Casein kinase II
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
Nspl. 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 Nspl 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 Nspl 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 Nspl backgrounds
provide evidence that both substitutions in Tik affect
proper CK2 functions. How might one interpret the
effects on Nspl? 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. Nspl only
perturbs the latter. Specifically, Nspl 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 Nspl perpetuates throughout retinal histogenesis,
and elicits the rough and reduced eye of Nspl. Consistent
with the notion that this allele renders R8’s sensitive to inhibitory
N signaling, the retinal defect of Nspl 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 Nspl 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 Nspl 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 Nspl. 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 Nspl, 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 Nspl, 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 Nspl. 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 Nspl. 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).
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 (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 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 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. 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 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).
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).
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).
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 rhd1-12 or the synthetic lethal interaction between e(r)p2 and the Notch allele Nnd-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).
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, CK2αTik (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 timUL 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 timUL 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 Dbtg 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 timUL 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 timUL mutant, near CK2 sites inhibits CK2 phosphorylation of the target Ser/Thr residue. timUL and Tik show allele-specific genetic interactions such that in timUL mutants, Tik period lengthening is partially suppressed. These results are consistent with the hypothesis that timUL 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 timUL 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 timUL 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 timUL, so the possibility that there is also an early-night defect in timUL cannot be ruled out. Therefore it is possible that timUL 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 timUL argue that timUL 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).
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).
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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