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).

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).

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).

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).

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).

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).

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).

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).

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).

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 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).

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).

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).

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).