InteractiveFly: GeneBrief

CkIIalpha and CKIIbeta : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Casein kinase II (CkII) is a heterotetramer composed of two types of subunits: an alpha subunit that is catalytically active and a beta subunit that serves as a regulatory subunit. In many species, including higher vertebrates, the alpha subunit consists of two forms: alpha and alpha1. Alpha1 subunits have not been detected in either Drosophila or Xenopus. The beta subunit is inactive by itself but it stimulates the catalytic activity of alpha 5 to 10 fold. In addition, beta causes stabilization of alpha against heat denaturation and proteolysis and can change the specificity of alpha for its interaction with substrates and inhibitors. CkII can be moderately activated by polyamines such as spermine, spermidine and by polylysine. The most potent inhibitor is heparin. CkII can use either ATP or GTP, and the enzyme targets serines and threonines immersed in acidic motifs of proteins and peptides (Allende, 1995).

The list of CkII's targets is long and varied (Allende, 1995). It includes

• Enzymes involved in nucleic acid synthesis such as RNA polymerase II;
• DNA topoisomerase II;
• transcription factors including c-Myc, N-myc, Serum response factor, C-jun, p53, Retinoblastoma and cAMP regulatory element binding protein (CREB);
• signal transduction proteins including Protein kinase C, the regulatory subunit of Protein kinase A and Calmodulin;
• several protein synthesis factors;
• cytoskeletal and structural proteins such as beta-Tubulin, Myosin heavy chains, and Spectrin;
• other nuclear proteins such as HMG1, HMG14, and FK506-binding protein.

Known targets in Drosophila include Cactus (Packman, 1997), Engrailed (Bourbon, 1995), Dishevelled (Willert, 1997), Antennapedia (Jaffe, 1997), Drosophila Max (see Myc) (Gallant, 1996), and Tropoisomerase II (Ackerman, 1988).

The regulation of CkII is enigmatic; CkII does not seem to be regulated by many of the well-known second messenger molecules. Nevertheless, CkII activity is often increased in cells that are actively proliferating and embryonic tissues or organs contain higher concentrations of CkII than do their adult counterparts. It has also been shown that CkIIalpha is phosphorylated by cdc2 kinase in cells arrested in mitosis and that p24cdc2 kinase can be phosphorylated by CkII during G1. This suggests that the cell cycle is regulates and is regulated by CkII. The biological function served by the beta subunit in the regulation of the alpha subunit activity is unknown (Allende, 1995).

One example of the role of CkII in modification of a Drosophila protein is found in the interaction of CkII with Antennapedia. The in vivo activity of this HOX protein is modified by phosphorylation due to CkII. Antp has four putative CkII target sites. Sites 1 and 2 are found in the amino-terminal portion of the protein, whereas sites 3 and 4 are clustered close to the homeodomain in the C-terminal tail. Antp with alanine substitutions at its CkII target sites produces altered thoracic and abdominal development. Ubiquitous expression of Antp in flies produces an inhibition of head involution, the elimination of dorsal head structures, a transformation of T1 into a second thoracic segment (T2), and the appearance of one to two partial T2 denticle belts in the head segment. Embryos that express Antp with altered CkII target sites (alanine replacing serine or threonine) exhibit additional phenotypes including an absence of Keilin's organs, shortened denticle belts, and a failure of germ-band retraction. Embryos that express altered Antp show a disorganized CNS with irregularly spaced or fused horizonal commissures and gaps in the longitudinal commissures. CkII sites 1 and 4 appear to be the most important in terms of the altered phenotypes produced (Jaffe, 1997).

The novel functions that result from mutationally removing CkII sites suggest that altered Antp is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of Antp that contains acidic amino acid substitutions at its CkII target sites is greatly reduced, mimicking a constitutively phosphorylated Antp protein. This hypoactive form of Antp, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. These results suggest that phosphorylation of Antp by CkII is important for preventing inappropriate activities of this homeotic protein during embryogenesis. The information provided however does not address the mechanism by which phosphorylation alters Antp's properties. Thus phosphorylation appears to modulate Antp's properties, restricting its activity to an appropriate level (Jaffe, 1997).

Dishevelled protein is also targeted by CkII. Immunoprecipitated Dsh protein is associated with Casein Kinase II. Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CkII in vitro and in vivo, suggesting that CkII is at least one of the kinases that phosphorylates Dsh. Overexpression of frizzled2, a Wingless receptor, also stimulates phosphorylation of Dsh, Dsh-associated kinase activity, and association of CkII with Dsh. It is not known whether association of CkII with unphosphorylated Dsh occurs first, or whether phosphorylation of Dsh promotes association with CkII. Unphosphorylated Dsh clearly has some affinity for CkII, however, in vivo phosphorylated Dsh is associated with more CkII than is underphosphorylated Dsh. This suggests a model in which CkII can bind with low affinity to underphosphorylated Dsh and effect its phosphorylation. The phosphorylated Dsh then has a higher affinity for CkII, leading to an increase in the amount of Dsh-CkII complex. Phosphorylation of Dsh in response to the Wg signal, leads to the phosphorylation of Dsh but this is insufficient for the transduction of the Wg signal to Armadillo. Thus the function of the phosphorylation of Dsh by CkII is unknown (Willert, 1997).

Clearly CkII plays an important metabolic role in the regulation of enzyme activity, perhaps linked to the cell cycle. Nevertheless, an understanding of the regulatory inputs into CkII and an understanding of the global role of CkII in regulation of cell function awaits future work, especially the induction of CkII mutation.

REGULATION

Functional dissection of Timekeeper (Tik) implicates opposite roles for CK2 and PP2A during Drosophila neurogenesis

Repression by E(spl)M8 during inhibitory Notch signaling (lateral inhibition) is regulated, in part, by protein kinase CK2, but the involvement of a phosphatase has been unclear. Timekeeper (Tik), a unique dominant-negative (DN) mutation in the catalytic subunit of CK2, was used in a Gal4-UAS based assay for impaired lateral inhibition. Specifically, overexpression of Tik elicits ectopic bristles in N1 flies and suppresses the retinal defects of the gain-of-function allele N^spl. Functional dissection of the two substitutions in Tik (M¹⁶¹K and E¹⁶⁵D), suggests that both mutations contribute to its DN effects. While the former replacement compromises CK2 activity by impairing ATP-binding, the latter affects a conserved motif implicated in binding the phosphatase PP2A. Accordingly, overexpression of microtubule star (mts), the PP2A catalytic subunit closely mimics the phenotypic effects of loss of CK2 functions in N¹ or N^spl flies, and elicits notched wings, a characteristic of ^N mutations. These findings suggest antagonistic roles for CK2 and PP2A during inhibitory N signaling (Kunttas-Tatli, 2009).

Inhibitory N signaling is vital for stereotyped patterning of sense organs such as the eye and the bristles. This signaling pathway is required for proper SOP/R8 selection and involves cell-cell communications. Specifically, the future SOP/R8 cell expresses the highest levels of the N ligand, Delta, which activates N in all cells of the PNC, but the future SOP/R8. This, in turn, elicits expression of the E(spl) repressors, a family of homologous basic-helix-loop-helix (bHLH) proteins. These bHLH proteins, along with the corepressor Groucho, then antagonize ASC/Ato. As a result, cells that receive N signaling are redirected from adopting the default (SOP/ R8) neural fate. This model reflects the findings that loss of inhibitory N signaling leads to excess SOP and R8 specification, which manifest as ectopic bristles and rough eyes, respectively. It is, therefore, important to fully define the mechanisms that regulate this critical step in neural patterning (Kunttas-Tatli, 2009).

Earlier studies suggested that transcription of E(spl) and the ensuing rise in protein levels was, perhaps, sufficient for restriction of the R8/SOP fate. Accumulating evidence, however, suggests that phosphorylation of E(spl) proteins is important for repression. Evidence has so far been obtained for M8 and its structurally related repressor Hairy, and in either case phosphorylation by CK2 augments repression in the eye and/or the bristle. It has, however, remained unclear whether protein phosphatases act to oppose CK2 functions. The characterization of such a regulation would open the possibility that phosphorylation and repression by E(spl) (inhibitory N signaling) is dynamically controlled in vivo. A role for PP2A has been implicated in studies showing ectopic bristle defects upon increased dosage of the regulatory subunits widerborst (wdb) or twins (tws) and in screens for modifiers of N. However, interactions between PP2A and alleles of N, such as N^spl have not yet been described. These studies provide new insights into the genetic behaviors of Tik and its revertant allele TikR, and implicate a tripartite regulatory nexus, involving CK2, PP2A and inhibitory N signaling (Kunttas-Tatli, 2009).

Both Tik and TikR lack CK2 kinase activity (in vitro). The severe clock defect of Tik/1 flies is, however, not observed in TikR/1 animals, and in this sense TikR meets the criteria of a revertant allele. These studies suggest that the TikR protein is not only devoid of kinase activity, but more importantly is deficient for binding CK2b, a prerequisite for CK2-holoenzyme formation and for proper functions in vivo. The most parsimonious interpretation is that misfolding of TikR prevents its incorporation into the holoenzyme. It seems reasonable to, therefore, suggest that the ability of Tik to incorporate into and 'poison' the endogenous holoenzyme (by binding CK2b) underlies its strong DN effects in vivo. However, it has been generally thought that these effects of Tik primarily reflect the M¹⁶¹K, but not the E¹⁶⁵D, substitution. These studies on site-specific variants, suggest that these substitutions have additive effects on activity and N signaling, and Tik is likely to therefore be a 'double hit (Kunttas-Tatli, 2009).

The studies in N¹ and N^spl backgrounds provide evidence that both substitutions in Tik affect proper CK2 functions. How might one interpret the effects on N^spl? Unlike the bristle, where N signaling occurs only after the specification of the bristle PNCs, the development of patterned founding R8 photoreceptors requires N signaling in a biphasic manner in the MF of the developing third instar eye disc. At the anterior margin of the MF, N elicits ato expression (for R8 specification), whereas in the MF it drives expression of E(spl) enabling refinement of a single R8 cell from the PNCs. N^spl only perturbs the latter. Specifically, N^spl renders R8 precursors hypersensitive to inhibitory N signaling, and consequently impairs R8 differentiation. These impaired R8’s are defective in the presentation of signals such as Hedgehog and Decapentaplegic, whose activities are necessary for ato expression at the anterior margin of the MF. As a result, the reduced ato expression in the MF of N^spl perpetuates throughout retinal histogenesis, and elicits the rough and reduced eye of N^spl. Consistent with the notion that this allele renders R8’s sensitive to inhibitory N signaling, the retinal defect of N^spl are strongly suppressed by conditions that attenuate E(spl) activity, such as halved dosage of Delta or E(spl), or by reduced CK2 activity (Kunttas-Tatli, 2009).

The dominant-negative effects of CK2a-M¹⁶¹K and CK2a-E¹⁶⁵D in N¹ and in N^spl animals are likely to involve the ability of either variant to robustly interact with CK2b and efficiently incorporate into the endogenous holoenzyme, in a manner akin to wild type CK2alpha. It is suggested that incorporation of the former variant attenuates endogenous CK2 activity. In contrast, the dominant-negative effects of the E¹⁶⁵D substitution might not involve impaired CK2 kinase activity, but instead reflect its ability to perturb the interaction of endogenous CK2 with PP2A, an interaction that is increasingly suspect in the regulation of this protein phosphatase. These possibilities are addressed below (Kunttas-Tatli, 2009).

The effects of CK2alpha-M¹⁶¹K in N¹ or in N^spl are easier to reconcile given its position in the ATP-binding site. This substitution substantially impairs kinase activity, and consequently ectopic CK2alpha-M¹⁶¹K mimics the neural defects of knockdown of this enzyme by RNAi. It would therefore seem to be the case that ectopic CK2alpha-M¹⁶¹K binds CK2beta, efficiently incorporates into the endogenous CK2-holoenzyme and attenuates activity, and this lowered activity impairs phosphorylation of, and repression by, endogenous E(spl). If so, this will reduce the 'strength' of inhibitory N signaling and elicit ectopic bristles in N¹, and suppress the eye/R8 defects of N^spl. The effects of CK2alpha-M¹⁶¹K in these three developmental contexts are consistent with this model (Kunttas-Tatli, 2009).

However, the behavior of the E¹⁶⁵D substitution was unexpected. The suggestion that this substitution exerts a negative impact on CK2 functions is supported by multiple findings, in addition to the extraordinary conservation of Glu165 in metazoan CK2alpha subunits. First, CK2alpha-E¹⁶⁵D elicits ectopic bristles in N¹ and suppresses the retinal defects of N^spl, and these effects are observed with multiple independent insertions and with multiple drivers. Second, CK2alpha-E¹⁶⁵D restores eye size and the hexagonal phasing of the facets in N^spl, akin to Tik or CK2alpha-M¹⁶¹K. Third, CK2alpha-E¹⁶⁵D appears to restore Ato expression anterior to the MF and increases the number of Sens-positive R8 cells at its posterior margin. Therefore, its effects closely correlate, in time and space, to R8 cell specification, which is defective in N^spl. Together, these results suggest that the E¹⁶⁵D substitution impairs CK2 functions. These functions, however, might not involve perturbed kinase activity per se, but may instead be related to the interaction of this enzyme with PP2A (Kunttas-Tatli, 2009).

Studies with mts overexpression are of interest, because this is the first demonstration that increased dosage of the PP2A catalytic subunit elicits developmental defects that are hallmarks of loss of N functions. Specifically, mts overexpression elicits ectopic bristles and notched wings in N¹ flies, and suppresses the retinal defects of N^spl. Furthermore, its effects on restored ommatidial phasing and eye size (facet numbers) are comparable to those seen with Tik, CK2alpha-M¹⁶¹K or CK2alpha-E¹⁶⁵D. These studies lead to the suggestion that interaction of PP2A with CK2 down-regulates phosphatase activity, perhaps by competing with the regulatory subunit such as Wdb, which is essential for target recognition and dephosphorylation. Such a mechanism would reflect the mutually exclusive binding of the catalytic (Mts) subunit of PP2A with Wdb or SV40 t-antigen. If so, ectopic Mts would override the binding capacity of endogenous CK2, and upon recruiting Wdb attenuate repression by E(spl) through dephosphorylation (Kunttas-Tatli, 2009).

This model could account for the dominant-negative effects of CK2alpha-E¹⁶⁵D. In this case, ectopic CK2alpha-E¹⁶⁵D would bind CK2beta, incorporate into the endogenous CK2-holoenzyme, and impair PP2A binding and downregulation. Its effects should therefore mimic Mts overexpression, a proposal that is consistent with the findings. If so, overexpression of CK2-E¹⁶⁵D probably leads to enhanced PP2A activity. In contrast, the effects of ectopic CK2alpha-M¹⁶¹K more likely reflect a negative influence on CK2 activity itself, and suggest that this variant may represent a more precise dominant-negative construct of CK2 (Kunttas-Tatli, 2009).

The possibility arises that a precise regulation of repression by E(spl) proteins involves a balance between the opposing activities of CK2 and PP2A, perhaps involving direct interactions. Indeed, direct interactions between CK2 and PP2A have been identified by proteomic analysis in the mouse model and in cultured cells. While consensus sequences for kinases are easier to identify computationally and biochemically, similar analysis with phosphatases has been less forthcoming. For example, in the case of Period (Per), the central clock protein, coordinated activities of CK2, CK1, and PP2A are required for proper function. While Per is phosphorylated by CK2 and CK1 in vitro and in vivo, evidence for its dephosphorylation by PP2A is lacking especially as it relates to its site preference(s). In the future it will be important to determine whether E(spl) proteins are direct targets of PP2A, and if so how a balance between PP2A and CK2 activities regulates repression. PP2A may play a similar role in the regulation of mammalian Hes6 (the homolog of fly M8), given its phosphorylation by CK2. A reversible switch could be important in neural patterning to confer a rapid and precise temporal control over the onset of repression, or prevent a protracted block to the neural fate once resolution of the PNC has occurred and the SOP’s and R8’s have been selected (Kunttas-Tatli, 2009).

CK2-alpha regulates the transcription of BRP in Drosophila

Development and plasticity of synapses are brought about by a complex interplay between various signaling pathways. Typically, either changing the number of synapses or strengthening an existing synapse can lead to changes during synaptic plasticity. Altering the machinery that governs the exocytosis of synaptic vesicles, which primarily fuse at specialized structures known as active zones on the presynaptic terminal, brings about these changes. Although signaling pathways that regulate the synaptic plasticity from the postsynaptic compartments are well defined, the pathways that control these changes presynaptically are poorly described. In a genetic screen for synapse development in Drosophila, this study found that mutations in CK2α lead to an increase in the levels of Bruchpilot (Brp), a scaffolding protein associated with the active zones. Using a combination of genetic and biochemical approaches, this study found that the increase in Brp in ck2α mutants is largely due to an increase in the transcription of brp. Interestingly, the transcripts of other active zone proteins that are important for function of active zones were also increased, while the transcripts from some other synaptic proteins were unchanged. Thus, these data suggest that CK2α might be important in regulating synaptic plasticity by modulating the transcription of Brp. Hence, it is proposed that CK2α is a novel regulator of the active zone protein, Brp, in Drosophila (Wairkar, 2013).

Drosophila Mbm is a nucleolar Myc and CK2 target required for ribosome biogenesis and cell growth of central brain neuroblasts

Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy sensing organs into growth promoting processes such as the sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila. Yet, the molecular function of Mbm has been unknown so far. This study shows that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which among other functions relays information from nutrient dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2, which has an impact on localization of the protein. It is concluded that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which together with CK2-mediated signals enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth (Hovhanyan, 2014).

A fundamental issue during development of a multicellular organism is to coordinate cell proliferation, the availability of nutrients, and cell growth. Prominent examples are neuroblasts, the progenitor cells of the Drosophila melanogaster central nervous system, which proliferate in a highly regulated manner during development. Upon selection and specification, central brain neuroblasts proliferate until the end of embryogenesis, when they enter a quiescent state until resuming proliferation with the beginning of larval development. Notable exceptions are the neuroblasts generating the mushroom bodies, a paired neuropil structure in the central brain involved in learning and memory processes, which proliferate throughout development. Depending on the neuroblast lineage, proliferation stops at late larval or pupal stages by terminal differentiation or apoptosis. The embryonic and larval waves of neurogenesis correlate with changes in neuroblast size. Embryonic neuroblasts decrease in size with each cell division until they enter quiescence; resumption of proliferation at the larval stage is preceded by cell growth. In contrast to embryonic neuroblasts, larval neuroblasts maintain their cell size until the end of the proliferation period, which is again accompanied by a decrease in cell size. Exit of neuroblasts from quiescence, and thereby activation of proliferation, depends on the nutritional status of the whole animal and is governed by the insulin receptor (InsR)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, triggered by insulin-like peptide-producing glia cells, which receive nutritional signals from the fat body). Maintaining InsR signaling in combination with blocking of apoptosis is sufficient for long-term survival and proliferation of neuroblasts even in the adult fly. On the other hand, cellular nutrient sensing is mediated by the target of rapamycin (TOR) pathway, which, together with the InsR pathway, regulates cell growth through a variety of effector proteins at the levels of gene expression, ribosome biogenesis, and protein synthesis. Whereas neuroblast reactivation requires the interconnected InsR-PI3K and TOR pathways, neuroblast growth at larval stages is maintained even under nutrient restriction, by anaplastic lymphoma kinase (Alk)-mediated but InsR-independent activation of the PI3K pathway in combination with a direct influence of Alk on TOR effector proteins (Cheng, 2011; Hovhanyan, 2014 and references therein).

Cell growth requires protein synthesis, which depends on a sufficient supply of functional ribosomes. Ribosome biogenesis takes place in the nucleolus and involves transcription of single rRNA units and their processing and modification into 18S, 28S, and 5.8S rRNAs, which assemble with multiple ribosomal proteins to separately form the small and large ribosomal subunits. Upon transport to the cytoplasm, both subunits mature before they build up functional ribosomes. In general, one key downstream effector of TOR signaling is the transcription factor Myc, which controls cell growth in part by regulating ribosome biogenesis through transcriptional control of rRNA, ribosomal proteins, and proteins required for processing and transport of ribosomal components. Genomewide analyses of Drosophila Myc transcriptional targets emphasized the role of Myc as a central regulator of growth control but also identified many target genes with unknown molecular functions of the corresponding proteins. One of the Myc-responsive genes with an unknown function was mushroom body miniature (mbm). The original hypomorphic mbm¹ allele was identified in a screen for viable structural brain mutants and showed a pronounced reduction in the size of the adult mushroom body neuropil, which was due at least in part to a reduction in the number of intrinsic mushroom body neurons. More severe allelic combinations indicated a general requirement for Mbm in brain development and uncovered a neuroblast proliferation defect as a major cause of the phenotype. However, which step requires Mbm for neuroblast proliferation remains elusive. Homology searches provided no clue about the molecular function of Mbm. Structural features of Mbm include several stretches enriched in certain amino acids, a putative nuclear localization signal, and two consecutive CCHC zinc knuckles. This report describes Mbm as a new nucleolar protein. Mbm is highly expressed in neuroblasts and is required for proper cell growth but not for processes controlling asymmetric cell division. Corresponding to the observed cell size defect, evidence is provided that small but not large ribosomal subunit biogenesis is impaired in the mutant, which could be a consequence of defective rRNA processing. Mbm is a transcriptional target of Myc and requires posttranslational modification by casein kinase 2 (CK2) for full functionality, as revealed by mutation of identified CK2 phosphorylation sites. These results provide a new link between Myc and growth control of neuroblasts and also establish a function of CK2 in neuroblasts (Hovhanyan, 2014).

This study identified Mbm as a new component of the nucleolus which has no obvious homologue outside the Drosophilidae. In contrast to the tripartite organization of vertebrate nucleoli in a fibrillar center, a dense fibrillar component (DFC), and a granular component, nucleoli of Drosophila neuroblasts often appear as a homogenous structure at the ultrastructural level, sometimes with intermingled fibrillar and granular components. In neuroblasts, Mbm colocalizes with fibrillarin and Nop5. In vertebrates, the methyltransferase fibrillarin is associated with Nop56/58 (corresponding to Drosophila Nop5) as part of the C/D type of small nucleolar ribonucleoprotein (snoRNP) complex required for rRNA processing in the DFC. Indeed, this study observed an aberrant rRNA intermediate in mbm on Northern blots, implicating a requirement of Mbm in rRNA processing. More specifically, based on the retention of RpS6 in the nucleoli of mbm neuroblasts, a function of Mbm is proposed in small ribosomal subunit biogenesis. The complementary phenotype, failure of large ribosomal subunit nucleolar-to-cytoplasmic transport, was observed in Drosophila upon knockdown of nucleostemin 1 (NS1). Yet the molecular function of Mbm remains elusive at this point because of its unique domain composition, with two zinc knuckle structures, several clusters of acidic or basic amino acids, and arginine/glycine-rich sequence stretches. For example, proteins containing arginine/glycine (RGG) repeats are found in a variety of RNP complexes, including snoRNPs. For a detailed biochemical analysis of Mbm function in ribosome biogenesis, cellular systems that are more accessible than neuroblasts are required, as these represent only a minor fraction of all brain cells. However, despite expression of Mbm in tissue culture S2 cells, neither cell size nor proliferation defects were detected under knockdown conditions (Hovhanyan, 2014).

Metabolic labeling of mbm neuroblasts indicated lowered protein synthesis rates, which could have been due to the lack of sufficient numbers of functional ribosomes. mbm larval brains reach nearly wild-type size, with a delay of several days, indicating that protein synthesis is maintained to at least some degree in neuroblasts. Since the process of asymmetric cell division itself is not affected in mbm flies, this provides one likely explanation for impaired neuroblast growth and proliferation. The importance of sufficient cell growth for repeated division of neuroblasts has been documented for mutations in signaling components. The comparison of the relative protein expression levels of Mbm and other nucleolar proteins in different cell types showed a more pronounced expression of Mbm in neuroblasts. This is confirmed by a comparative transcriptome analysis between neuroblasts and neurons. Altogether, the data suggest a more neuroblast-specific function of Mbm in ribosome biogenesis. Indeed, whereas most ribosomal subunit components are required in all cells, different isoforms are expressed for some components, with one isoform being required in all cells and the other isoform being required more specifically in stem cell lineages. Whereas loss of the generally required components causes early lethality, loss of specifically expressed isoforms is associated with a decrease in neuroblast size and an underproliferation phenotype. This emphasizes the specific needs of neuroblasts in ribosome biogenesis and cell growth as rate-limiting steps for self-renewal (Hovhanyan, 2014 and references therein).

The identification of Mbm as a transcriptional target of Myc provides a potential link to systemic and cell-intrinsic growth control of neuroblasts mediated by the InsR-PI3K-Akt and TOR pathways. In contrast to other tissues, where Myc is a downstream effector of these pathways, information is still largely missing in the case of neuroblasts. Myc is expressed in neuroblasts, and upon knockdown, mild effects on neuroblast size but not neuroblast number were observed. Consistent with the role of Drosophila Myc in expression of many genes involved in ribosome biogenesis, removal of Myc function in single neuroblasts caused corresponding decreases in Mbm and Nop5 levels (Hovhanyan, 2014).

Mbm function is dependent on posttranslational modification by the protein kinase CK2. CK2 is a promiscuous kinase expressed in all eukaryotic cells, with a vast array of substrates with pleiotropic functions. However, CK2 not only acts as a heterotetrameric α₂β₂ holoenzyme but also exists as free populations of both subunits, with independent functions. Pronounced nuclear or nucleolar localization of CK2 subunits was observed in vertebrate cells. In the nucleolus, CK2 participates in rRNA transcription by phosphorylating different components of the RNA polymerase I transcription machinery. Proteins involved in ribosome biogenesis, such as B23 (also known as nucleophosmin), are also CK2 phosphorylation targets. CK2 modulates the ability of B23 to act as a molecular chaperone, its mobility rate and compartmentalization in the nucleolus, and its shuttling between the nucleolus and the nucleoplasm. Mbm and Nopp140 are the only described nucleolar phosphorylation targets of CK2 in Drosophila. This correlates with the observed nucleolar accumulation of CK2α in neuroblasts and the copurification of Mbm and CK2α. Although Mbm proteins with mutated CK2 phosphorylation sites showed cytoplasmic accumulation, nucleolar localization was still evident. Yet they were largely unable to complement the loss of endogenous Mbm function, indicating that phosphorylation by CK2 not only is a localization determinant but also is important for proper functioning of Mbm in the nucleolus. CK2 is often considered a constitutively active kinase which is not regulated by second messenger signaling cascades. However, there is increasing evidence for regulation of CK2 at the levels of the holoenzyme, the dynamics of localization of individual subunits under different conditions, and interactions with small molecules such as polyamines. Overexpression of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis and a known Myc target gene, increases CK2 phosphorylation activity toward nucleolar B23 in mouse keratinocytes. It would be interesting to test for a regulatory influence of CK2 on Mbm function under different conditions (Hovhanyan, 2014).

In summary, Mbm is considered to be part of the Myc and CK2 regulatory networks for coordination of neuroblast growth and proliferation; however, more information about the molecular function of Mbm at the level of small ribosomal subunit biogenesis is still required (Hovhanyan, 2014).

Protein Interactions

In vitro experiments have shown that Stellate can interact with the catalytic ß subunit of casein kinase 2 enzyme, altering its activity (Bozzetti, 1995).

Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. Like mammalian IkappaBalpha, the PEST domain of Drosophila Cactus is phosphorylated by Casein kinase II. The site of modification has been localized to a single residue, Ser468 in the PEST domain: no evidence for additional phosphorylation sites are found. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by Casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis (Packman, 1997).

Regulated proteolysis of Cactus, the cytoplasmic inhibitor of the Rel-related transcription factor Dorsal, is an essential step in the patterning of the Drosophila embryo. Signal-induced Cactus degradation frees Dorsal for nuclear translocation on the ventral and lateral sides of the embryo, establishing zones of gene expression along the dorsoventral axis. Cactus stability is regulated by amino-terminal serine residues necessary for signal responsiveness, as well as by a carboxy-terminal PEST domain. Drosophila casein kinase II (CKII) has been identified as a Cactus kinase: CKII specifically phosphorylates a set of serine residues within the Cactus PEST domain. These serines are phosphorylated in vivo and are required for wild-type Cactus activity. Conversion of these serines to alanine or glutamic acid residues differentially affects the levels and activity of Cactus in embryos, resulting in a longer half-life for endogenous Cactus. As a result, Cactus accumulates above wild-type levels, interfering with signal transduction. Mutation modification of the PEST domain does not inhibit the binding of Cactus to Dorsal. Taken together, these data indicate that wild-type axis formation requires CKII-catalyzed phosphorylation of the Cactus PEST domain. CKII phosphorylation may play a general role in PEST domain function (Liu, 1997).

The Engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos: phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled Engrailed protein. The primary embryonic protein kinase modifying Engrailed protein is Casein kinase II (CkII). Analysis of mutant proteins reveals that the in vitro phosphoacceptors are mainly clustered in a region outside The Engrailed homeodomain and identifies serines 394, 397, 401, and 402 as the targets for CkII phosphorylation. CkII-dependent phosphorylation of an N-truncated derivative of Engrailed protein purified from bacteria increases its DNA binding 2-4-fold (Bourbon, 1995).

The dishevelled gene of Drosophila encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. Immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity shows it to be identical to Casein kinase II. Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by Ck2 in vitro and in vivo, suggesting that Ck2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulates phosphorylation of Dsh, Dsh-associated kinase activity, and association of Ck2 with Dsh, thus suggesting a role for Ck2 in the transduction of the Wg signal. The Wg signal leads to the phosphorylation of Dsh but this is insufficient for the transduction of the Wg signal to Armadillo. Thus the function of the phosphorylation of Dsh by CkII is unknown (Willert, 1997).

Wingless (Wg) treatment of the Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg receptor, Frizzled2. However, a line of S2 cells obtained from another source express both Frizzled-2 and Frizzled. Thus, this cell line was designated as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and Shotgun protein levels and by hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induces the same changes in Dsh, Arm, and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells is phosphorylated as a consequence of expression of Frizzled-2 or mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as sensitive to Wg as are clone 8 cells, but theycan grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of Wg signaling (Yanagawa, 1998).

Thus expression of Dfz2 or Mfz6 induces phosphorylation of Dsh in S2 cells and a small proportion of Dsh protein is phosphorylated in S2R+ and clone 8 cells. These results suggest that expression of frizzled family proteins induces the basal phosphorylation of Dsh. In this regard, Casein kinase 2 (CK2), which binds to the PDZ domain of Dsh, is known to be the major kinase responsible for phosphorylation of Dsh upon Dfz2 overexpression in S2 cells. Therefore, CK2 may take part in the basal phosphorylation of Dsh in Dfz2/S2, Mfz6/S2, clone 8 and S2R+ cells not stimulated with soluble Wg. In addition, Frizzled overexpression leads to translocation of Dsh from cytoplasm to plasma membrane. Overexpression of rat frizzled-1 has been shown to result in recruitment of Xwnt-8 and XDsh to the plasma membrane in Xenopus embryos. Thus, it is possible that Dfz2 or Mfz6 expression induces translocation of at least a part of Dsh to the plasma membrane in S2 cells and that this Dsh translocation in some way stimulates Dsh phosphorylation by CK2. However, it is not clear whether CK2 also participates in Wg-induced hyperphosphorylation of Dsh or whether other kinase(s) are activated by the binding of Wg to Dfz2 in clone 8, S2R+, and Dfz2/S2 cells and that these other kinases induce the hyperphosphorylation. In view of the reports indicating association (probably indirect) between frizzled family proteins and Dsh and the binding of Dsh to CK2, it is attractive to speculate that Wg binding induces aggregation of Fz2 receptors, which, in turn, brings the Dsh-CK2 or other kinase complexes close together, and this aggregation stimulates the Dsh phosphorylation by CK2 or other kinases in these Dsh-kinase complexes. This could explain how Wg induces Dsh hyperphosphorylation in clone 8, S2R+, and Dfz2/S2 cells. However, it is noteworthy that Dfz2 overexpression leads to marked phosphorylation of Dsh, but not to elevation of Arm, in S2 cells, indicating that phosphorylation of Dsh, at least by Dfz2 overexpression, cannot activate the Wg signaling pathway by itself. Clearly, further detailed experiments are necessary to evaluate the function of Dsh phosphorylation in Wg signaling (Yanagawa, 1998 and references).

The in vivo activity of the HOX protein Antennapedia (Antp) is modified because of phosphorylation by the serine/threonine kinase casein kinase II (CkII). Antp has four putatitive CkII target sites. Sites 1 and 2 are found in the amino-terminal portion of the protein, whereas sites 3 and 4 are clustered close to the homeodomain in the C-terminal tail. Using an in vivo assay a form of Antp that has alanine substitutions at its CkII target sites has (in addition to wild-type Antp functions) the ability to alter severely thoracic and abdominal development. Ubiquitous expression of Antp in flies produces an inhibition of head involution, the elimination of dorsal head structures, a transformation of T1 into a second thoracic segment (T2), and the appearance of one to two partial T2 denticle belts in the head segment. Embryos that express Antp with altered CkII target sits (alanine replacing serine or threonine) exhibit additional phenotypes including an absence of Keilin's organs, shortened denticle belts, and a failure of germ-band retraction. Occasionally, individual denticles within the abdominal belts appear small and thoracic-like. 25-30% of embryos exhibit defects at the posterior terminus including reduced anal pads, anal tuft and filzkörper (a prominent telson structure). Antp with altered CkII target sites reduces or eliminates expression of rhomboid in ventral-lateral clusters (normally associated with tracheal pits and precursors of the chordotonal organs) and also induces ectopic dpp expression between dorsal and ventral rows. Embryos that express altered Antp show a disorganized CNS with irregularly spaced or fused horizonal commissures and gaps in the longitudinal commissures. Distal-less expression is reduced in the head segment and eliminated or nearly eliminated in the thoracic segment of 6- to 9-hour embryos expressing altered Antp. dpp is inappropriately expressed in 7- to 10-hr embryos in the visceral mesoderm, and empty spiracles, normally activated by Abd-B in parasegment 8, is greatly diminish. CkII sites 1 and 4 apppear to be the most important in terms of the altered phenotypes produced (Jaffe, 1997).

The novel functions of altering CkII sites with alanine suggest that this form of Antp is not suppressed phenotypically by the more posterior homeotic proteins. In contrast, the in vivo activity of a form of Antp that contains acidic amino acid substitutions at its CkII target sites is greatly reduced, thereby mimicking a constitutively phosphorylated Antp protein. This hypoactive form of Antp, but not the alanine-substituted form, is also reduced in its ability to bind to DNA cooperatively with the homeodomain protein Extradenticle. These results suggest that phosphorylation of Antp by CkII is important for preventing inappropriate activities of this homeotic protein during embryogenesis. The information provided however does not address the mechanism by which phosphorylation alters Antp's properties (Jaffe, 1997).

DMax, the Drosophila homolog of mammalian Max, consists of 161 amino acids, compared with the human Max9, which has 160. The greatest sequence similarity is within the bHLH leucine zipper domain with a 67% identity. All residues contacting DNA are conserved. The N-termini of dMax and human Max are highly conserved, including two Casein kinase II phophorylation sites that negatively regulate DNA binding. DMax interacts strongly with dMyc and with itself, but dMyc does not self associate. DMyc alone does not bind the CACGTG site but dMyc-dMax does. The dMyc-dMax heterodimer is also able to activate transcription (Gallant, 1996).

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identify casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II:

1. The only amino acyl residue modified in the enzyme is serine.
2. Partial proteolytic maps of topoisomerase II, which have been labeled with either (32P) phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified Casein kinase II are indistinguishable from one another.
3. Phosphorylation in cell homogenates is inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum.
4. Cell homogenates are able to employ(gamma-32P)GTP as a phosphate donor nearly as well as (gamma-32P)ATP.

Although topoisomerase II is phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification is always sensitive to anti-Casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by Casein kinase II in the Drosophila embryonic Kc cell system (Ackerman, 1988).

The self-aggregation behavior of casein kinase II from Drosophila has been analyzed by velocity sedimentation and electron microscopy. The results indicate that self-aggregation involves the formation of linear polymers or filaments approximately 10 nm in diameter. In the presence of 1 mM EDTA filament length is inversely proportional to total ionic strength over a range from 0.05 to 0.28; filaments as long as 0.5 micron are observed at the lower ionic strengths. Similar results are obtained in the presence of 10 mM MgCl2, but two additional ionic strength-dependent phenomena are superimposed: (1) at subphysiological ionic strength side-to-side aggregation of filaments occur, which results in enzyme precipitation; (2) at physiological ionic strength a time- and temperature-dependent increase in filament length occurs that generated polymers up to 5 microns long. No side-to-side aggregation occurs under the latter conditions. Filamentous forms of the kinase can be readily reconverted to the standard alpha 2 beta 2 tetramer by the addition of high salt. Filamentous casein kinase II is observed over a pH range from 6.8 to 8.0, at enzyme concentrations ranging from 6 to 150 micrograms/ml, in the presence of ATP, and at MgCl2 concentrations from 1 to 10 mM. However, time-dependent growth of long filaments is not observed at Mg2+ concentrations below 10 mM. The conditions under which filaments are observed in vitro suggest that they may also exist in vivo (Glover, 1986).

Casein kinase II is composed of two catalytic (alpha) and two regulatory (beta) subunits: the amino acid sequences of the alpha and beta subunits are highly conserved between species. To examine whether heterologous casein kinase II could be formed, recombinant alpha and beta subunits from human and Drosophila were reconstituted from inclusion bodies. Casein kinase II containing either human alpha and Drosophila beta or Drosophila alpha and human beta subunits exhibits enzymatic properties similar to those of the homologous holoenzymes with regard to specific activity, salt optima, and autophosphorylation. However, renaturation and reconstitution of casein kinase II is dependent on the type of beta subunits and the redox conditions, with the Drosophila beta subunits requiring more reduced conditions. Chimeric beta subunits prepared from human and Drosophila cDNA reveal that the N-terminal region is responsible for the requirement for the reduced redox state during renaturation. The N-terminal region also affects solubility and electrophoretic mobility of the beta subunit (Lin, 1994).

Casein kinase II is unique when compared to other protein kinases; it utilizes GTP with almost the same effectiveness as ATP and exists as an active holoenzyme that does not need to be activated by dissociation of regulatory subunits or unfolding of regulatory domains. In vitro, the activity of casein kinase II is inhibited by acidic compounds and stimulated by basic compounds. Casein kinase II activity is inhibited by 2,3-bisphosphoglycerate and stimulated by polyamines at levels which are physiological in red cells. To examine the effects of autophosphorylation of the beta subunit on activity, two mutants of the Drosophila beta subunit have been constructed in which Ser-4 or Ser-(2-4) are changed to alanine residues. Analysis of autophosphorylation with wild-type and mutant recombinant holoenzymes reveals Ser-2 and Ser-3 as the major autophosphorylation sites. Autophosphorylation does not affect the phosphorylation of casein, but reduces the rate of phosphorylation of glycogen synthase by 30%, elongation factor I by 50-70%, and calmodulin by 20-40%. The data indicate that autophosphorylation of the beta subunit can negatively regulate the phosphotransferase activity of casein kinase II with physiological substrates. To examine regulation of casein kinase II activity by the beta subunit, recombinant alpha and beta subunits from human and Drosophila were expressed in Escherichia coli. Upon formation of the holoenzyme, the beta subunit stimulates the catalytic activity 4- to 5-fold. The catalytic alpha subunit contains the eleven conserved subdomains characteristic of all protein kinases (Jakobi, 1994).

Both calf and Drosophila contain a type II casein kinase with similar molecular structure and catalytic activity. Purified calf thymus casein kinase II is composed of three subunits of Mr = 44,000 (alpha), 40,000 (alpha'), and 26,000 (beta), whereas the Drosophila enzyme is composed of two subunits of Mr = 36,700 (alpha) and 28,200 (beta). The native form of the enzyme is an alpha 2 beta 2 tetramer. Polyclonal antibodies prepared against each enzyme react with both the alpha and beta subunits of the homologous enzyme and cross-react with both subunits of the heterologous enzyme. Reaction of polyclonal antibodies with proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis establishes that no significant difference in subunit molecular weight exists between the purified enzymes and the enzyme present in initial cell extracts. Each antibody effectively inhibits the in vitro activity of the homologous enzyme and causes a slight inhibition in the activity of the heterologous enzyme. Peptide maps derived from purified subunits indicate that the alpha and beta subunits are unique and that there is extensive primary sequence homology between the corresponding subunits of the calf and Drosophila enzyme. Casein kinase II from both sources phosphorylates the same subunits of calf thymus RNA polymerase II and an identical set of proteins in a complex mixture of acid-soluble proteins from Drosophila tissue culture cells. The striking similarity in molecular structure and catalytic activity between the calf and Drosophila enzyme suggests that casein kinase II has been highly conserved in evolution (Dahmus, 1984).

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 (Glover, 1983).

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme is very similar to that of the native enzyme, and is fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme is estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrates a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation show that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme is tetrameric (like the native enzyme), whereas the structure of the alpha subunit is monomeric. At lower ionic strength the recombinant holoenzyme has a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displays a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates (Birnbaum, 1992).

Casein kinase II is composed of catalytic (alpha) and regulatory (beta) subunits that 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 yeast 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. Casein kinase II activity was characterized 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 (Bidwai, 1992).

The Drosophila melanogaster DmCK2beta transcription unit encodes for functionally non-redundant protein isoforms

Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2α subunit and/or a regulatory CK2β subunit may exist. The phosphotransferase activity of members of the protein kinase CK2α family is thought to be independent of second messengers but is modulated by interaction with CK2β-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2α subunit and three genes encoding for CK2β-like proteins are present. The X-linked DmCK2β transcription unit encodes for several CK2β protein isoforms due to alternative splicing of its primary transcript. This study addresses the question whether CK2β-like proteins are functionally redundant. In vivo experiments show that variations of the very C-terminal tail of CK2β isoforms encoded by the X-linked DmCK2β transcription unit influence their functional properties. In addition, CK2β-like proteins encoded by the autosomal D. melanogaster genes CK2βtes and CK2β′ (see Casein kinase II ß2 subunit ) cannot fully substitute for a loss of CK2β isoforms encoded by DmCK2β (Jauch, 2006).

Despite more than 50 years of research on CK2, in vivo regulation of this pleiotropic protein kinase remains enigmatic. Biochemical experiments suggested that CK2 holoenzyme activity depends on N-terminal phosphorylation of its regulatory subunits. In addition, binding of polyamines to the acidic loop of the β-subunit was found to modulate CK2 holoenzyme phosphotransferase activity in vitro. It remains an unanswered question whether these modulating processes work in vivo. The identification of the two Drosophila CK2β-like proteins CK2β′ and CK2βtes that interact with CK2α points towards a mechanism to regulate CK2α in vivo by the usage of tissue specific regulatory subunits that may differ in their regulatory properties, but the restricted expression pattern of these two CK2β-like proteins would limit this assumed regulatory mechanism to the male testis. This limitation could be overcome by the use of CK2β isoforms generated by the translation of alternatively spliced DmCK2β mRNAs. All of the identified CK2β variants interact with CK2α, a prerequisite for a differential regulation of this serine/threonine kinase. However, the existence of different CK2β-like proteins in Drosophila does not rule out the possibility of functional redundancy, which was observed for a CK2β isoform and CK2βtes in ion-homeostasis rescue experiments in CKB mutant yeast strains. Based on ectopic expression experiments and the experiments designed to rescue the lethality of a DmCK2β null allele full functional redundancy among CK2β isoforms in one case and CK2βtes and CK2β′ in the other can be ruled out. This does not exclude that these CK2β-like proteins have some overlapping functions, but the assumed common functions are not sufficient to confer the same properties to these evolutionary closely related members of the Drosophila CK2β-like protein family. Furthermore, in overexpression and rescue experiments full functional redundancy was observed only between two CK2β isoforms, namely CK2β-VIIa and CK2β-VIIc. Taken together, these experiments point to specific functions of different CK2β isoforms that might be employed to fine-tune the CK2 holoenzyme (Jauch, 2006).

The very C-terminal tail of human CK2β is disordered in the crystal structure of recombinant human CK2. Furthermore, truncation of these amino acids has no influence on the biochemical properties of recombinant CK2. Therefore, the conclusion has been drawn that these amino acid residues are not important for holoenzyme function and formation. Since only the very C-terminal tail varies among Drosophila CK2β isoforms that are functionally non-redundant, this CK2β domain is likely to have a so far not recognized in vivo function. It might be used as a domain that allows integration of interacting proteins to the CK2 holoenzyme in a CK2β isoform specific manner. Thereby, the catalytic activity or substrate specificity and the localization of the Drosophila holoenzyme could be influenced. Alternatively, variations in the C-terminal tail might influence the formation of higher-order structures of the CK2 holoenzyme. In the crystals of human CK2, trimeric rings of the CK2 holoenzyme have been observed. It remains to be determined whether other higher eukaryotes also generate CK2β isoform diversity by alternative splicing (Jauch, 2006).

Phosphorylation of heterochromatin protein 1 by casein Kinase II is required for efficient heterochromatin binding in Drosophila

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein with a dose-dependent effect on heterochromatin mediated position-effect silencing. It is multiply phosphorylated in vivo. Hyperphosphorylation of HP1 is correlated with heterochromatin assembly. HP1 is phosphorylated by casein kinase II in vivo at three serine residues located at the N and C termini of the protein. Alanine substitution mutations in the casein kinase II target phosphorylation sites dramatically reduce the heterochromatin binding activity of HP1, whereas glutamate substitution mutations, which mimic the charge contributions of phosphorylated serine, apparently have wild-type binding activity. It is proposed that phosphorylation of HP1 promotes protein-protein interaction between HP1 and target binding proteins in heterochromatin (Zhao, 1999).

The methods used to identify HP1 phosphorylation sites involved direct comparison of the in vivo and in vitro tryptic peptide map by high concentration PAGE, rHP1 phosphopeptide sequencing, and radioactivity detection of each amino acid derivative. For all three sites common to phosphorylated recombinate HP1 (rHP1) and HP1 derived from whole flies (dHP1), the targets are good fits to CKII consensus motifs, which, together with the sensitivity of rHP1 phosphorylation to spermine, heparin, and anti-Drosophila CKII serum, strongly suggests that HP1 is a substrate for CKII. CKII is a ubiquitous cyclic nucleotide-independent protein kinase that appears not to directly mediate known signaling pathways. CKII activity has been found to increase in response to some mitogens, and its substrates include a number of transcription factors involved in growth control. Because CKII is found both in the nucleus and the cytoplasm, and because alanine substitution has no effect on nuclear targeting, HP1 phosphorylation by CKII could occur in either compartment (Zhao, 1999 and references).

CKII consensus target sites are found at the N and/or C terminus of HP1 homologs from Drosophila virilis, Schizosaccaromyces pombe, mealybug, mouse, and human. Not all HP1 homologs have CKII targets at both ends (some have neither), but in several such cases the homologous position is occupied by glutamate. Little or nothing is known about the functional homology between Drosophila melanogaster HP1 and its structural homologs in other species, but such apparent structural conservation suggests functional conservation. Nevertheless, the data presented here showing that CKII phosphorylation is required for efficient heterochromatin targeting by the unique D. melanogaster HP1 suggest that such structural conservation is likely to be functionally significant. CKII phosphorylation could contribute to HP1 heterochromatin binding by promoting a conformational shift that permits (1) additional kinases to phosphorylate internal targets in, for example, the HP1 linker region between the chromo domains, or (2) the exposure of sites for protein-protein interactions. Either of these results could facilitate heterochromatin assembly. This interval is serine/threonine-rich and includes two consensus targets for protein kinase A and one for protein kinase C (Zhao, 1999 and references).

Phosphorylation on both the N- and the C-terminal CKII sites is required for heterochromatin binding. The most basic HP1 isoforms in vivo are phosphorylated at CKII sites. Thus, CKII phosphorylation does not directly account for the hyperphosphorylation that accompanies the appearance of heterochromatin in the early embryonic development. Indeed, it probably accounts for the maternally loaded HP1 isoforms seen in unfertilized oocytes. Nevertheless, the mutational analysis shows that CKII phosphorylation is essential for heterochromatin binding. CKII is an ubiquitous eukaryotic serine/threonine protein kinase that phosphorylates more than 100 substrates, many of which control cell division or signal transduction. These substrates include a striking number of nuclear proteins involved in DNA replication and transcription. CKII modifies protein-DNA binding and protein-protein interaction. In Drosophila, CKII is present in both the cytoplasmic and nuclear compartments. CKII phosphorylation enhances the DNA binding activity of the Engrailed protein and modulates Antennapedia activity and dorsoventral patterning. Drosophila DNA topoisomerase II is stimulated by CKII phosphorylation. Significant HP1 phosphorylation still occurs in vivo in tissues treated with sufficient cycloheximide to block all detectable nascent protein synthesis. This turnover of phosphate uncoupled from a new synthesis suggests that HP1 phosphorylation could regulate its chromatin association, an example being the dynamic dissociation and reassociation of HP1 that reportedly takes place during mitosis. Alternatively, phosphorylation-dephosphorylation may be regulated during decondensation of heterochromatin to permit DNA replication in late S phase (Zhao, 1999).

Casein kinase II interacts with and phosphorylates the basic helix-loop-helix proteins m5, m7, and m8

Drosophila Casein kinase II (DmCKII) is composed of catalytic (alpha) and regulatory (beta) subunits associated as an alpha2beta2 heterotetramer. Using the two-hybrid system, a D. melanogaster embryo cDNA library has been screened for proteins that interact with DmCKIIalpha. One of the cDNAs isolated in this screen encodes E(spl) region transcript m7 (m7), a basic helix-loop-helix (bHLH)-type transcription factor encoded by the Enhancer of split complex [E(spl)C], which regulates neurogenesis. m7 interacts with DmCKIIalpha but not with DmCKIIbeta, suggesting that this interaction is specific for the catalytic subunit of DmCKII. In addition to m7, DmCKIIalpha also interacts with two other E(spl)C-derived bHLH proteins, m5 and m8, but not with other members, such as m3 and mC. Consistent with the specificity observed for the interaction of DmCKIIalpha with these bHLH proteins, sequence alignment suggests that only m5, m7, and m8 contain a consensus site for phosphorylation by CKII within a subdomain unique to these three proteins. Accordingly, these three proteins are phosphorylated by DmCKIIalpha, as well as by the alpha2beta2 holoenzyme purified from Drosophila embryos. In line with the prediction of a single consensus site for CKII, replacement of Ser(159) of m8 with either Ala or Asp abolishes phosphorylation, identifying this residue as the site of phosphorylation. m8 forms a direct physical complex with purified DmCKII, corroborating the observed two-hybrid interaction between these proteins. Substitution of Ser(159) of m8 with Ala attenuates interaction with DmCKIIalpha, whereas substitution with Asp abolishes the interaction. These studies constitute the first demonstration that DmCKII interacts with and phosphorylates m5, m7, and m8 and suggest a biochemical and/or structural basis for the functional equivalency of these bHLH proteins that is observed in the context of neurogenesis (Trott, 2001).

All E(spl)C-derived proteins are structurally conserved. The sequence alignment emphasizes conservation of the HLH domain, helices III and IV (also known as Orange domain), a motif in the vicinity of the C terminus with a high PEST score, and the WRPW motif. The seven E(spl) proteins have been aligned with emphasis on residues N-terminal to the basic domain and those comprising the region from helix IV to the C terminus (C-domain) to determine whether some structural features were unique only to m5, m7, and m8. No sequences in the N terminus were found that were conserved among and/or unique to these three proteins. In contrast, analysis of the C-domain indicates that only these three proteins contain a consensus site for phosphorylation by CKII, ¹⁵⁶SDNE in m5, ¹⁶⁸SDNE in m7, and ¹⁵⁹SDCD in m8, immediately following the highly conserved sequence, (I/L)SP(V/A)SSGY, in a region that is characterized by a high PEST score. Although PEST-rich sequences act as cis-acting signals that regulate protein turnover and have been suggested to be activated via phosphorylation, the role of this motif in m5/7/8 is currently unknown. This conserved Ser in m5/7/8 conforms to the requirement that it must contain an acidic residue at the n+1 and n+3 positions to be a target for CKII. It should be noted, that although mß also contains a site for phosphorylation by CKII (¹⁹⁵SEDE), m is neither preceded by the (I/L)SP(V/A)SSGY sequence nor contained within its PEST motif. Interestingly, the cytology and spatial organization of the E(spl)C locus of Drosophila hydei exhibits an extraordinary level of conservation relative to that of D. melanogaster (Maier, 1993). Because the DNA sequence of only D. hydei m8 is currently available, this protein was compared with m5, m7, and m8 from D. melanogaster. Remarkably, D. hydei m8 also contains the CKII site following the (I/L)SP(V/A)SSGY sequence, both of which are contained within a region with a high PEST score (Trott, 2001).

One question raised by the sequence alignment was whether the presence of the consensus CKII site in m5, m7, and m8 correlates with their phosphorylation. Therefore GST-m5, GST-m7, GST-m8, and GST-mC, a noninteracting member, were subjected to phosphorylation using two isoforms of CKII, i.e. the monomeric alpha subunit purified from a yeast expression system, and the alpha₂ß₂ holoenzyme purified from embryos. The former isoform mimics the two-hybrid analysis, whereas the latter mimics the in vivo environment. The results demonstrate that m5, m7, and m8 are phosphorylated by both isoforms of CKII and corroborate their observed two-hybrid interaction with DmCKIIalpha. At a quantitative level, however, the rates of phosphorylation of the three E(spl) proteins are different for both enzyme isoforms, such that m5 > m7 = m8. What mechanism can account for the observed differences? Detailed kinetic analysis of CKII suggests that whereas DmCKIIalpha and the holoenzyme display virtually identical k_m values for the protein substrate, the K_cat can differ 5-50-fold in a substrate-dependent manner. Furthermore, studies with peptides suggest that whereas the acidic residues at n+1 and n+3 are absolutely required for phosphorylation, additional acidic residues C-terminal to the n+3 position further increase the K_cat with marginal effects on the k_m. These criteria, therefore, make it possible to predict the relative rates of phosphorylation of m5/7/8. In this regard, although m7 and m8 fit the consensus, m5 is probably the best because it contains an additional Asp at the n+4 position. The rank order for phosphorylation is, therefore, predicted to be m5 > m7 = m8. The analysis presented in this study essentially reflects this prediction. Because the gel analysis described in this study inherently reflects a semiquantitative assessment of phosphorylation, kinetic analysis will be necessary to determine whether the observed differences in phosphorylation of m5 versus m7/8 are due to differing catalytic efficiencies. That CKII interacts with and phosphorylates these proteins is consistent with the observation that this kinase has been found to exist in a complex with some of its in vivo substrates, such as Topoisomerase II, HSP90, ANTP, and Dishevelled, to name a few (Trott, 2001).

These results raise the likely prospect that DmCKII interacts with m5/7/8 when these proteins are in the nonphosphorylated state and that the complexes dissociate upon phosphorylation. It is not possible to extrapolate the two-hybrid and biochemical results to the situation in the epidermal precursors in the developing Drosophila embryo with certainty. However, given the requirements of CKII for cell cycle progression, it is likely that epidermal progenitors, which are expressing E(spl) proteins, also contain CKII. A direct test of this proposal in the developing embryo still remains a difficult task due to restricted expression of m5/7/8 and the absence of isoform-specific antibodies. At a functional level, the data indicate that interaction and/or phosphorylation of m5/7/8 is unlikely to affect their DNA binding properties (which require the basic region), their ability to heterodimerize with proneural proteins (which requires the HLH domain), or their ability to interact with Groucho (which requires the WRPW motif). What function could then be ascribed to interaction and/or phosphorylation? The structural and functional properties common to m5/7/8, and by extension those in D. hydei m8, provide the basis for a likely possibility. As mentioned above, all three proteins contain a PEST-rich motif that harbors an invariant Ser residue that is phosphorylated by CKII. In this regard, a mutation that removes sequences encompassing the PEST-rich region and the resident CKII site acts as a dominant-negative allele with regard to suppression of bristle development (Giebel, 1997). That this variant of m8 behaves as a dominant-negative, rather than a loss-of-function (as one would have predicted), suggests that the mutant protein might sequester endogenous wild-type m8, and possibly m5 and m7 as well, thus leading to enhanced neurogenesis. Thus, this region negatively regulates the activity of m8, in line with its ability to homodimerize or heterodimerize with m5 and m7 (Alifragis, 1997; Gigliani, 1996). These results and their interpretations are consistent with the proposal that this region of m5/7/8 may influence the stability of these proteins in vivo. A collective theme that emerges is that phosphorylation, in at least a restricted class of proteins, regulates protein stability via activation of PEST motifs. These studies further implicate the PEST motif in m5, m7, and m8 as a target for regulation via CKII-mediated phosphorylation. Future studies employing expression of epitope-tagged m8 and its nonphosphorylatable and/or constitutively phosphorylated variants in transgenic flies will be needed to clarify the role of this motif (Trott, 2001).

In summary, these data demonstrate that select members of the E(spl)C, i.e. m5, m7, and m8, physically interact with DmCKII and are phosphorylated by this enzyme at an invariant Ser residue that is contained within a motif unique to these three isoforms. The suggestion that these three proteins are more functionally related and that the C-terminal domain of m8 acts to negatively regulate function in vivo implicates the PEST motif and its resident CKII phosphorylation site. These data strengthen the contention for the presence of a new functional motif in these transcriptional repressors and raise the possibility that CKII may regulate neurogenesis via posttranslational modification of these proteins (Trott, 2001).

A role for casein kinase 2alpha in the Drosophila circadian clock

Circadian clocks drive rhythmic behavior in animals and are regulated by transcriptional feedback loops. For example, the Drosophila proteins Clock (Clk) and Cycle (Cyc) activate transcription of period (per) and timeless (tim). Per and Tim then associate, translocate to the nucleus, and repress the activity of Clk and Cyc. However, post-translational modifications are also critical to proper timing. Per and Tim undergo rhythmic changes in phosphorylation, and evidence supports roles for two kinases in this process: Doubletime (Dbt) phosphorylates Per, whereas Shaggy (Sgg) phosphorylates Tim. Yet Sgg and Dbt often require a phosphoserine in their target site, and analysis of Per phosphorylation in dbt mutants suggests a role for other kinases. The catalytic subunit of Drosophila casein kinase 2 (CK2alpha) is shown to be expressed predominantly in the cytoplasm of key circadian pacemaker neurons. CK2alpha mutant flies show lengthened circadian period, decreased CK2 activity, and delayed nuclear entry of Per. These effects are probably direct, since CK2alpha specifically phosphorylates Per in vitro. It is proposed that CK2 is an evolutionary link between the divergent circadian systems of animals, plants and fungi (Lin, 2002).

To identify new components of circadian clocks, about 2,000 ethylmethane-sulphonate (EMS)-mutagenized per^s (short-period allele of per) lines were screened for circadian behavioral defects. A dominant mutant, Timekeeper (Tik), was identified. Tik homozygotes do not live to adulthood. Tik heterozygotes exhibit behavioral rhythms approximately 1.5 h longer than per^s. In a per⁺ or per^l (long-period allele of per) background, Tik lengthens the period of the behavioral rhythm by 3 h, reflecting an allele-specific interaction between per and Tik. A spontaneous partial revertant, TikR, was identified and this revertant could not be separated from Tik by recombination. The change in period in Tik mutants (about 3 h) exceeds that of nearly all heterozygous circadian mutants in Drosophila, suggesting that it might identify a protein of central importance in the circadian clock mechanism (Lin, 2002).

Tik maps to a region around the chromosomal centromere. Sequencing of the CK2alpha coding region from the wild-type parental strain and the initial Tik mutant identified two sequence changes, both resulting in coding changes: Met161Lys and Glu165Asp. The change from the non-charged Met 161 to the charged Lys occurs near the catalytic loop and within a hydrophobic binding pocket for ATP. The TikR mutant that was proposed to be a new Tik revertant allele was also sequenced. In addition to the two original Tik coding changes, an additional in-frame, 27-base-pair (bp) deletion was identified coupled to an in-frame, 6-bp insertion, resulting in a deletion of 7 amino acids (234–240) and another amino acid change (Arg242Glu). These mutations occur in residues that are highly conserved with their human counterpart (Lin, 2002).

Immunohistochemistry was performed on adult whole-mount dissected brains with an antiserum directed against a CK2 peptide sequence common to both fly and mammalian CK2alpha (anti-CK2alpha). Anti-CK2alpha specifically labels the cytoplasm and axonal projections of neurons in the lateral protocerebrum. To determine whether these neurons are the well-described pacemaker lateral neurons, double labelling was performed with an antiserum against Pigment-dispersing factor (Pdf). This neuropeptide is specifically localized to small and large lateral neurons critical to behavioral rhythms. Consistent with its proposed circadian role, CK2 co-localizes with Pdf in these adult neurons. CK2alpha is also probably expressed in the eyes, as CK2 activity is modestly reduced (about 20%) in eyes absent mutants. Of note, CK2alpha and Pdf also co-localize to neuronal termini, indicating that CK2 may regulate directly Pdf processing or release. CK2alpha seems to be constitutively cytoplasmic as a function of the time of day. The tissue specificity of CK2alpha expression suggests that it has a specific function in circadian clocks (Lin, 2002).

To ascertain CK2 function on Per and Tim, their levels and phosphorylation were examined in heterozygotes. Flies were entrained in a light/dark cycle and were transferred to constant darkness (DD). During the early subjective day (circadian time, CT, 0–12) levels of Per in the CK2alpha^Tik mutants are increased and show delayed disappearance. Furthermore, there seems to be a modest increase in less-phosphorylated forms of Per. During the subjective night, Per phosphorylation and to a lesser extent accumulation are delayed relative to wild type. An increase in the level of Per and Tim in CK2alpha^Tik mutants is delayed by approximately 3 h relative to wild type, consistent with the period-lengthening effect of CK2alpha^Tik mutants. Disappearance of Tim is also more strongly affected than accumulation in CK2alpha^Tik mutants. The Per and Tim profiles of CK2alpha^TikR heterozygotes are largely unchanged, consistent with its heterozygous behavioral and biochemical phenotypes (Lin, 2002).

To examine the phenotype of homozygous CK2alpha mutants, immunofluorescence for Per was performed in third-instar larval brains. Per staining in wild-type larvae is predominantly nuclear by Zeitgeber time (ZT) 21 (three hours before 'lights on'). In CK2alpha^Tik and CK2alpha^TikR homozygotes, however, Per nuclear entry is significantly delayed, with Per predominantly cytoplasmic at ZT21. By ZT1, the Per staining pattern in the CK2alpha mutants remains distinct from the wild-type nuclear pattern, appearing to be present in both cytoplasmic and nuclear compartments. These data demonstrate a strong effect of CK2alpha on Per nuclear entry and are consistent with the cytoplasmic expression of CK2alpha. Furthermore, no significant differences between CK2alpha^Tik and CK2alpha^TikR were observed, consistent with biochemical data on recombinant proteins and their recessive lethality. These data support the model that CK2alpha^TikR is a strong loss-of-function allele, lacking the strong dominant behavioral and biochemical effects of CK2alpha^Tik (Lin, 2002).

Bacterially expressed and purified CK2alpha can phosphorylate Per in vitro. Notably, this effect is specific, since no significant phosphorylation of the circadian transcription factor Cyc is observed. Tim (amino acids 1–1159) is phosphorylated by CK2alpha to a lesser extent than Per. These data are consistent with the direct regulation of Per and Tim by CK2 (Lin, 2002).

Taken together, this analysis indicates a dedicated and direct role of CK2 in the Drosophila circadian clock mechanism. CK2alpha^Tik has one of the strongest phenotypes of any heterozygous circadian rhythm mutant. The modest nature of the biochemical defect further supports the hypothesis that the circadian clock is highly sensitive to CK2 activity. The CK2alpha expression pattern indicates that it has a specific role in circadian rhythms. Its localization to neuronal termini raises the possibility that CK2 links the clock to circadian outputs (Lin, 2002).

It is suggested that CK2 directly phosphorylates Per and Tim in vivo, promoting their transition to the nucleus. The evidence for this pattern is compelling for Per. Allele-specific interactions between per and CK2alpha alleles support the model of a direct interaction. Given the cytoplasmic expression of CK2alpha, this phosphorylation may serve as a signal for nuclear entry and subsequent degradation, explaining the delayed nuclear entry and disappearance of these proteins in CK2alpha^Tik mutants (Lin, 2002).

This study also establishes an evolutionary connection between animal, plant and fungal circadian systems -- genetic studies have revealed clock components in plants (Arabidopsis) and fungi (Neurospora). These studies suggest that clocks have arisen several times in evolution; however, studies in both Arabidopsis and Neurospora have linked CK2 to circadian timekeeping. CK2 is therefore a gene involved in circadian rhythms that is shared between all three phylogenetic kingdoms. Indeed, the fly enzyme (amino acids 7–322) shares 77% and 72% identity with the Arabidopsis and Neurospora enzymes, respectively. It is proposed that the conserved role for CK2 is driven by the need to avoid mutagenic ultraviolet light. CK2 has a pivotal role in the response to ultraviolet radiation from yeast to humans. Consistent with this model, cryptochromes, components of plant and animal circadian systems, are homologous to ultraviolet-dependent DNA repair enzymes (Lin, 2002).

In vivo circadian function of casein kinase 2 phosphorylation sites in Drosophila PERIOD

Phosphorylation plays a key role in the precise timing of circadian clocks. Daily rhythms of phosphorylation of the Drosophila circadian clock component Period (Per) were first described more than a decade ago, yet little is known about their phosphorylation sites and their function in circadian behavior. Serines 151 and 153 in Per are shown to be required for robust in vitro phosphorylation by the casein kinase 2 (CK2) holoenzyme, a cytoplasmic kinase shown to be involved in circadian rhythms. Mutation of these sites in transgenic flies results in significant period lengthening of behavioral rhythms, altered Per rhythms, and delayed Per nuclear localization in circadian pacemaker neurons. In many respects, mutation of these phosphorylation sites phenocopies mutation of the catalytic subunit of CK2. It is proposed that CK2 phosphorylation at these sites triggers Per nuclear localization (Lin, 2005).

Evidence is provided of a function for Per CK2 phosphorylation sites in circadian clock function and behavior in Drosophila. These phosphorylation sites were initially identified in vitro, and site-directed mutagenesis studies focused attention on three key serines in the N terminus required for robust in vitro phosphorylation of Per by CK2 alpha and the CK2 holoenzyme. This mutant form is still phosphorylated by another kinase, CK1epsilon, and is still able to directly interact with CK2, suggesting that these serines represent in vitro phosphorylation targets for CK2. Mutation of these sites in vivo results in a long circadian period, altered Per rhythms, and delayed Per nuclear entry. Overall, these phenotypes significantly overlap with those observed previously in CK2 mutants (Lin, 2005).

These data contribute significantly to the role of phosphorylation in the regulation of core clock components and in turn the effects on circadian behavior. Despite considerable progress in defining clock components and clock kinases, remarkably little is known about the target phosphorylation sites essential for their function, especially in metazoans. Recent work has defined key serines that are important for CK1epsilon regulation of mouse Per nuclear localization and phosphorylation in cultured cell lines. Although critical serines have been defined, these studies were not performed in the context of a functioning circadian clock. Thus, it is not known whether these sites are essential for clock function. Also, because the studies were performed in cultured cells, it is not clear whether these putative phosphorylation sites are important for circadian behavior. In contrast, this study shows functioning of critical serine targets of CK2 in an in vivo functioning circadian system and on circadian behavior (Lin, 2005).

Although these data argue for a role for CK2 in the phosphorylation of Ser151-153, they cannot exclude the effects of CK2 elsewhere on the Per protein or on other proteins in the circadian clock. Several potential CK2 sites have been identified in Per and in other clock proteins using Phosphobase, raising the possibility that CK2 may work through multiple sites on Per. The in vitro conditions (e.g., absence of Tim) may not fully replicate the in vivo situation and thus miss these other important sites. CK2 action also may not occur exclusively through Per. In vitro phosphorylation of Tim by CK2alpha as well as effects on in vivo Tim rhythms have been reported. Thus, CK2 may affect multiple targets to regulate clock function (Lin, 2005).

Consistent with this hypothesis, period lengthening of perS149-151-153A mutants was observed in a Tik mutant background. in which catalytic activity of CK2alpha is reduced by 50%. Under natural conditions, it is proposed that some fraction of these serines is phosphorylated. In the Tik mutant, it is expected that the activity is reduced but not absent both at this cluster and at other clock-relevant CK2 sites. In the S149-151-153A mutants, these residues cannot be phosphorylated, a more severe situation than that seen in wild type or even Tik. In wild type, normal CK2 phosphorylation elsewhere in Per or in other clock components partially compensates for the loss of these phosphorylation sites. Nonetheless, when CK2 is impaired, as in Tik mutants, an additional period lengthening is still observed, caused by the combination of a complete loss of phosphorylation of these key sites by alanine mutation and reduction of clock-relevant CK2 phosphorylation elsewhere (Lin, 2005).

Moreover, these data do not exclude a function for other kinases in the phosphorylation of Ser149-151-153. Serine 149 also represents a potential GSK3/SGG site. Although in vitro data fail to show GSK3 phosphorylation at this site, it is possible that it is a true in vivo target. Given the similarity of perS149-151-153A and CK2 mutant phenotypes, it is proposed that Ser149-151-153 represents one cluster of these in vivo phosphorylation sites that is phosphorylated, at least, by CK2 (Lin, 2005).

Based on these findings, it is proposed that the distinct CK2 sites present in D. melanogaster Per but not in other closely related species, such as D. pseudoobscura, may represent the basis for species-specific aspects of circadian function. Such variation has been proposed to underlie the process of allochronic speciation. Temporal isolation through the use of species-specific circadian programs may serve as a barrier to gene flow and thus facilitate speciation. Indeed, species-specific differences in locomotor activity and mating behavior have been observed between D. melanogaster and other Drosophila species. By using cross-species rescue of per⁰¹ behavior, many of these changes have been attributed to variation in the per gene, i.e., the species of per determines the nature of diurnal, circadian, and/or mating behavior (Lin, 2005).

It is proposed that variation in phosphorylation sites may underlie these species differences in behavior. A comparison of different Drosophila Pers reveals that Ser149-151-153 is part of one of the nonconserved modules of Per. This small region within the larger module is well conserved only within the melanogaster subgroup. Of note, this cluster is conserved in D. ananassae, in which the remainder of the module is not as well conserved, consistent with an underlying function. The single change of Ser153 to glutamate, a phosphomimetic residue, is also consistent with a function in phosphorylation (Lin, 2005).

Species specificity of clock function has also been noted at the molecular level in terms of Per nuclear localization. Of note, Per is predominantly cytoplasmic in the putative pacemaker neurons of the silkmoth (Antherea pernyi), beetle, and hawkmoth. It is interesting that the CK2 sites described in this study are also not conserved with Antherea Per, and mutation of these serines alters Per nuclear entry, a process apparently present in melanogaster but not the silkmoth. It is proposed that nuclear entry triggered by CK2 phosphorylation of Per may be a species-specific aspect of the clock program (Lin, 2005).

Drosophila CK2 phosphorylates Deadpan, raising the possibility that Dpn repressor functions might be regulated by phosphorylation

In Drosophila, protein kinase CK2 regulates a diverse array of developmental processes. One of these is cell-fate specification (neurogenesis) wherein CK2 regulates basic-helix-loop-helix (bHLH) repressors encoded by the Enhancer of Split Complex (E(spl)C). Specifically, CK2 phosphorylates and activates repressor functions of E(spl)M8 during eye development. This study describes the interaction of CK2 with an E(spl)-related bHLH repressor, Deadpan (Dpn). Unlike E(spl)-repressors which are expressed in cells destined for a non-neural cell fate, Dpn is expressed in the neuronal cells and is thought to control the activity of proneural genes. Dpn also regulates sex-determination by repressing sxl, the primary gene involved in sex differentiation. Dpn is weakly phosphorylated by monomeric CKalpha, whereas it is robustly phosphorylated by the embryo holoenzyme, suggesting a positive role for CK2beta. The weak phosphorylation by CK2_ is markedly stimulated by the activator polylysine to levels comparable to those with the holoenzyme. In addition, pull down assays indicate a direct interaction between Dpn and CK2. This is the first demonstration that Dpn is a partner and target of CK2, and raises the possibility that its repressor functions might also be regulated by phosphorylation (Karandikar, 2005).

A subset of E(spl)-repressors, i.e., M5, M7, and M8, robustly interact with CK2alpha (Trott, 2001). In addition, these three proteins are equivalently phosphorylated by monomeric CK2alpha or the holoenzyme at a conserved CK2 site that is located in close proximity to the C-terminal Groucho binding WRPW motif. Furthermore, deletion of the CK2 site (SDCD) or replacement of the CK2 phosphoacceptor in M8 with Asp abolishes interaction, suggesting that the CK2-site might, by itself, confer interaction. Given the overall structural conservation of the HES family, i.e., E(spl), Dpn, and Hairy, the sequence of Dpn was analyzed to determine the presence of CK2 sites and their positional conservation, if any. This analysis revealed the presence of two potential sites, i.e., S9DDD and S408DCS411LDE. While the N-terminal site satisfies the requirement for an Asp/Glu at the n+1 and n+3 positions, the C-terminal site is lacking Asp/Glu at the n + 1. However, it is noted that a number of substrates where the n + 1 position is not an Asp/Glu have been identified. In Dpn, the first site is adjacent to the basic domain and harbors a single potential phosphoacceptor (Ser9). In contrast, the second site is located in the vicinity of the Groucho binding WRPW motif and contains two potential phosphoacceptors (Ser408 and Ser411) that might be subject to hierarchical phosphorylation by CK2. Interestingly, the second site localizes to a region of Dpn which, although hypervariable amongst HES members, is positionally conserved in a number of repressors (M5/7/8, Hes6, etc.). In the case of M8 and its murine homolog Hes6, this site is targeted by CK2 in vitro, and its perturbation dramatically affects Hes6 and M8 repressor activity in vivo (Karandikar, 2005).

Given the strong two hybrid interaction of E(spl)M5/7/8 with CK2, and the fact that interaction required integrity of the CK2 site, it was asked whether Dpn is also a partner of CK2. However, in an explicit test it was observed that strength of the (two hybrid) interaction between LexA-Dpn and AD-CK2alpha appears marginal when compared to that between LexA-M8 and AD-CK2alpha. This result is surprising because Dpn contains two CK2 sites, both of which are significantly more acidic than the single site in M5/7/8. It was reasoned that the significantly attenuated Dpn-CK2alpha interaction might reflect attenuated expression and/or instability of Dpn in yeast, or, perhaps, its ability to act as a repressor in yeast. An alternative possibility is that this interaction also requires CK2beta. If so, a direct biochemical route might be more informative to assess targeting of Dpn by CK2 (Karandikar, 2005).

To test if Dpn is a CK2 target an in vitro phosphorylation assay was performed. GST and GST-Deadpan were subjected to phosphorylation using purified monomeric CK2alpha or CK2 holoenzyme. The former isoform is relevant to the two hybrid analysis, whereas the latter isoform mimics the environment most likely to be encountered in vivo and thus might be considered to be physiologically more relevant. The results indicate that GST-Dpn is phosphorylated weakly by monomeric CK2alpha, whereas it was robustly phosphorylated by the embryo-holoenzyme. No phosphorylation of the GST affinity tag was observed for either isoform of CK2. These results suggest that phosphorylation of Dpn by CK2 is positively influenced by the beta subunit, and might explain its 'weak' interaction with CK2alpha in yeast. It is not considered likely that Dpn interacts exclusively via CK2beta, because CK2alpha exhibits phosphorylation of this bHLH protein, albeit weakly. The more likely scenario is that the Dpn interacts with CK2 via a binding site encompassing both subunits, i.e., the holoenzyme. Because this is the in vivo conformation of CK2 strengthens the notion that Dpn is a CK2 target. Comparative kinetic analysis with the two isoforms will be needed to address how CK2beta enhances interaction and phosphorylation of Dpn (Karandikar, 2005).

Although the phosphorylation analysis suggests that Dpn interacts preferentially with the holoenzyme, two hybrid analysis with this isoform per se has been precluded because yeast strains that express equivalent amounts of CK2alpha and CK2beta are currently unavailable. Therefore the ability of Dpn to form a direct complex with embryo-CK2 or CK2alpha was assessed. GST-alone and GST-Dpn were purified, immobilized on glutathione-sepharose, and tested for complex formation with the two isoforms of CK2. The presence of CK2 in the bound (pellet) and unbound (supernatant) fractions was assessed by Western blotting using an antisera that recognizes both (alpha and beta) subunits of CK2. Incubation of GST-Dpn beads withCK2alpha resulted in a minor amount of immunoreactive material in the pellet. In contrast, incubation of GST-Dpn beads with embryo-CK2 resulted in significantly greater amounts of immunoreactive material in the pellet, demonstrating that Dpn and CK2-holoenzyme interact directly. These binding data appear to qualitatively mirror the phosphorylation data, and it is estimated that ~20% of the holoenzyme interacted with Dpn. Given the experimental conditions of these assays, CK2-holoenzyme contributed half the amount of catalytic subunit compared to CK2alpha alone, suggesting that complex formation appears to be relatively efficient for the holoenzyme. These results demonstrate that the Dpn-CK2 interaction is direct. In addition, complex formation occurs in the absence of MgATP, in line with previous analysis of the interaction of this enzyme with M5/7/8, ZFP35, etc (Karandikar, 2005).

The observations of a direct CK2-Dpn complex and its preferential phosphorylation by the holoenzyme, suggest a positive role for CK2beta. The marginal ability of CK2alpha to phosphorylate Dpn, and the fact that CK2beta mediates activation by polybasic effectors, led to an assessment of whether phosphorylation was responsive to polybasic activation. The marginal phosphorylation of Dpn by CK2alpha was unaffected by either spermine or protamine, but was dramatically stimulated by poly(DL)lysine. The stimulatory effects of poly(DL)lysine are not due to non-specific phosphorylation, because GST is not phosphorylated in its presence. In contrast, phosphorylation of Dpn by embryo-CK2 was unresponsive to further activation by these effectors. These results suggest that phosphorylation of Dpn by embryo-CK2 is unresponsive to further activation, and support the notion that substrates that are efficiently phosphorylated, e.g., the RII subunit of PKA, topoisomerase II, etc., are generally refractory to these activators (Karandikar, 2005).

While the mechanism by which Dpn functions during neurogenesis remains to be resolved, its role(s) during sex determination is much better understood. In either case, however, one common feature of its functions is antagonism of ASC, whereby Dpn represses transcription of ASC via DNA-binding. In line with this, ectopic expression of dpn reduces ASC activity, suggesting a negative interaction between these two loci. It is noteworthy that a similar function is ascribed to HES repressors as well, although in their case DNA-binding as well as direct interactions with proneural factors (ASC and Atonal) are known to be required for antagonism (Karandikar, 2005).

How might phosphorylation of Dpn regulate its in vivo functions? It is difficult to propose this with certainty based solely on in vitro analysis. However, based on the extensive body of genetic and molecular analysis on Dpn to date, and the emerging notion that CK2 profoundly influences the activity of the related repressor, E(spl)M8, during eye development (Karandikar, 2004), some possibilities can be predicted. As stated above, CK2 phosphorylation regulates repressor activity of M8 and replacement of the phosphoacceptor with Asp generates a dominant allele that is severely exacerbated for its antineurogenic functions. A similar CK2 dependent mechanism might also underlie the interaction of M8 with the ASC-bHLH activator, Lethal of Scute. In a similar vein, it is conceivable that phosphorylation of Dpn might augment its ability to antagonize ASC-derived bHLH activators by either modulating DNA binding or direct protein-protein interactions. CK2 is known to regulate DNA-binding as well as protein-protein interactions (Karandikar, 2005).

The development of nervous system is regulated by the interplay between proneural proteins and their repressors. A general strategy during neurogenesis appears to be the conferring of neural potential on a field of cells, from which arises a precise pattern of neural and accessory cell fates through this interplay and, as such, this mechanism also appears to be involved in other cell fate decisions. It is increasingly becoming apparent that cell fate choice is unlikely to be based simply on the levels of an activator versus its cognate repressor. Rather, this interplay must also be modulated in a spatial and temporal context. In such a scenario, regulation of protein turnover, presence or absence of cofactors, and regulatory modifications, are among those factors that might provide a means to achieve 'fine tuning' of this interplay. In this context, protein kinases and/or phosphatases might provide a simple bistable mechanism to 'fine tune' the developmental outcome. Such a mechanism is beginning to emerge for regulation of repression by E(spl)M8 and its mammalian counterpart, Hes6. In both, phosphorylation by CK2 regulates their ability to interact with and antagonize proneural factors. Given the expanding repertoire of HES proteins that are targeted by CK2, it would not come as a surprise that a similar mechanism might also be employed for regulation of another HES member, Dpn (Karandikar, 2005).

Among the HESmembers that are CK2targets, Dpn differs from E(spl) in a number of ways. While E(spl) transcription [by Su(H)] occurs in response to an activated Notch receptor, Dpn has been thought to be Notch-independent, although it contains binding sites for Su(H) in a region that recapitulates PNS/CNS specific expression. Furthermore, E(spl) repressors block proneural proteins in cells undergoing lateral inhibition, whereas Dpn achieves a similar outcome but in neural cells. The remarkable conservation of CK2 by itself, and its ability to modulate the activity of repressors in different developmental contexts might be indicative of its selection as a general modulator of cell fate determination (Karandikar, 2005).

Regulation of the Drosophila melanogaster protein, enhancer of rudimentary, by casein kinase II

The Drosophila melanogaster gene enhancer of rudimentary, e(r), encodes a conserved protein, ER whose Xenopus homolog has been identified as a cell type-specific transcriptional repressor, probably interfering with a positive cofactor of HNF1-dependent gene regulation (Pogge von Strandmann, 2001). Most ER homologs share two casein kinase II (CKII) target sites. In Drosophila, these sites are T18 and S24. A third CKII site, T63, has been seen only in drosophilids. The conservation of these CKII sites, particularly T18 and S24, suggests a role for these residues in the function of the protein. To test this hypothesis, these positions were mutated either to alanine as a nonphosphorylated mimic or to glutamic acid as a phosphorylated mimic. The mutations were tested individually or in double or triple combinations for their ability to rescue either a wing truncation characteristic of the genotype e(r)^p1 r^hd1-12 or the synthetic lethal interaction between e(r)^p2 and the Notch allele N^nd-p. All of the substitutions as single mutations rescued both mutant phenotypes, arguing that individually the phosphorylation of the three residues does not affect ER activity. The double mutants T18A-S24A and T18E-S24E and the triple mutants T18A-S24A-T63A and T18E-S24E-T63E failed to rescue. Together the data support the following model for the regulation of ER by CKII. ER that is unphosphorylated at both T18A and S24 is inactive. CKII activates ER by phosphorylating either T18 or S24. Further phosphorylation to produce the doubly phosphorylated protein inactivates ER (Gelsthorpe, 2006).

Timeless is an important mediator of CK2 effects on circadian clock function in vivo

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of Period and Timeless proteins govern period length. Yet little is known about the kinases that phosphorylate Tim in vivo. It has been shown previously that the protein kinase CK2 phosphorylates Tim in vitro. This study identified a role for CK2 in Tim regulation in vivo. Induction of a dominant-negative CK2α, CK2α^Tik (Tik), increases Tim protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic Tim localization. Exposure to light and subsequent Tim degradation results in an increase in the fraction of the transcriptional repressor Per that is nuclear and suppression of per and tim RNA levels. Tim protein, but not tim transcript, levels are elevated in Tik mutants in a per⁰¹ background. In contrast, Tik effects on Per are undetectable in a tim⁰¹ background, suggesting that Tim is required for CK2 effects on Per. To identify potential CK2 target sites, Tim phosphorylation rhythms were assayed in a deletion mutant that removes a conserved serine-rich domain. It was found that Tim protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a tim^UL mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that Tim is an important mediator of CK2 effects on circadian rhythms (Meissner, 2008).

Evidence is presented that CK2 operates through Tim to control circadian clock function in vivo. Expression of the dominant-negative CK2α allele, Tik, elevates trough levels of Tim protein and RNA during constant darkness and alters Tim subcellular localization. Tik effects on Per are undetectable in a tim⁰¹ mutant, whereas Tik effects on Tim are evident in a per⁰¹ background, suggesting direct Tim effects. Behavioral period effects of Tik are reduced in the tim^UL mutant that disrupts a putative CK2 site. The effects on Tim metabolism as well as the genetic requirement of tim for CK2 effects indicate CK2 primarily operates through Tim to regulate the circadian clock (Meissner, 2008).

One potential model consistent with these data is that CK2 regulates Tim abundance and in turn, promotes negative feedback. Elevated trough Tim levels are accompanied by elevated trough tim transcript levels in DD, suggesting impaired turnover, negative feedback, and/or tim transcriptional regulation. Effects on Tim are evident in per⁰¹ flies but are not accompanied by changes in tim transcript levels, suggesting a direct effect on Tim protein, perhaps by regulating Tim stability, although a translational effect cannot be ruled out. Importantly, the per⁰¹ data argue strongly against a direct effect of CK2 on CLK/CYC-driven transcription of tim. The ability of light to degrade Tim in Tik-expressing flies implies that Tim levels can be regulated by two distinct pathways, a light/CRY-dependent pathway and a CK2-dependent pathway. During light/dark entrainment, light robustly degrades Tim in Tik-expressing flies. This degradation is accompanied by a sharp suppression in per and tim transcript levels, suggesting that it is excessive Tim levels, rather than direct Per effects, that abrogate negative feedback in Tik-expressing flies. per and tim RNA oscillations remain phase delayed during LD in homozygous Tik-expressing flies, suggesting that Tik also affects the timing of Per repression (Meissner, 2008).

Some CK2 effects on Per may be mediated by CK2 effects on Tim. CK2 effects on Per levels require Tim. CK2 is localized to the cytoplasm where Per/Tim dimers are likely present. CK2 regulates Per and Tim nuclear entry, a process that likely depends on the Per/Tim dimer. Although effects on Per require Tim, Per is likely a direct in vivo CK2 substrate. CK2 robustly and specifically phosphorylates Per in vitro and in-vitro-defined sites have clear in vivo functions. Tik mutants can also strongly effect Per mobility on Western blots; Per mobility is highly dependent on phosphorylation. These data are most consistent with the idea that CK2 targets the Per/Tim dimer in the cytoplasm and phosphorylates Per to promote nuclear entry. Additional experiments will be required to test the dimer hypothesis. Based on S2 cell experiments, it has been proposed that CK2 phosphorylation of Per promotes its intrinsic repressor activity independent of its effects on nuclear localization. The observation that light-induced Tim degradation results in a suppression of per and tim RNA in Tik-expressing flies suggests that elevated Tim levels block Per repression. Nonetheless, remaining alterations in transcript levels suggest that the freed Per repressor may not be entirely functional, consistent with additional CK2 effects on Per (Meissner, 2008).

Tim may be an in vivo CK2 substrate. CK2 has been shown to phosphorylate Tim in vitro. In vivo, Tik increases Tim levels in the absence of Per, suggesting CK2 effects on Tim may be direct. Importantly, increases in Tim protein are not accompanied by increases in transcript, indicating CK2 regulates Tim posttranscriptionally. The notion is favored that CK2 acts to phosphorylate Tim in vivo, consistent with published in vitro phosphorylation experiments (Meissner, 2008).

One potential argument against the hypothesis that CK2 phosphorylates Tim is the finding that low-mobility Tim accumulates to high levels in timGAL4-62; UAS-Tik homozygotes compared with wild-type controls. Interestingly, this result is similar to the observation that in Dbt^g mutants, low mobility forms of Per accumulate that are even lower in mobility than those in wild-type, although Per is widely established as a DBT substrate. In both cases, phosphorylation-induced mobility changes cannot be attributed solely to a single kinase. It is also possible that elevated Tim levels may render low mobility forms more visible in Tik-expressing flies. Thus, the presence of low-mobility forms does not exclude CK2 as an in vivo kinase for Tim (Meissner, 2008).

Although no phosphorylation sites have been identified in Tim, deletion of a small Tim serine-rich domain (Tim 260-292) reduces or eliminates significant circadian mobility changes. This domain is conserved among insects and may contain multiple phosphorylation sites that are responsible for regulating period length and rhythms in Tim levels and mobility. CK2 may be one of several kinases that phosphorylates Tim serine-rich domain. In addition, although this domain is critical for shifts in Tim mobility, there are likely phosphorylation sites for CK2 outside of this region. When the bacterial expression construct used previously to generate Tim protein for CK2 in vitro phosphorylation assay was sequenced, it was discovered that the construct lacks amino acids 260-292. The possibility cannot be excluded that loss of rhythmic mobility changes may be secondary to reduced Tim levels in serine-rich domain mutants or may not be linked to CK2. Nonetheless, the in vivo data and sequence conservation raise the possibility that the serine-rich domain may be a kinase substrate (Meissner, 2008).

The Tim serine-rich domain contains four predicted CK2 sites. One of these sites, Ser²⁷⁹, is potentially altered in tim^UL mutants by the Glu²⁸³Lys point mutation. CK2 preferentially phosphorylates serine or threonine residues located 2-5 residues N-terminal to acidic amino acid residues such as glutamate or aspartate. The placement of basic residues, such as lysine as in the tim^UL mutant, near CK2 sites inhibits CK2 phosphorylation of the target Ser/Thr residue. tim^UL and Tik show allele-specific genetic interactions such that in tim^UL mutants, Tik period lengthening is partially suppressed. These results are consistent with the hypothesis that tim^UL is a CK2 site mutant. This prediction suggests that Ser²⁷⁹ plays a very important role in regulating circadian period length in flies. It will be of interest to test the hypothesis that CK2 promotes Tim degradation (Meissner, 2008).

It has been claimed that tim^UL shows a late-night specific defect principally based on the phase response curve to light and persistent nuclear Tim levels. CK2, however, is mostly restricted to the cytoplasm and thus would be predicted to act on cytoplasmic Per and/or Tim proteins during the early night. Although it is true that tim^UL shows profound late-night defects, on closer examination, there are alterations in the phase response curve to light during the early night. In addition, Tim protein was not examined in pacemaker neurons during the early night in tim^UL, so the possibility that there is also an early-night defect in tim^UL cannot be ruled out. Therefore it is possible that tim^UL also displays early-night defects, as is proposed for CK2 effects. Second, it is possible that phosphorylation of Tim by CK2 in the cytoplasm could influence the function of phospho-Tim in the nucleus. Third, although CK2 is principally observed in the cytoplasm, the possibility that small but functional levels of CK2 are present in the nucleus cannot be ruled out. Regardless, the allele-specific, nonadditive genetic interaction between Tik and tim^UL argue that tim^UL is important for CK2 effects on circadian behavior (Meissner, 2008).

The data are consistent with the speculation that Tik expression reduces CK2 phosphorylation of Tim. This results in retention of Per in the cytoplasm, thus reducing Per-mediated repression. Light-mediated degradation of Tim can then liberate Per (pending some additional events) to enter the nucleus and repress CLK/CYC. Although additional experiments will be needed to test this model, the data demonstrate that it is likely that CK2 effects through Tim will play an important role in circadian clock function (Meissner, 2008).

Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo

Hairy is a repressor that regulates bristle patterning, and its loss elicits ectopic bristles (neural hyperplasia). However, it has remained unknown whether Hairy is regulated by phosphorylation. This study describes the interaction of protein kinase CK2 and Hairy. Hairy is robustly phosphorylated by the CK2-holoenzyme (CK2-HoloE) purified from Drosophila embryos, but weakly by the catalytic CK2alpha-subunit alone, suggesting that this interaction requires the regulatory CK2beta-subunit. Consistent with this, Hairy preferentially forms a direct complex with CK2-HoloE. Importantly, genetic interactions were demonstrated between CK2 and hairy (h). Thus, flies trans-heterozygous for alleles of CK2alpha and h display neural hyperplasia akin to homozygous hypomorphic h alleles. In addition, similar phenotypes are elicited in wild-type flies upon expression of RNAi constructs against CK2alpha/beta, and these defects are sensitive to h gene dosage. Together, these studies suggest that CK2 contributes to repression by Hairy (Kahali, 2008. Full text of article).

The functioning of the Drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orb^F303, which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach this study identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, it was found that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, it was shown that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase (Wong, 2011).

While only two Orb phosphoisoforms are resolved on SDS-PAGE gels, a combination of phosphatase treatment, analysis of phosphoisoforms in different mutants, and fractionation on Phos-Tag gels indicate that Orb must be phosphorylated at multiple sites on Tyr, Ser and/or Thr residues. At least seven distinct isoforms are resolved on Phos-Tag gels, four faster migrating species and three slower migrating species. Since the degree of retardation in this gel system depends largely upon the number of phosphate residues, the set of more rapidly migrating 'hypophosphorylated' isoforms are expected to have between zero and three phosphate residue, while the set of more slowly 'hyperphosphorylated' isoforms are expected to have four or more phosphate residues. Mobility in the Phos-Tag gel is also influenced by the location of the phosphorylated amino acid, and several bands appear to be doublets. Thus, there may be isoforms that have the same number of phosphorylated residues, but differ in which amino acids are modified. Further studies will be required to determine the number and location of the phosphorylated residues associated with each of the different isoforms (Wong, 2011).

Several lines of evidence argue that phosphorylation is critical for orb activity. One comes from the dramatic effects of the orb^F303 mutation: the 'hyperphosphorylated' isoforms are absent and the two more slowly migrating 'hypophosphorylated' isoforms are largely missing as well. While this would link phosphorylation to Orb function, it is not immediately clear why the Tyr742 mutation has such drastic effects. The simplest model is that Tyr742 must be phosphorylated to generate the other phosphoisoforms. However, Tyr742 is predicted to be on the buried side of the second RRM α-helix and should have essentially no solvent accessibility. Thus, unless it is modified during translation, a scenario in which phosphorylation of this Tyr is obligatory for subsequent phosphorylation elsewhere seems unlikely. A more likely possibility is that the second Orb^F303 RRM domain does not fold properly and this weakens or eliminates RNA binding. In the absence of RNA-binding, it is possible that oogenesis might arrest at a point prior to when most phosphoisoforms are generated. Arguing against this is the fact that the fast and slow phosphoisoforms are found in mutants in other genes that cause even earlier oogenesis arrest. Another possibility is that phosphorylation of the sites in the Orb protein that generate the collection of more slowly migrating isoforms requires prior binding to target RNAs. This idea is suggested by the structural changes that are induced when proteins that have two RRM domains interact with RNA. For example, when Sxl binds to its target RNAs, the two RRM domains rearrange so that they clamp around the RNA, while the linker region separating the two domains is converted from an unordered structure into a distorted but spatially fixed helix. In addition to stabilizing RNA:protein interactions, this rearrangement alters the ability of Sxl to physically interact with other splicing co-factors. If the Orb RRM domains and linker region also undergo similar conformational changes upon RNA binding, this could provide a mechanism for coupling binding to phosphorylation. Since the linker region separating the two RRM domains would be an obvious target for binding dependent conformational changes that could potentially modulate phosphorylation, it is intriguing that a tryptic peptide (which contains two potential CK2 sites) from this linker region is phosphorylated in vivo. Unfortunately, it was not possible to test this mechanism properly folded wild type (or mutant) Orb protein that had RNA-binding activity could not be generated (Wong, 2011).

Another line of evidence arguing that Orb activity is linked to its phosphorylation status is the difference in the spectrum of proteins associated with the hypo- and hyperphosphorylated isoforms. While Me31B and PABP are in complexes with both isoforms, Bruno appears to interact primarily with the 'hypophosphorylated' isoforms. This interaction fits with the striking difference in the distribution of Bruno and Orb proteins on sucrose gradients and with the limited co-localization of the two proteins to sponge bodies near the anterior of the oocyte. In contrast to Bruno, the poly(A) polymerase Wisp, which is needed to activate translation, interacts preferentially with the 'hyperphosphorylated' isoforms. Although a precursor-product relationship remains to be established for orb target mRNAs, this specificity would be consistent with a model in which hypophosphorylated isoforms are in complexes with mRNAs that are translationally repressed. Translational activation would then depend upon phosphorylation of the hypophosphorylated isoforms and reorganization of the Orb complex. Bruno and/or other repressive factors would be displaced from the complex, while the Wisp poly(A) polymerase would be recruited and could potentially begin extending the poly(A) tails. Supporting a model of this type, preliminary studies indicate that like ck2, mutations in wisp dominantly enhance the HD19G orb^F343/+ DV polarity defects, while mutations in the Bruno gene arrest have the opposite effect (Wong, 2011).

The conclusion that the two isoforms are incorporated into complexes that differ substantially in their composition, and likely also their function, is supported by sucrose gradient fractionation of ovary extracts. 'Hypophosphorylated' isoforms are found mostly near the top of the gradient in comparatively small complexes (<80S). This region of the gradient is also greatly enriched in the translational repressors Bruno and Me31B, while these repressors are largely absent from the more rapidly sedimenting fractions that contain the polysomes. In contrast, 'hyperphosphorylated' isoforms are found in 80S complexes and polysomes. As would be expected if the hyperphosphorylated Orb in these big complexes is directly associated with ribosomes, rather than with some other type of very large RNP, a large collection of ribosomal proteins and translation initiation/elongation factors are found in Orb immunoprecipitates.

The kinase most closely tied to CPEB phosphorylation in vertebrates is Aurora, which has been shown to phosphorylate the Ser174 residue in the N-terminal half of the Xenopus CPEB. However, Aurora's role in Orb phosphorylation is uncertain as this residue is not conserved in Orb and no genetic interactions were detected between aurora mutations and orb. Though these results don't exclude a role for Aurora, they suggest that other kinases may phosphorylate Orb. Using a proteomics approach two candidate Orb kinases were identified, SRPK2 and CK2, and this study has focused on CK2 (Wong, 2011).

Like orb, ck2 is needed during oogenesis for the formation of the DV polarity axis of the egg and embryo. Chorion defects characteristic of disruptions in the grk signaling pathway are observed in eggs laid by females heterozygous for the dominant negative ck2α^Tik allele or homozygous for the very weak loss-of-function ck2β^and allele. Moreover, the genetic interactions between ck2 and orb in DV polarity would argue that these defects arise, at least in part, because ck2 is required for orb function in this particular signaling pathway. The most compelling of these interactions is between orb and the strong loss-of-function ck2β^mbuδA26-2L allele. Unlike ck2α^Tik, eggs laid by ck2β^mbuδA26-2L/+ females have no apparent DV polarity defects; however, when this mutation is introduced into a background partially compromised for orb activity, a very strong interaction is observed and almost three quarters of the eggs laid by trans-heterozygous females have chorion defects (Wong, 2011).

In addition to the genetic interactions in DV polarity, it was found that ck2 has a direct impact on orb autoregulation. Orb is required for localizing and activating the on-site translation of orb mRNA in the developing oocyte. Strikingly, both of these autoregulatory activities are disrupted in females that are only partially compromised for ck2. orb mRNA is not properly localized in vitellogenic stage egg chambers. In addition, the accumulation of Orb protein is reduced compared to wild type. Since the females harboring these ck2 mutant combinations are viable and morphologically normal, it would appear that like DV polarity, these orb regulatory activities are especially sensitive to reductions in ck2 activity (Wong, 2011).

The effects of ck2 on orb function correlate with changes in the phosphoisoform profile. In ck2 backgrounds that have modest effects on polarity and/or orb activity, there is a small shift towards the hypophosphorylated isoforms. In backgrounds that have stronger effects on orb activity and/or show synergistic genetic interactions with orb, the changes in phosphoisoform profile are more pronounced. This is, perhaps, most evident in females heterozygous for the amorphic ck2β^mbuδA26-2L allele. In addition to the hypo- and hyperphosphorylated isoforms visible on regular SDS-PAGE gels, these females have an Orb species that has a similar mobility to dephosphorylated Orb after λ phosphatase-treatment (Wong, 2011).

While these findings indicate that ck2 is required both for orb activity and to generate the normal array of Orb phosphoisoforms, the fact that this kinase has been implicated in many cellular processes raises the possibility that the effects on orb are an indirect consequence of pleiotropic defects in oogenesis induced by ck2 mutations. Unfortunately, this possibility can not be excluded; however, arguing against it is the fact that all of the experiments were done under conditions in which ck2 activity is only partially compromised. Though this might not eliminate pleiotropic effects, it should certainly minimize them and at the same time reveal cellular processes that are especially sensitive to reductions in ck2 activity and thus most likely to be intimately connected to ck2 function. DV polarity, orb activity and Orb phosphorylation would fit into this category. As for how ck2 impacts orb activity and the Orb phosophoisoform profile, the simplest explanation is that it is directly responsible for phosphorylating Orb. Consistent with this idea, there are twelve potential CK2 sites, of which nine are conserved even in distantly related Drosophila species. Of the conserved sites, two are in the linker region separating the two Orb RRM domains and, as mentioned above, could be potential candidates for RNA binding dependent phosphorylation. The seven remaining sites are in short conserved sequence blocks in the otherwise poorly conserved N-terminal half of the Orb protein. Interestingly, six of these are in a closely spaced cluster. The physical association between CK2 and Orb would also support a direct mechanism. On the other hand, it is also possible that ck2 acts indirectly through intermediate kinases. In this case, the activity of this kinase cascade would have to be especially sensitive to changes in ck2 levels. However, even in this indirect scenario, the effects of ck2 mutations on orb activity and the phosphoisoform profile provide further evidence linking the regulatory functions of the Orb protein to its phosphorylation status (Wong, 2011).

Whether the effects of ck2 on orb are direct or indirect, there are indications that other kinases must phosphorylate Orb. For one, there are likely to be phosphorylated tyrosine residues since the Orb phosphoisoforms are not completely collapsed by the Ser/Thr specific Protein Phosphatase 1, but are collapsed by λ phosphatase. Consistent with this possibility, preliminary experiments indicate that Orb is recognized by phosphotyrosine antibodies. Secondly, other Ser/Thr kinases might be needed to activate Orb. Studies have demonstrate that srpk2 mutations disrupt oogenesis and have DV polarity phenotypes. Though the reported phenotypes seem different from those of well-characterized orb mutants, this study found that a P-element-induced mutation in srpk2 dominantly enhanced the orb DV polarity defects. Finally, since known orb target mRNAs exhibit different patterns of localization and translation, they are likely to be associated with a unique set of regulatory proteins and depend upon different signaling cascades for translational activation. Thus, a more likely scenario is that CK2 is just one of several potential Orb kinases, and that translation of different orb target mRNAs might require the deployment of specific constellations of modifying enzymes (Wong, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

CkII is present ubiquitously throughout embryogeneis (M. Abu-Shaar and R. S. Mann, personal communication to Jaffe, 1997).

Drosophila CK2 regulates lateral-inhibition during eye and bristle development

Lateral inhibition is critical for cell fate determination and involves the functions of Notch (N) and its effectors, the Enhancer of Split Complex, E(spl)C repressors. Although E(spl) proteins mediate the repressive effects of N in diverse contexts, the role of phosphorylation has been unclear. This study implicates a common role for the highly conserved Ser/Thr protein kinase CK2 during eye and bristle development. Compromising the functions of the catalytic (α) subunit of CK2 elicits a rough eye and defects in the interommatidial bristles (IOBs). These phenotypes are exacerbated by mutations in CK2 and suppressed by an increase in the dosage of this protein kinase. The appearance of the rough eye correlates, in time and space, to the specification and refinement of the ‘founding’ R8 photoreceptor. Consistent with this observation, compromising CK2 elicits supernumerary R8’s at the posterior margin of the morphogenetic furrow (MF), a phenotype characteristic of loss of E(spl)C and impaired lateral inhibition. Compromising CK2 elicits ectopic and split bristles. The former reflects the specification of excess bristle SOPs, while the latter suggests roles during asymmetric divisions that drive morphogenesis of this sensory organ. In addition, these phenotypes are exacerbated by mutations in CK2 or E(spl), indicating genetic interactions between these two loci. Given the centrality of E(spl) to the repressive effects of N, these studies suggest conserved roles for this protein kinase during lateral inhibition. Candidates for this regulation are the E(spl) repressors, the terminal effectors of this pathway (Bose, 2006).

Neurogenesis reflects the outcome of a complex balance between the activities of transcription factors that favor this cell fate (ASC/Ato) and those that oppose it (E(spl)). It is increasingly apparent that formation of the eye and bristle are predicated on a similar mechanistic framework, even though the proneurals that participate in these two developmental programs are distinct. For example, the PNCs in the eye (the R8 cell) require ato, while those in the bristle (macrochaetes and IOBs) require ASC. Nevertheless, one common feature of the resolution of PNCs in the eye and the bristle is the centrality of E(spl)C, since loss of E(spl)C leads to exaggerated neurogenesis in both contexts. In the eye it leads to excess R8’s, rough eyes, and duplicated IOBs, while in the bristles this manifests as ectopic, split and missing bristles. Extensive analyses have identified the genes involved with these developmental programs, the feedback loops that reinforce proneural expression in R8’s/SOPs, and the role of E(spl)C for lateral inhibition. In contrast, it has remained unclear how phosphorylation contributes to the dynamics of this process (Bose, 2006).

It has been thought that transcription of E(spl) is, by itself, necessary and sufficient for lateral inhibition. This model emerged from studies on bristle development, where ectopic E(spl) proteins extinguished the SOPs, whereas loss of E(spl) favored this cell fate. This model needs qualification because similar outcomes have not been recapitulated in the eye. In this context, loss of E(spl) demonstrably compromises lateral inhibition and elicits excess R8’s. However, ectopic expression of E(spl) members does not block the R8 fate, and consequently the eye displays the normal hexagonal packing of the ommatidia; the only defect is loss of the IOBs whose developmental program bears similarities to that of the macrochaetes. In contrast, R8 formation is blocked by the truncated M8* protein encoded by the E(spl)D allele, or by the CK2 phophomimetic variant M8SD. It is important to note that the eye defect of E(spl)D requires N^spl, a recessive allele that attenuates ato, but not E(spl), expression. The inability of M8* to recruit Gro, which compromises repression, thus necessitates a sensitized background, one conferred by N^spl. Accordingly, M8SD (which binds Gro) elicits eye defects independent of N^spl (Karandikar, 2004). Based on the observation that both M8* and M8SD display exacerbated and equivalent interactions with Ato, it was proposed that CK2 phosphorylation switches M8 into an active repressor by uncovering the Orange domain, and it is this regulation that is bypassed by the E(spl)D mutation (Karandikar, 2004). Given that the Orange domain mediates binding to other proneurals as well, this regulation by CK2 should have been more general to lateral inhibition. These studies suggest just such a role in the eye and the bristle (Bose, 2006).

This work supports the notion that CK2 is a participant in lateral inhibition. Compromising CK2 by a number of independent routes, i.e., in wild type and backgrounds mutant for CK2 and E(spl), elicits neural defects in the eye and bristle. These include rough eyes due to the specification of excess 'founding' R8 cells, and ectopic bristles (macrochaetes and IOBs) due to the specification of excess SOP’s. These phenotypes are hallmarks of impaired lateral inhibition, and have been previously described for loss of function of the E(spl)C. Evidence is provided for genetic interactions between CK2 and E(spl). While these studies provide multiple lines of evidence, the absence of suitable antibodies have precluded a formal demonstration that E(spl) repressors are, in fact, phosphorylated in cells undergoing lateral inhibition. Nevertheless, the congruence of the results utilizing CK2-RNAi or CK2-DN in conjunction with extant mutants and cell fate in imaginal discs, together, constitute a plausible argument supporting a role for this protein kinase (Bose, 2006).

These studies also suggest secondary roles for CK2 in the bristle lineage. In contrast to R8 patterning, the roles of N and E(spl) are different during bristle morphogenesis. In the case of the macrochaete or the IOB, N and E(spl) are re-deployed following SOP selection. Specifically, the SOP gives rise to the pI neuroblast that undergoes two asymmetric divisions to generate four cell types characteristic of the sensillum; socket, shaft, sheath and neuron, and these divisions are dependent on N- and E(spl)-inhibitory signaling. Thus loss of E(spl) following SOP selection manifests as split bristles (aberrant division of the pIIa cell) or missing bristles (aberrant division of the pI cell). The split bristles described thus suggest a role for CK2 during the socket-to-shaft sister cell fate. In contrast, while the missing bristles suggest a role for CK2 during the pIIa-vs-pIIb fates, this phenotype could result from loss of the SOP itself, a possibility if CK2 levels become rate limiting for cell division. Despite the fact that the timing of the asymmetric divisions of pI, pIIa and pIIb are well known, CK2-RNAi or CK2-DN are not suitable for dissecting the roles of CK2 at these later steps of bristle development. Conditional alleles of CK2, e.g., temperature-sensitives, will be necessary to better define its roles during specification of these sister-cell fates. One major question that emerges from these studies is why is phosphorylation necessary, given that not all members of the E(spl)C are targets of CK2. It is thought that evolutionary principles, the diversities and/or affinities of interactions between E(spl)C and ASC/ato, and their spatial expression patterns, perhaps, offer insights (Bose, 2006).

Of the seven E(spl) proteins, three (M8, M5, and M7) are targeted by CK2, and these are also the most closely related. Among all E(spl) members, two regions largely account for length heterogeneity and divergence. These are sequences between HLH and Orange and those between Orange and WRPW, the CtD. However, within the CtD of M8 (and M5 and M7 as well) is a highly invariant sequence, the phosphorylation domain (P-domain) that harbors the CK2 site. Given the phylogenetic relationships of these species, it is noteworthy that over a period of ~50 million years the P-domain and the CK2 site have been remarkably conserved. For example, of all M8 homologs, only D. pseudoobscura, D. grimshawii and D. hydei harbor a Glu residue, in place of Asp, at the n + 3 position of the CK2 phosphoacceptor. While it has not been experimentally confirmed that these homologs are phosphorylated, the possibility is high because this change still conforms to the consensus (S/T-D/E-x-D/E) for recognition by CK2. The virtually identical consensus site that is present in mammalian Hes6 is targeted by CK2 (Gratton, 2003) in vivo (Bose, 2006).

The mechanisms by which E(spl) proteins mediate repression have been intensely studied. In essence, E(spl) proteins repress ASC/Ato. Repression was initially thought to involve binding to a DNA sequence, the N-box. This, however, is not the case, because E(spl) proteins neutralized for DNA-binding still function as potent repressors. Furthermore, no N-box has been found in the regulatory region of ato, while that in sc is dispensable for repression in non-SOPs. It is now thought that direct (protein–protein) interactions between E(spl) and proneurals are more critical for repression, the protein–tether model (Giagtzoglou, 2003). In this model, repression by E(spl) occurs via direct interactions with enhancer bound proneurals, rather than by activator sequestration. This model is consistent with direct interactions between E(spl) and ASC/Ato proteins. It was, in fact, the analyses of various binary combinations that were the first to suggest that these interactions are regulated and non-redundant, two aspects that appear relevant to the current findings (Bose, 2006).

Analysis of M8 and its E(spl)D encoded variant, M8* provided the first hint that these antagonistic interactions are regulated. For example, it has been reported that, in addition to Ato, M8* interacts with a much higher affinity with Ac, Sc, and Ase. A similar case is described for M8SD, which interacts with Ato or L’sc with affinities significantly higher than M8 or its non-phosphorylatable variant M8SA (Karandikar, 2004). It is noteworthy that phosphorylation of mammalian Hes6 by CK2 is also a pre-requisite for its interactions with Hes1 (Gratton, 2003). Thus CK2 phosphorylation influences antagonistic interactions between the E(spl) and the ASC/Ato. Because these studies have employed two hybrid, instead of direct protein, approaches the possibility that these are kinetic effects remains open. This interpretation is consistent with the observations that a 2× dosage of a UAS-mδ construct interferes with Ato and blocks eye development in the wild type, whereas that of m7, m5 or m8 requires N^spl. Together, these findings argue that E(spl)-ASC interactions are of variable strengths and are isoform-specific. Given that only a subset of E(spl) and ASC members are expressed in the eye and wing disc, the possibility thus arises that distinct domains of ASC define sub-regions of the proneural field. In this context, E(spl) members might have been selected based on their affinities and/or specificities for these proneural factors. Thus the type of E(spl) repressors that are deployed might reflect the combinations and levels of proneurals, with CK2 playing an integrative role. The currently available techniques preclude a distinction between these possibilities (Bose, 2006).

It is presently unclear if/how CK2 activity is modulated during neurogenesis. Expression of this enzyme appears to be constitutive in the eye and wing disc (Karandikar, 2004). Holoenzyme formation, proposed to be a dynamic process in vivo, represents an attractive regulatory mechanism, given that CK2β modulates substrate recognition and that the fly CK2β gene encodes for non-redundant isoforms of this regulatory subunit. Alternatively, CK2 might be regulated by assembly into multiprotein complexes and/or via interactions with protein phosphatases. Such a coordinated function has been described for regulation of Period, the central component of the circadian clock, by CK2 and the phosphatase PP2A. Future studies aimed at the identification of protein phosphatase(s) that counteract the phosphorylation of E(spl)m8/5/7 by CK2, or multiprotein complexes containing E(spl) and/or CK2 will be required to better define the regulatory dynamics of this process during eye and bristle development (Bose, 2006).

EFFECTS OF MUTATION

Analysis of a spatial orientation memory in Drosophila

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Here we show that Drosophila melanogaster possesses a similar spatial memory during locomotion. Using a new detour setup, this study shows that flies can remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, it was found that the GABAergic (stainable with antibodies against GABA) ring neurons of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. We also find that the protein kinase S6KII (ignorant) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies (Neuser, 2008).

A role for CK2 in the Drosophila circadian oscillator

The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, it has been shown that Casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the ß subunit of CK2 and is predicted to perturb CK2ß subunit dimerization. It is associated with reduced ß subunit levels, indicative of a defect in alpha:ß association and production of the tetrameric alpha2:ß2 holoenzyme. Consistent with a direct action on the clock mechanism, it has been shown that CK2ß is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator (Akten, 2003).

It is of interest that the Andante mutation affects the nuclear entry of clock proteins in the small LN_v population, but seems to have no effect on the large LN_v neurons. Such a differential effect suggests that CK2 is important for oscillator function in one population but not the other. Indeed, the small LN_v cells have been shown to be critical for the clock regulation of activity rhythms; the small cells send projections to a region of the dorsal brain implicated in clock output, and there is a circadian rhythm in the release of the clock output factor pigment dispersing factor (PDF) in these dorsal projections. Furthermore, it has been reported that the small but not the large LN_v cells show Tim rhythmicity in constant darkness (DD). Finally, there are differences between the large and small LN_v cells, with regard to the timing of Per and Tim nuclear entry. As CK2 deficits seem only to affect nuclear entry in the small LN_v neurons, it is possible that this kinase regulates differences between the two neuronal populations (Akten, 2003).

Although it is likely that CK2 acts within clock cells to help specify period length, these studies indicate neither the cellular compartment in which the kinase acts nor the molecular substrates of the enzyme that are relevant for clock function. A delay in the nuclear accumulation of clock proteins, as seen in Andante, suggests that the kinase functions in the cytoplasm to promote nuclear entry. This function might be mediated by direct phosphorylation of a clock protein (such as Per or Tim) or by activation of a second kinase such as GSK-3/Shaggy, which has been implicated in promoting the nuclear entry of Tim. The elevated levels of Per and Tim observed in Andante suggest a decreased turnover of the clock proteins, and this might arise because of a defect in the targeted degradation of one or both proteins within the nucleus. Similar to the Doubletime kinase, CK2 might act both in the cytoplasm and nucleus of clock cells to determine the timing of nuclear entry and/or stability of clock proteins (Akten, 2003).

These studies of CK2 in Drosophila suggest that this kinase might have an important role in the regulation of circadian period in other animal species. Previous studies in the plant Arabidopsis and the fungus Neurospora have also implicated CK2 activity in circadian oscillator function. The Arabidopsis study showed that overexpression of the CK2ß3 subunit is associated with a shortening of circadian period, a result similar to that obtained in this study of Drosophila CK2ß. The Neurospora study showed abnormal phosphorylation of the Frequency (Frq) protein in a Neurospora CK2alpha mutant, but circadian behavior (such as conidiation rhythms) could not be examined in that study because of reduced viability. Although neither of these previous reports characterize a mutant with decreased CK2 activity, the results are consistent with studies of Andante and indicate an evolutionarily conserved role for CK2 in circadian oscillator function (Akten, 2003).

Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. This study shows that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, a physical interaction was demonstrated between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. It is proposed that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism (Akten, 2009).

Drosophila CK2 promotes Wts to suppress Yki activity for growth control

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. This study reports that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant induced Ex upregulation and overgrowth phenotype while it cannot affect wts mutant. Consistent with this, knockdown of CK2 upregulates Hpo pathway target expression. It was also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in developing discs induces severe growth defect as well as caspase3 (Drice) signal. Taken together, these results uncover a dual role of CK2: while its major role is promoting cell survive, it may potentially be a growth inhibitor as well (Hu, 2014).

EVOLUTIONARY HOMOLOGS

Regulation of the Neurospora circadian clock by casein kinase II

Phosphorylation of clock proteins represents an important mechanism regulating circadian clocks. In Neurospora, clock protein FREQUENCY (FRQ) is progressively phosphorylated over time, and its level decreases when it is extensively phosphorylated. To identify the kinase phosphorylating FRQ and to understand the function of FRQ phosphorylation, a FRQ-phosphorylating kinase was purified and identified as casein kinase II (CKII). Disruption of the catalytic subunit gene of CKII in Neurospora results in hypophosphorylation and increased levels of FRQ protein. In addition, the circadian rhythms of frq RNA, FRQ protein, and clock-controlled genes are abolished in the CKII mutant. These data suggest that the phosphorylation of FRQ by CKII may have at least three functions; it decreases the stability of FRQ, reduces the protein complex formation between FRQ and the WHITE COLLAR proteins, and is important for the closing of the Neurospora circadian negative feedback loop. Taken together, these results suggest that CKII is an important component of the Neurospora circadian clock (Yang, 2002).

CKII (CK2) is also involved in the function of the Arabidopsis circadian clock. In vitro, CKII subunits of Arabidopsis have been shown to interact and phosphorylate the circadian clock-associated 1 (CCA1) protein. The phosphorylation of CCA1 by CKII-like activity in vitro affects the formation of a DNA-protein complex containing CCA1. In addition, overexpression of a regulatory subunit of CKII results in the shortening of the periods of clock-controlled genes in Arabidopsis. Because the Neurospora WC complex binds to DNA and FRQ interacts with the WCs, it is possible that the CKII phosphorylation of FRQ influences the formation of the DNA-WC complex (Yang, 2002).

In Drosophila, the phosphorylation of Tim appears to have at least two different functions. The phosphorylation of Tim by Sgg promotes the nuclear transfer of the Per/Tim complex, whereas a tyrosine-linked phosphorylation of Tim by an unknown kinase is implicated in the proteasome-mediated degradation of Tim. Dbt of Drosophila and the casein kinase Iepsilon in mammals phosphorylate Per and may trigger its turnover. Although both CKI and CKII are Ser/Thr kinases, their structures and substrate specificities are significantly different, suggesting that they did not evolve from a common ancestral protein (Yang, 2002).

Unlike Dbt and Sgg in Drosophila and CKII in Saccharomyces, both of which are required for the survival of their respective organisms, CKII of Neurospora is not essential for the survival of Neurospora. However, the Neurospora CKII is important for the normal growth and development of the fungus. As a protein serine/threonine kinase that is ubiquitously found in eukaryotes, CKII is known to phosphorylate many substrates and regulate many processes in eukaryotic cells. However, in Neurospora, it is thought that the clock defects in the cka^RIP mutant are probably not caused by its growth and developmental defects. (1) In genetic screening for clock mutants, it has been found that most of Neurospora growth and developmental mutants have normal functional clocks. (2) It was well known that the growth rate of Neurospora is not an influencing factor in the function of the clock. (3) The molecular phenotypes of CKII are very similar to those of the frq null strain. These results suggest that the loss of clock function in the cka mutant appears to be the result of loss of the fully functional FRQ protein. (4) In this study, CKII was identified as a potential clock component due to its role of phosphorylating FRQ, one of the central components of the Neurospora clock, in vitro and in vivo. Based on these data, it is thought that the role of CKII in the Neurospora circadian oscillator may be unrelated to its function in cell growth and development. However, the possibility that the growth and developmental defects indirectly contributed to the clock phenotypes observed in the cka^RIP strains cannot be ruled out. It is also not known whether CKII can affect the clock by regulating the activity of other unknown clock genes in Neurospora (Yang, 2002).

CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. This study shows that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a^L strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt^L mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ~32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a^L strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a^L strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka^RIP strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka^RIP strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop (He, 2006).

The eukaryotic circadian clocks are composed of auto- regulatory circadian negative feedback loops including both positive and negative elements. In Neurospora, Drosophila, and mammals, the positive elements are all heterodimeric complexes, consisting of two PER-ARNT-SIM (PAS) domain-containing transcriptional factors that bind to the cis-elements in the promoter of the negative elements to activate their transcription. In contrast, the negative elements repress their own transcription by inhibiting the activity of the positive elements through their physical interactions. It is unclear how negative elements inhibit the activity of positive elements to close the circadian negative feedback loops. Since the identification of the Drosophila doubletime (dbt) gene, which encodes for a casein kinase I (CKI) homolog, it has become clear that post-translational protein phosphorylation is essential for the function of circadian clocks. Despite the evolutionary distance, remarkable conservation of post-translational regulation exists among different eukaryotic systems from fungi to human (He, 2006).

In the filamentous fungus Neurospora crassa, the core circadian negative feedback loop consists of four essential components: WHITE COLLAR-1 (WC-1), WC-2, FREQUENCY (FRQ), and a FRQ-interacting RNA hecliase FRH. WC-1 and WC-2, two PAS domain-containing transcription factors, form a heterodimeric WC complex (D-WCC) in the dark, which binds to the Clock (C)-box in the frq promoter to activate frq transcription. Thus, WCC is the positive element in the Neurospora circadian negative feedback loop. In contrast, FFC, the complex formed by FRQ and FRH functions as the negative element. To repress the transcription of frq, FFC possibly mediates the inhibition of the WCC activity through their physical interaction. In a wild-type strain, this circadian negative feedback loop generates robust daily rhythms of frq RNA and FRQ protein. When the circadian negative feedback loop is disrupted by mutation of frq or down-regulation of frh, frq RNA levels stay at constant higher levels, resulting in arrhythmici- ties. In addition to their role in the circadian negative feedback loop in the dark, WC-1and WC-2 are also essential components for the light responses and light resetting of the clock, with WC-1 being the blue-light photoreceptor (He, 2006).

In addition to the repression of D-WCC activity in the dark, FRQ promotes the accumulation of WC-1 and WC-2, forming positive feedback loops that are inter-locked with the negative loop, a feature that is shared by animal circadian systems. In Neurospora, these positive feedback loops have been shown to be important for the robustness and function of the clock. It was recently shown that the phosphorylation of the PEST-2 region of cytoplasmic FRQ is important for the accumulation of WC-1 but not WC-2 (He, 2006).

FRQ, WC-1, and WC-2 are regulated by phosphorylation events. After its synthesis, FRQ is immediately phosphorylated and becomes progressively more phosphorylated over time before its degradation through the ubiquitin–proteasome pathway mediated by FWD-1. Thus, in the dark, FRQ is not only robustly rhythmic in quantity, but also in its phosphorylation states. CK-1a (casein kinase 1a), CKII (casein kinase II), and CAMK-1 are the three identified FRQ kinases. However, only CKII's role in mediating FRQ phosphorylation is firmly established in vivo. In vitro, CKII is one of the main kinases that phosphorylate FRQ. In strains in which either the CKII catalytic subunit (cka) or one of its regulatory subunits (ckb1) is disrupted, FRQ is both hypophosphorylated and more stable, and the clock function is either completely abolished (cka mutant) or oscillates with a severely damped amplitude (ckb1 mutant). Furthermore, in the cka mutant strain that has no CKII activity, frq mRNA levels are constantly high, which is reminiscent of the frq RNA levels in strains with a disrupted circadian negative feedback loop. These data suggest that CKII not only promotes FRQ degradation, but it is also required for the repressor function of FRQ. The mechanism by which CKII carries this latter function is not known (He, 2006).

CK-1a is one of the two Neurospora CKI homologs and it can phosphorylate the PEST-1 and PEST-2 domains of FRQ in vitro. The deletion of the PEST-1 domain resulted in the increased stability of FRQ and a long period rhythm. More importantly, CK-1a was found to associate with FRQ, suggesting that it may phosphorylate FRQ in vivo. However, in vivo evidence for the involvement of CK-1a in the clock was not available because CK-1a is essential for cell survival in Neurospora (He, 2006).

Similar to FRQ, both WC-1 and WC-2 are phosphorylated both in the dark and in a light-dependent manner. Their phosphorylation plays important roles in regulating WCC activity. Five major in vivo WC-1 phosphorylation sites, located immediately downstream from its DNA-binding domain, have been identified. Mutation of these light-independent sites suggested that they are critical for circadian clock function and they negatively regulate the D-WCC activity. The importance of WC phosphorylation in the circadian clock was later confirmed by the surprising observation that the WC phosphorylation is FRQ dependent. In the frq-null strain, both WC-1 and WC-2 are hypophosphorylated. In a wild- type strain, WC-2 exhibits a robust circadian rhythm of its phosphorylation profile when analyzed on two-dimensional electrophoresis. Importantly, the activation of frq transcription correlates with the hypophosphorylation of the WCs. Consistent with these data, it has been shown that dephosphorylation of the Neurospora WCC significantly promotes its binding to the C-box. Together, these results suggest a model in which FFC inhibits the WCC activity by promoting the phosphorylation of WC proteins. Interestingly, PER-dependent phosphorylation of CLK has also been observed in Drosophila, suggesting a common mechanism that closes the circadian negative feedback loops (He, 2006).

The kinase(s) recruited by FRQ to phosphorylate the WC proteins has not been identified. How WCC activity is affected by the phosphorylation mediated by this kinase(s) is also not known. In this study, it is shown that like CKII, CK-1a phosphorylates FRQ in vivo to promote its degradation. More importantly, both kinases mediate the FRQ-dependent phosphorylation of WCC, which inhibits its activity to close the circadian negative feedback loop. In addition to CK-1a's role in the negative feedback loop, it is also required for the function of the circadian positive feedback loops by increasing WC levels (He, 2006).

Protein kinase A and casein kinases mediate sequential phosphorylation events in the circadian negative feedback loop

Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. In contrast, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. By using whole-cell isotope labeling and quantitative mass spectrometry methods, this study shows that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. The cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop (Huang, 2007).

A role for casein kinase 2 in the mechanism underlying circadian temperature compensation

Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. Casein kinase 2 (CK2) was found to be a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the β1 and α subunits of CK2, respectively. Reducing the dose of these subunits, particularly β1, significantly alters temperature compensation without altering the enzyme's Q10. By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles effectively phenocopying CK2 mutants (Mehra, 2009).

Casein kinase II regulation of yeast TFIIIB is mediated by the TATA-binding protein

The highly conserved protein kinase casein kinase II (CKII) is required for efficient Pol III transcription of the tRNA and 5S rRNA genes in Saccharomyces cerevisiae. TFIIIB is the CKII-responsive component of the Pol III transcription machinery. Dephosphorylation of TFIIIB eliminates its ability to complement CKII-depleted extract, and a single TFIIIB subunit, the TATA-binding protein (TBP), is a preferred substrate of CKII in vitro. Recombinant TBP purified from Escherichia coli is phosphorylated efficiently by CKII and, in the presence of a limiting amount of CKII, is able to substantially rescue transcription in CKII-deficient extract. These results establish that TBP is a key component of the pathway linking CKII activity and Pol III transcription and suggest that TBP is the target of a CKII-mediated regulatory mechanism that can modulate Pol III transcription (Ghavidel, 1997).

Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin

Heterochromatin protein 1 (HP1) recruits various effectors to heterochromatin for multiple functions, but its regulation is unclear. In fission yeast, a HP1 homolog Swi6 recruits SHREC, Epe1, and cohesin, which are involved in transcriptional gene silencing (TGS), transcriptional activation, and sister chromatid cohesion, respectively. This study shows that Casein kinase II (CK2) phosphorylates Swi6. Loss of CK2-dependent Swi6 phosphorylation alleviates heterochromatic TGS without affecting heterochromatin structure. This is due to the inhibited recruitment of silencing complex SHREC to heterochromatin, accompanied by an increase in transcriptional activator Epe1. Interestingly, loss of phosphorylation does not affect cohesion. These results indicate that CK2-dependent Swi6 phosphorylation specifically controls TGS in heterochromatin (Shimada, 2009).

Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae

The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the genes CKA1 and CKA2, which together are essential for the animal's viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2ts strains arrest within a single cell cycle and exhibit a dual arrest phenotype consisting of 50% unbudded and 50% large-budded cells. The unbudded half of the arrested population contain a single nucleus and a single focus of microtubule staining, consistent with arrest in G1. Most of the large-budded fraction contain segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirms that CKII is required in G1, at a point that must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicates that CKII is not required for completion of previously initiated DNA replication but confirm that the enzyme is again required for cell cycle progression in G2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle (Hanna, 1995).

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock has remained unknown. This study shows that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortens the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduces phytochrome induction of an Lhcb gene. Finally, the photoperiodic flowering response, which is influenced by circadian rhythms, is diminished in the transgenic lines, and the plants flower earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis (Sugano, 1999).

Casein kinase II from Caenorhabditis elegans. Cloning, characterization, and developmental regulation of the gene encoding the beta subunit

Complementary DNAs encoding the beta subunit of Casein kinase II (CkII beta) from the nematode Caenorhabditis elegans were cloned and sequenced. The predicted beta subunit polypeptide comprises 234 amino acid residues and has a Mr of 26,452. CkII beta is not homologous with other types of proteins. In synchronously developing C. elegans the abundance of the 1.3-kilobase mRNA for CkII beta varies in parallel with the level of mRNA encoding the catalytic subunit (alpha) of CkII. Thus, the developmental expression of CkII subunits is controlled coordinately and pretranslationally. CkII beta and CkII alpha mRNAs are enriched 5-10-fold in C. elegans embryos relative to their concentrations at several other stages of nematode development. A 3.8-kilobase pair segment of C. elegans DNA that contains the CkII beta gene and an extensive 5'-flanking region was cloned and sequenced. The CkII beta gene is divided into 6 exons by introns ranging from 49 to 533 base pairs in length. The first exon encodes 88 nucleotides of 5'-untranslated mRNA. Exon 2 (72 base pairs) contains the initiator Met codon and only 5 additional codons. Exons 3-6 encode 52, 63, 64, and 49 amino acid residues, respectively. The 5' terminus of CkII beta mRNA is modified post-transcriptionally by trans-splicing with a leader sequence of 22 nucleotides. The CkII beta gene maps to a position on C. elegans chromosome 2 that is in close proximity to the lin-11 gene (Hu, 1991).

Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line

Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. This study describes a large-scale RNAi screen that identified kin-10, which encodes the beta subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. A role or kin-10's regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. It is proposed that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision (Wang, 2014).

Cloning and expression of genes coding for protein kinase CK2 alpha and beta subunits in zebrafish

cDNA clones coding for the alpha and beta subunits of CK2 in zebrafish (Danio rerio) have been isolated. One contains the complete coding sequence for the beta subunit of CK2 while the alpha clone is truncated and lacks 183 nucleotides of the 5' coding region. Comparison of the deduced amino acid sequences shows an extremely high degree of evolutionary sequence conservation for these two proteins. Northern analysis of the mRNAs coding for the alpha subunit indicates that this messenger is present in 1 h embryos as a 3.6 Kb and a 1.9 Kb species, both of which decrease in 24-h embryos. In the case of beta, the major mRNA species of approximately 1.7 Kb maintained its level during the period of embryogenesis studied. In situ hybridization of early embryos, using antisense RNAs against alpha and beta mRNAs demonstrates temporal and tissue specific expression patterns. The alpha mRNA decreases after blastula, when it is evenly distributed. The beta mRNA is maintained at high levels between 4 and 24 h of development, showing in 18 h embryos a higher concentration in the developing neural tube and in the embryonic optic and otic vesicles (Daniotti, 1994).

The beta subunit of CKII negatively regulates xenopus oocyte maturation

In Xenopus oocytes, CKIIbeta, associates with both CKIIalpha and the serine/threonine kinase, Mos. As a key regulator of meiosis, Mos is necessary and sufficient to initiate oocyte maturation. The binding of CKIIbeta to Mos represses Mos-mediated mitogen-activated protein kinase (MAPK) activation; the ectopic expression of CKIIbeta inhibits progesterone-induced Xenopus oocyte maturation. Oocytes with a reduced content of CKIIbeta (achieved by using antisense oligonucleotide to CKIIbeta) are more sensitive to low doses of progesterone and show accelerated MAPK activation and germinal vesicle breakdown. Ectopic expression of a Mos-binding fragment of CKIIbeta suppresses the effect of antisense oligonucleotide. These results suggest that the endogenous CKIIbeta normally sets a threshold level for Mos protein, which must be exceeded for Mos to activate the MAPK signaling pathway and induce oocyte maturation (Chen, 1997).

Expression of casein kinase 2 during mouse embryogenesis

The expression and distribution of Casein kinase 2 subunits in mouse embryos at different developmental stages was investigated: expression at the mRNA level of CK2 alpha- and beta-subunits (by in situ hybridization) and distribution at the protein level by immunohistochemistry using CK2-alpha-antibodies (expression) and CK2-beta-specific antibodies (distribution). In general both methods gave similar results. In earlier stages of mouse embryonic development (day 10.5 after coitus) CK2 is expressed at a higher level in neuroepithelia than in all other tissues. From day 11.5 on, high expression of CK2 is detected in all epithelia. From day 16.5 on, all tissues and anlagen of the fetus involved in organogenesis reveal a higher CK2 expression as compared with secondary mesenchyma. The only difference between in situ hybridization and immunocytochemistry is observed in the skin. Transcripts of CK2 are found mostly in the basal layer of the epidermis and in the nuclei of keratinocytes, whereas CK2 protein is almost exclusively found in the cytoplasm of epidermal cells (Mestres, 1994).

Regulation of protein kinase CK2 isoform expression during rat brain development

Protein kinase CK2 is very abundant in rat brain when compared with other rat tissues. The enzyme is an oligomeric protein with the structures three possible heterotrimeric structures: alpha2 beta2, alpha alpha'beta2 and alpha'2 beta2. The alpha and alpha' subunits are catalytic and have a high degree of homology, whereas the beta subunit seems regulatory. There is a significant increase in the amount of alpha' subunit during the late postnatal neocortical maturation period. The increased alpha' expression occurs at a time parallel to synaptogenesis. As for its distribution, the alpha' subunit of CK2 is much more abundant in neurons (particularly in large size neurons) than it is in glia. These results are consistent with a hypothetical role for CK2 isoforms containing alpha' subunits in the regulation of specific functions in fully differentiated neurons (Diaz-Nido, 1994).

Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat

The activity and cellular localization of hepatic Casein kinase II (CkII) was examined during late fetal development in the rat. Cultured fetal hepatocytes display constitutive CKII activity that is not further activated by growth factor exposure. Similarly, fetal liver CKII shows approximately fivefold greater activity than adult liver. The fetal hepatic activity is, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver show similar CKII activity. Immunoreactive CKII coincides with kinase activity. However, a higher (five- to sixfold) CKII specific activity is found in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. It is hypothesized that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes (Gruppuso, 1995).

A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposited in the EMBL database under the accession numbers R08806 and Z17360, for the ribosomal protein L5 and for A-Raf kinase. All isolated clones except the one for CK2 beta show no interaction with the catalytic alpha subunit of CK2. A-Raf kinase is a new interesting partner of CK2 beta. The isolated A-Raf clone represents amino acids 268-606, but also a full length A-Raf clone interacts with CK2 beta. At the site of CK2 beta, residue 175 and amino acids between residues 194 and 200 are likely to be involved in direct interaction (Boldyreff, 1996).

The regulatory subunit of protein kinase CK2 is a specific A-Raf activator

Two protein kinases that are involved in proliferation and oncogenesis but so far have been thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library an interaction between A-Raf and CK2beta subunit has been detected. This binding is specific, as no interaction between CK2beta and B-Raf or c-Raf-1 is observed. Regions critical for this interaction are located between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity is enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell (Hagemann, 1997).

Regulation of protein phosphatase 2A by direct interaction with casein kinase 2alpha

Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase Casein kinaseII (CkII) binds to PP2A in vitro and in mitogen-starved cells; binding requires the integrity of a sequence motif common to CK2alpha and SV40 small t antigen. Overexpression of CK2alpha results in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2alpha inhibits the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2alpha may regulate the deactivation of the mitogen-activated protein kinase pathway (Heriche, 1997).

Interaction of CkII with p53, MDM2, p21 and Bid: Involvement of CkII in the cell cycle

p53 is an essential component of cellular growth control. Some of its distinct biological functions are regulated by interaction with cellular proteins. p53 binds to the regulatory subunit of protein kinase CK2. Using C-terminal protein fragments of p53 it has been demonstrated that the region between amino acids 287 and 340 on the polypeptide chain of p53 is critical for the binding of p53 to the beta-subunit of CK2. Neither phosphorylation at the p34cdc2 site (aa315) nor at the CK2 site (aa392) is necessary for binding of p53 to the beta-subunit of CK2. Using deletion mutants of the CK2 beta-subunit, it has also been shown that an internal region between amino acids 72 and 149 of the CK2 beta-subunit is necessary for binding to p53. This study defines new functional regions on the polypeptide chains of p53 and of protein kinase CK2 (Appel, 1995).

Casein kinase 2 inhibits the renaturation of complementary DNA strands mediated by p53 protein

The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. This activity is inhibited in the presence of the serine/threonine protein kinase CK2. This inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the beta subunit of the oligomeric kinase. Accordingly, the isolated alpha subunit of the kinase is without effect. In addition, after phosphorylation by CK2, phosphorylated p53 loses its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, these results suggest that either protein-protein interaction or phosphorylation of this domain might control the base pairing of complementary sequences promoted by p53 in processes related to DNA replication and repair (Filhol, 1996).

p21WAF1/CIP1 interacts with protein kinase CK2

p21WAF1/CIP1, which belongs to a class of regulatory proteins that interact with cyclin dependent kinases is a potent inhibitor of these kinases. The inhibition of the cyclin dependent kinases induces an arrest of cells in the G phase of the cell cycle. In addition p21WAF1/CIP1 associates with PCNA (Drosophila homolog: Mutagen-sensitive 209) and inhibits DNA replication. p21WAF1/CIP1 binds to the regulatory beta-subunit of protein kinase CK2 but not to the catalytic alpha-subunit. Binding of p21WAF1/CIP1 down regulates the kinase activity of CK2 with respect to the phosphorylation of the beta-subunit of CK2, casein and the C-terminus of p53. This study demonstrates a new binding partner for the regulatory beta-subunit of protein kinase CK2 which regulates the activity of the holoenzyme (Gotz, 1996).

The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation

The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzes the incorporation of twice as much phosphate into the MDM2 protein as is obtained with the holoenzyme. Polylysine stimulates MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the alpha-subunit-catalyzed MDM2 phosphorylation which is reduced by about 66% when polylysine is added. Full length p53 mimics the polylysine effect in all respects; that is, stimulation of phosphate incorporation by CK2 holoenzyme and inhibition in the presence of the catalytic CK2 alpha-subunit. This is also the case with a peptide representing a C-terminal fragment of the tumor suppressor gene product p53 (amino acids 264-393, which also harbor the CK2beta interaction site at amino acids 287-340). Stimulation by p53(264-393) is on average close to twofold that of CkII, and inhibition in the case of the alpha-subunit-catalyzed MDM2 phosphorylation is about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21(WAF1/CIP1), known to be a potent inhibitor of cyclin-dependent protein kinases, also leads to a significant reduction of phosphate incorporation into MDM2, indicating that p21(WAF1/CIP1) does not exclusively inhibit cell cycle kinases. These data add new insight into the autoregulatory loop which include p21(WAF1/CIP1), MDM2 protein, CK2 and p53 (Guerra, 1997).

Conformation-dependent phosphorylation of p53

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 [(His)p53], human p53 phosphorylation was determined by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and the beta subunit of Casein kinase II (CkIIbeta) as well as by kinases present in whole cell extracts (WCEs). PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. JNK interacts with p53, and JNK phosphorylates truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 [p53(249)] or 273 [p53(273)]. Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. A comparison of truncated forms of p53 reveals that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region residues 97-117 increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrate that mutant p53(249) exhibits significant changes within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, and for the phosphorylation pattern generated by each kinase (Adler, 1997).

Casein kinase II is required for transition of G0/G1, early G1, and G1/S phases of the cell cycle

Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with many cellular functions, including participation in mitogenic signaling by cytoplasmic nuclear translocation. To examine whether cell compartment-specific availability is a requirement for CKII function during cell cycle progression, antibodies against CKII beta, the regulatory subunit of CKII, were microinjected into the cytoplasm or the nucleus of G0-synchronized human primary fibroblasts (IMR-90) at the time of mitogenic stimulation or at various intervals thereafter. Significant inhibition of the stimulation is obtained with both cytoplasmic and nuclear injections. The inhibition is reversible, is not observed with control antibodies, and is abolished by co-injection of purified CKII holoenzyme. The inhibition differs, however, in extent, duration, and cell cycle phase between cytoplasmic and nuclear injections. After cytoplasmic injection, inhibition reachs 45-50% and is effective at two intervals within the first 2 h and at 12-16 h post-stimulation, i.e. at G0/G1 phase transition and at the G1/S phase boundary of the cell cycle. After injection into the nucleus, the inhibition is considerably stronger, reaching 80-85%, and is effective for the first 6 h post-stimulation, i.e. for the transition of G0/G1 phase and the adjoining first part of G1 phase. Cytoplasmic or nuclear injections within S phase affect neither DNA synthesis nor cell division. The data suggest that cell cycle transition from G0 to S phase requires the presence of a certain functional level of CKII at defined times and at defined cellular locations as follows: for transition of G0/G1 at both the nucleus and the cytoplasm, for transition of early G1 at the nucleus, and for transition of G1/S at the cytoplasm (Pepperkok, 1994).

Phosphorylation of Bid by casein kinases I and II regulates its cleavage by Caspase 8

BH3-only proapoptotic proteins of the Bcl-2 family such as Bad, Bid, Bim, or Bik transduce death stimuli from the cell surface to the central death machinery. Following apoptosis stimulation, these molecules translocate from the cytosol to mitochondria where they bind to membrane-based Bcl-2 family members. Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In type II cells, such as Jurkat cells or hepatocytes, death-inducing signaling complex (DISC) formation is strongly reduced compared to type I cells in which activation of large amounts of caspase 8 by the DISC enables direct activation of downstream caspases leading to irreversible cell damage. In type II cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerates Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delays apoptosis. When phosphorylated, Bid is insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis (Desagher, 2001).

CK2 is a component of the KSR1 scaffold complex that contributes to Raf kinase activation

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, the heterotetrameric protein kinase, casein kinase 2 (CK2), has been identified as a new KSR1-binding partner. Moreover, the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase (Ritt, 2006).

Carbon monoxide neurotransmission activated by CK2 phosphorylation of Heme Oxygenase-2

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. The biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. These findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter (Boehning, 2003).

Carbon monoxide is generated in neurons by the enzymatic cleavage of heme by heme oxygenase-2. Abundant evidence suggests that CO can function as a neurotransmitter. Like nitric oxide (NO), endogenously produced CO stimulates soluble guanylate cyclase (sGC) activity, increasing cGMP levels in target cells. HO2 and sGC expression overlap in many regions of the brain, including areas such as the olfactory bulb, which are devoid of the neuronal NO synthesizing enzyme nitric oxide synthase (nNOS). Olfactory receptor neurons (ORNs) generate micromolar quantities of CO, which are sufficient to activate endogenous sGC. Endogenously produced CO also regulates cGMP levels and odorant adaptation in olfactory receptor neurons of salamanders and invertebrates. Furthermore, HO inhibitors block odorant-induced cGMP production in rat ORNs (Boehning, 2003 and references therein).

The strongest evidence for a role of CO in neurotransmission comes from studies of nonadrenergic, noncholinergic (NANC) neurotransmission in the enteric nervous system. NANC transmission is substantially reduced by HO inhibitors and in mice with genomic deletion of HO2. Nitric oxide (NO) and CO appear to be coneurotransmitters in the gut; HO2 and neuronal NO synthase (nNOS) are both enriched in the myenteric neuronal plexus, and NANC neurotransmission, reduced in nNOS-deleted mice, is virtually abolished in mice with deletion of both HO2 and nNOS. Cyclic GMP levels are similarly reduced in intestinal preparations from nNOS and HO2 knockout mice (Boehning, 2003 and references therein).

Unlike conventional neurotransmitters, gases cannot be stored in synaptic vesicles. Therefore, release of NO or CO upon neuronal depolarization requires rapid activation of their biosynthetic enzymes. NO is generated by the calcium/calmodulin-dependent activation of nNOS during neuronal activity. Treatment of neuronal cultures with phorbol esters increases HO2 activity in a protein kinase C (PKC)-dependent manner. However, no direct molecular mechanism for depolarization-induced activation of HO2 during neuronal activity has been reported, which has led to skepticism about the physiologic role of CO as a neurotransmitter (Boehning, 2003 and references therein).

This study examines the molecular mechanisms by which neuronal depolarization leads to increased HO2 activity and subsequent CO synthesis. HO2 is regulated by a protein kinase cascade that ultimately leads to the direct phosphorylation and enzymatic activation of HO2 by the protein kinase CK2, resulting in CO-dependent neurotransmission. These results provide a mechanistic basis for the activation of CO biosynthesis following neuronal activity, as expected for a gaseous neurotransmitter/neuromodulator (Boehning, 2003).

CK2 is a member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis in Xenopus

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of β-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including β-catenin. The role of CK2 in dorsal axis formation was examined. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2α was able to block ectopic axis formation induced by XWnt8 and β-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis (Dominguez, 2004).

In conclusion, experiments carried out in this study demonstrate that CK2 plays an essential role in the complex process of dorsal axis determination. The current model of dorsal axis formation proposes that in the fertilized embryo, β-catenin degradation is promoted by GSK3β phosphorylation. When the dorsal determinants move dorsally during cortical rotation, they promote signaling that stabilizes β-catenin. In cells in culture, β-catenin is regulated by a destruction complex that involves kinases, phosphatases, and scaffold proteins. In the Xenopus embryo, it is not know how β-catenin is dorsally up-regulated, since the nature of the dorsal determinants is still unknown. However, recent experiments have proposed that the dorsal determinants might include dsh and GBP, two Wnt transducers implicated in dorsal axis formation. These translocate to the dorsal side of the embryo where GSK3β is down-regulated, β-catenin accumulates, and dsh is phosphorylated. CK2, expressed most highly in the medial part of the embryo where β-catenin up-regulation occurs, may contribute to Wnt signaling by phosphorylating and stabilizing β-catenin itself, perhaps through the major CK2 phosphorylation site at T393 identified in mammalian cells. Alternatively, CK2 may act upon dsh, GBP, or other as yet uncharacterized dorsal determinants. Indeed, CK2 inhibition in mammalian cells leads to dsh instability (Dominguez, 2004).

The lack of CK2 expression in the vegetal part of the embryo is consistent with a model in which the constitutively active CK2 kinase does not act until the physical process of cortical rotation brings the dorsal determinants to the waiting enzyme in the medial portion of the embryo. This model is consistent with vegetal cortex transplantation studies that suggest that it is the combination of the dorsal determinants plus uncharacterized factors in the cytoplasm of the equatorial (medial) part of the embryo that are required for dorsal determination. Future experiments will validate this hypothesis through a more detailed analysis of the signaling pathway and of biochemical studies of functional protein complexes and their localization in the developing embryo (Dominguez, 2004).

Characterization of mouse Dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, have been biochemically characterized in mouse and Drosophila cell culture systems. Treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with the finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induces hyperphosphorylation of Dsh. Mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of the Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II coimmunoprecipitates with Dvl proteins, and Dvl proteins are phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway (Lee, 1999).

Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. In vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites results in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion (Lickert, 2000).

Casein kinase-II regulates NMDA channel function in hippocampal neurons

Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. CKII is identified as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings is enhanced by CKII or spermine and is decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduce synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function (Lieberman, 1999).

Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability

The alpha catalytic subunit of casein kinase II (CkII) associates with and phosphorylates IkappaBalpha. Deletion mutants of IkappaBalpha localize phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduces phosphorylation of IkappaBalpha by the kinase in vitro. Cells that stably expresses wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant is eliminated in vivo. Mutation of the CkII sites in IkappaBalpha result in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CkII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha (Lin, 1996).

Distinct functional properties of IkappaB alpha and IkappaB beta

In vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their varying abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. The inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes (Tran, 1997).

Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity

Sp1 (see Drosophila Buttonhead), a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in the whole animal; this results in decreased DNA binding activity. Casein kinase II (CkII) is able to phosphorylate the C terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CkII may be responsible for the observed regulation of Sp1. Mutation of a consensus CkII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of this site and the CkII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CkII site at Thr-579 as well as additional sites within the C terminus. No gross changes in CkII subunit levels were seen during de-differentiation associated with liver regeneration. The serine/threonine phosphatase PP1 is identified as the endogenous liver nuclear protein able to dephosphorylate Sp1 but again no gross changes in activity are observed in the regenerating liver. Okadaic acid treatment of K562 cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are established by a balance between kinase and phosphatase activities (Armstrong, 1997).

DNA binding by cut homeodomain proteins is down-modulated by casein kinase II

The Drosophila and mammalian Cut homeodomain proteins contain, in addition to the homeodomain, three other DNA binding regions called Cut repeats. Cut-related proteins thus belong to a distinct class of homeodomain proteins with multiple DNA binding domains. Using nuclear extracts from mammalian cells, Cut-specific DNA binding is increased following phosphatase treatment, suggesting that endogenous Cut proteins are phosphorylated in vivo. Sequence analysis of Cut repeats reveals the presence of sequences that match the consensus phosphorylation site for casein kinase II (CKII). Therefore, an investigation was carried out to determine whether CKII can modulate the activity of mammalian Cut proteins. In vitro, a purified preparation of CKII efficiently phosphorylated Cut repeats, causing an inhibition of DNA binding. In vivo, overexpression of the CKII alpha and beta causes a decrease in DNA binding by Cut. The CKII phosphorylation sites within the murine Cut (mCut) protein have been identified by in vitro mutagenesis as residues Ser400, Ser789, and Ser972 within Cut repeats 1, 2, and 3, respectively. Cut homeodomain proteins are known to function as transcriptional repressors. Overexpression of CKII reduces transcriptional repression by mCut, whereas a mutant mCut protein containing alanine substitutions at these sites is not affected. Altogether these results indicate that the transcriptional activity of Cut proteins is modulated by CKII (Coqueret, 1998).

Identification of the in vivo casein kinase II phosphorylation site within the homeodomain of the cardiac tisue-specifying homeobox gene product Csx/Nkx2.5

Csx/Nkx2.5 (see Drosophila Tinman) serves critical developmental functions in heart formation in vertebrates and nonvertebrates. The putative nuclear localization signal (NLS) of Csx/Nkx2.5 has been identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 is mutated, a significant amount of the cytoplasmically localized Csx/Nkx2.5 is unphosphorylated. This is in contrast to the Csx/Nkx2.5 that is localized to the nucleus; that is serine- and threonine-phosphorylated, and suggests that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicates that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, a Csx/Nkx2.5 kinase has been identified whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggest that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increases the DNA binding activity of Csx/Nkx2.5. Serine-to-alanine mutation at the CKII phosphorylation site reduces transcriptional activity when the carboxyl-terminal repressor domain is deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, since this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes (Kasahara, 1999).

Casein kinase II is required for efficient transcription by RNA polymerase III

Casein kinase II (CkII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, the role of CkII was assessed in specific transcription by RNA polymerases I, II, and III. CkII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CkII activity but low in extracts from a temperature-sensitive mutant that has decreased CkII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CkII. An inhibitor of CkII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CkII. Polymerase III transcription in vivo is impaired when CkII is inactivated. These results demonstrate that CkII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III (Hockman, 1996).

Phosphorylation of the C-terminal domain of yeast topoisomerase II by casein kinase II affects DNA-protein interaction

Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme that is essential for cell proliferation. This homodimeric enzyme catalyzes the cleavage and re-ligation of double-stranded DNA required to separate replicated sister chromatids. Both biochemical and genetic studies show that its catalytic activity is required for chromosome condensation and segregation, and that its decatenation activity can be stimulated by a variety of protein kinases in vitro. In budding yeast, topoisomerase II is most highly phosphorylated in metaphase, and casein kinase II (CkII) has been shown shown to be the major kinase modifying topoisomerase II. The effects of phosphorylation of yeast topoisomerase II by CkII were investigated in vitro. The phosphorylation of the C terminus of topoisomerase II by CkII appears to increase the stability of the complex formed with linear DNA fragments, while dephosphorylation has the opposite effect. Rephosphorylation of phosphatase-treated topoisomerase II by chicken casein kinase II restores a stable protein-DNA complex using a linear DNA fragment. The enhanced stability of the topoisomerase II-DNA complex is also observed with relaxed circular DNA, but not with supercoiled minicircles, in agreement with published results using topoisomerase II from Drosophila. Limited proteolysis and probing with domain-specific antibodies shows that, with the exception of a weakly modified residue between amino acid residues 660 and 1250, all residues modified by casein kinase II are in the last 180 amino acid residues of yeast topoisomerase II (Dang, 1994).

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Two adaptation-defective mutants have been identified that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype is entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. One mutation resides in CDC5, which encodes a polo-like kinase (see Drosophila Polo), whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II. It is likely that Cdc5p promotes checkpoint adaptation by inhibiting or bypassing the checkpoint pathway. The Cdc5p polo-like kinase has a role in activating Cdc25C, a conserved tyrosine phosphatase that removes an inhibitory phosphate on Cdc2. It may be that CKII acts to inhibit some part of the cell cycle arrest machinery that is not essential for maintaining the checkpoint arrest but is extremely important for maintaining viability during arrest (Toczyski, 1997).

CDC5 and CKII control adaptation to the yeast DNA damage checkpoint

The presence of fibroblast growth factor-2 (Fgf-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Fgf-2 added to nuclear extract binds to protein kinase Ck2 and nucleolin, a Ck2 natural substrate. Added to baculovirus-infected cell extracts overexpressing Ck2 or its isolated subunits, Fgf-2 binds to the enzyme through its regulatory beta subunit. Using purified proteins, Fgf-2 is shown to directly interact with Ck2 and to stimulate Ck2 activity toward nucleolin. A mitogenic-deficient Fgf-2 mutant protein has an impaired ability to interact with Ck2 and to stimulate Ck2 activity using nucleolin as substrate. It is proposed that in growing cells, one function of nuclear Fgf-2 is to modulate Ck2 activity through binding to its regulatory beta subunit (Bonnet, 1996).

asein kinase II binds to and phosphorylates cytoplasmic dynein

A 27-kDa protein was isolated that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yields a sequence that completely matches the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. It is propose that CkII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin (Karki, 1997).

Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52

FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein; it possesses peptidyl prolyl isomerase activity and produces a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. An examination was performed to determine if FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. Deletion mutants of FKBP52 were expressed to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region has been identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region is phosphorylated by CK2, and the peptide competitively inhibits the phosphorylation of other substrates by CK2. The [32P]phosphate labeling of FKBP52-expressing cells reveals that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 does not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 is not affected by the phosphorylation. Most important, CK2-phosphorylated FKBP52 does not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases (Miyata, 1997).

Constitutive phosphorylation of the acidic tails of the high mobility group 1 proteins by casein kinase II alters their conformation, stability, and DNA binding specificity

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. HMG1 proteins are abundant components of chromatin. A subfamily of the HMG proteins containing the HMG1 box domain (HMG1-BD) is widely distributed in eukaryotic cells from yeast to man. The members of this group are thought to have various functions related to modulation of transcription, DNA integration, and recombination. Since these proteins have an ability to induce strong bends and unwind DNA, they are called architectural components of chromatin. The most abundant of the HMG1 box proteins are the HMG1 and HMG2 proteins. They are composed of one or two HMG1-BDs, which are primarily responsible for contacts with DNA. HMG1-BDs are amino- and/or carboxyl-terminally flanked by stretches of positively or negatively charged residues. These regions modulate the binding affinity of HMG1-BDs, but do not influence the extent of DNA distortion. Deletion of these regions, in particular those of the negatively charged carboxyl-terminal tails, alters binding specificity of the HMG1 proteins. Moreover, the C-terminal portion of the HMG1 proteins is important for stimulation of transcription and nuclear retention (Wisniewski, 1999 and references).

Essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses reveal that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, dephosphorylation weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, it has been proposed that the detected phosphorylation is important for the proper function and turnover rates of these proteins. Since the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, these results are probably of general significance (Wisniewski, 1999).

CK2 is a c-terminal IkappaB kinase responsible for NF-kappaB activation during the UV response

NF-κB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-κB by many stimuli depends on the IκB kinase (IKK) complex, which phosphorylates IκBs at N-terminal sites, the mechanism of NF-κB activation by ultraviolet (UV) radiation remained enigmatic, since it is IKK independent. UV-induced NF-κB activation has been shown to depend on phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IκB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IκBα degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-κB axis is an important component of the mammalian UV response (Kato, 2003).

Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. This study reports the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. The significance of CK2 phosphorylation was confirmed by reporter assay, and a mechanism involving the process of phosphorylation acting as a molecular switch is proposed Finally, it is proposed that the SPOC domain functions as a phosphorylation binding module (Mikami, 2013).

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Search PubMed for articles about Drosophila CkIIalpha and CKIIbeta

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