduction in CKII activity in PLD-over-expressing NIH3T3 cells was due to reduced protein level, but not mRNA level, of the CKIIβ subunit. This PLD-induced CKIIβ degradation was mediated by ubiquitin-proteasome machinery, but MAP kinase and mTOR were not involved in CKIIβ degradation. PLD isozymes interacted with the CKIIβ subunit. Immunocyto-chemical staining revealed that PLD and CKIIβ colocalize in the cytoplasm of NIH3T3 cells, especially in the perinuclear region. PLD binding to CKIIβ inhibited CKIIβ autophosphory-lation, which is known to be important for CKIIβ stability. In summary, the current data indicate that PLD isozymes can down-regulate CKII activity through the acceleration of CKIIβ degradation by ubiquitin-proteasome machinery.     

The phosphotransferase activity of casein kinase II is required for its physiological function in vivo.

We have previously shown that the inviability associated with disruption of both catalytic subunits of casein kinase II in Saccharomyces cerevisiae can be rescued by plasmids expressing the catalytic subunit of the Drosophila enzyme (Padmanabha et al., 1990, Mol. Cell. Biol. 10, 4089). Here we describe the construction of mutant forms of the Drosophila catalytic subunit in which residues known to be crucial for catalytic activity in other protein kinases have been altered by site-directed mutagenesis. Mutation of either Lys66 or Asp173, which correspond to Lys72 and Asp184 of cAMP-dependent protein kinase, respectively, yields a casein kinase II catalytic subunit which fails to rescue a yeast strain lacking both endogenous catalytic subunit genes. The data indicate that the phosphotransferase activity of casein kinase II is required for its physiological function in vivo.     

Protein Kinase C-Mediated Phosphorylation of Kvβ2 in Adult Rat Brain

The phosphorylation of Kvβ2 was investigated by different protein kinases. Protein kinase A catalytic subunit (PKA-CS) yielded the greatest phosphorylation of recombinant Kvβ2 (rKvβ2), with limited phosphorylation by protein kinase C catalytic subunit (PKC-CS) and no detectable phosphorylation by casein kinase II (CKII). Protein kinase(s) from adult rat brain lysate phosphorylated both rKvβ2 and endogenous Kvβ. The PKA inhibitor, PKI 6-22, fully inhibited PKA-mediated phophorylation of rKvβ2 yet showed minimal inhibition of kinase activity present in rat brain. The inhibitor Gö 6983, that blocks PKCα, PKCβ, PKCγ, PKCδ and PKCζ activities, inhibited rKvβ2 phosphorylation by rat brain kinases, with no inhibition by Gö 6976 which blocks PKCα and PKCβΙ activities. Dose-response analysis of Gö 6983 inhibitory activity indicates that at least two PKC isozymes account for the kinase activity present in rat brain. Τhus, while PKA was the most active protein kinase to phosphorylate rKvβ2 in vitro, Kvβ2 phosphorylation in the rat brain is mainly mediated by PKC isozymes.