Casein kinase II
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: 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).
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).
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).
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 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 km values for the protein
substrate, the Kcat 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 Kcat with marginal effects on the
km. 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).
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 pers (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 pers. In a per+ or perl (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 (234240) 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, 012) levels of Per in the CK2alphaTik 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 CK2alphaTik mutants is delayed by approximately 3 h relative to wild type, consistent with the period-lengthening effect of CK2alphaTik mutants. Disappearance of Tim is also more strongly affected than accumulation in CK2alphaTik mutants. The Per and Tim profiles of CK2alphaTikR 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 CK2alphaTik and CK2alphaTikR 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 CK2alphaTik and CK2alphaTikR were observed, consistent with biochemical data on recombinant proteins and their recessive lethality. These data support the model that CK2alphaTikR is a strong loss-of-function allele, lacking the strong dominant behavioral and biochemical effects of CK2alphaTik (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 11159) 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. CK2alphaTik 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 CK2alphaTik 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 7322) 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).
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).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D. The Interactive Fly resides on the
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).
Casein kinase II:
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
| References
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