From the Molecular Oncology Group, Departments of
** Medicine, §§ Oncology, and
Biochemistry, McGill University, Royal
Victoria Hospital, Montreal, Quebec H3A 1A1, Canada, and
¶ Department of Biochemistry, University of Western Ontario,
London, Ontario N6A 5C1, Canada
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ABSTRACT |
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The Drosophila and mammalian Cut
homeodomain proteins contain, in addition to the homeodomain, three
other DNA binding regions called Cut repeats. Cut-related proteins thus
belong to a distinct class of homeodomain proteins with multiple DNA
binding domains. Using nuclear extracts from mammalian cells,
Cut-specific DNA binding was increased following phosphatase treatment,
suggesting that endogenous Cut proteins are phosphorylated in
vivo. Sequence analysis of Cut repeats revealed the presence of
sequences that match the consensus phosphorylation site for casein
kinase II (CKII). Therefore, we investigated whether CKII can modulate
the activity of mammalian Cut proteins. In vitro, a
purified preparation of CKII efficiently phosphorylated Cut repeats
causing an inhibition of DNA binding. In vivo,
overexpression of the CKII and
caused a decrease in DNA binding
by Cut. The CKII phosphorylation sites within the murine Cut (mCut)
protein were identified by in vitro mutagenesis as residues
Ser400, Ser789, and Ser972 within
Cut repeat 1, 2, and 3, respectively. Cut homeodomain proteins were
previously shown to function as transcriptional repressors.
Overexpression of CKII reduced transcriptional repression by mCut,
whereas a mutant mCut protein containing alanine substitutions at these
sites was not affected. Altogether our results indicate that the
transcriptional activity of Cut proteins is modulated by CKII.
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INTRODUCTION |
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The cDNAs for several mammalian homologues of the Drosophila Cut homeodomain protein have recently been isolated (1-4). In humans, a Cut-related protein was independently identified either as a CCAAT displacement protein or as a protein that binds to an Sp1-like site within the c-Myc promoter and represses its expression (1, 2). Other mammalian Cut homologues isolated from dog, mouse, and rat were, respectively, termed Clox (Cut-like homeobox), Cux (Cut homeobox), and CDP-2, respectively (3, 4). More recently a second Cux gene, called Cux-2, was identified in mouse. In contrast to Cux-1, which is expressed in most tissues, Cux-2 was found to be expressed exclusively in nervous tissues (5). For matters of simplicity, the terms human and mouse Cut (hCut and mCut),1 or Cut when describing all Cut proteins, will be used in this manuscript.
Genetic evidence obtained in Drosophila indicates that Cut is an important determinant of cell fate (6). Viable as well as lethal Cut mutations have been described in Drosophila, and literature on this subject can be found as early as 1931 (7-13). Mutations result in defects in several tissues including the wings, legs, external sense organs, Malpighian tubules, tracheal system and some structures in the central nervous systems (10-13). In several tissues, defects caused by Cut mutations appear to result from the fact that some cells have enrolled in the wrong developmental program (6, 10-14). For example, in the peripheral nervous system, external sensory organs failed to develop in the absence of Cut. Instead, all precursor cells differentiated to form internal (chordotonal) sensory organs (15). In contrast, when Cut expression was artificially elevated in embryos, all precursor cells gave rise to external sensory organs (6). In mammals, the biological function of Cut proteins remains to be established; however, by analogy with other homeodomain proteins also conserved in evolution, it is likely that Cut proteins play an equally important role in determining cell type specificity.
At the molecular level, Cut proteins were found generally to function as transcriptional repressors. The human Cut protein (hCut) was found to bind to upstream regulatory sequences of the gp91-phox gene and the expression of this gene was shown to coincide with down-regulation of hCut binding activity upon differentiation of granulocytic cells (16-18). In co-transfection experiments, recombinant mammalian Cut proteins repressed transcription of reporter genes driven by the promoters of either the c-Myc, Ncam, or gp91-phox genes, as well as from a promoter in which Cut consensus binding sites had been inserted (2, 3, 16, 19). In two independent studies, however, Cut proteins appeared to function as transcriptional activators and it was suggested that the regulatory effect of Cut on transcription may vary depending on the proteins with which it interacts (4, 20). In particular, supershift assays using antibodies against pRb-related proteins suggested that Cut may interact with pRB and p107 (20).
From the analysis of their deduced amino acid sequences, mammalian Cut proteins are almost identical over their entire lengths (Fig. 1) (1-4). Sequence homology between Drosophila and mammalian Cut proteins is limited to five evolutionarily conserved domains, a region predicted to form a coiled-coil structure, three repeated regions called Cut repeats (CR), and a Cut-type homeodomain (HD) (Fig. 1) (1-4, 15). The high degree of conservation of Cut repeats suggested that they may have an important biochemical function. Indeed, we and others have demonstrated that Cut repeats can function as specific DNA binding domains (21-24). Moreover, we showed that at least Cut repeat 3 can cooperate with the Cut homeodomain to bind to DNA (CR3HD) (23, 24). Thus, Cut proteins form a novel class of homeodomain proteins that contain multiple DNA binding domains. Another region at the carboxyl terminus does not show amino acid sequence homology; however, both in Drosophila and mammals this region is rich in alanine and proline amino acids. We have identified two active repression domains within the carboxyl-terminal domain of hCut (R1 and R2) and we have shown that mammalian Cut proteins can down-regulate gene expression via two mechanisms, active repression and competition for binding site occupancy with activators like Sp1 and C/EBP (25).
Casein kinase II (CKII) is a serine/threonine kinase composed of
catalytic and
subunits and regulatory
subunits which combine to form a tetrameric holoenzyme (26, 27). CKII is conserved
among eukaryotes and disruption of the genes encoding the catalytic
subunits of CKII in Saccharomyces cerevisiae is lethal (28).
Microinjection of CKII antibodies into mammalian cells have
demonstrated that CKII is necessary both in the cytoplasm and the
nucleus for cell cycle progression (29). Although there are indications
that the activity of CKII is required at different stages in the cell
cycle, it is not clear whether CKII is itself regulated during the cell
cycle (29-31). Little is known about the regulation of CKII; however,
the enzyme does not seem to be modulated by cyclic nucleotides,
calcium, or lipids (31). CKII has been shown to phosphorylate a large
number of transcription factor. Depending on the particular protein
involved, phosphorylation by CKII has been found to affect
transcription factor activity by modulating either nuclear transport
(large T), DNA binding, positively (MEF2C) or negatively (c-Jun)
transactivation, repression (p53), or protein stability (I
B
)
(32-36).
Sequence comparison of Cut repeats from Drosophila and mammalian Cut proteins revealed the presence of conserved sequences that match the consensus phosphorylation site for protein kinase C and CKII. In a previous study, we demonstrated that protein kinase C can phosphorylate Cut repeats, in vitro and in vivo, causing a reduction in DNA binding and transcriptional repression (37). In the present study, we have investigated whether CKII can phosphorylate Cut repeats and modulate their activity.
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MATERIALS AND METHODS |
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Preparation of Bacterial Fusion Proteins-- Plasmid vectors expressing glutathione S-transferase (GST) Cut fusion proteins were introduced in Escherichia coli DH5. Induction of expression and purification of GST proteins were done as described previously (23, 38).
In Vitro Phosphorylation Reactions--
In vitro
phosphorylation reactions were performed by incubating 50 ng of GST
fusion proteins at 37 °C for 30 min in a 20-µl volume containing 2 µl of solution A (200 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 50 mM MgCl2,
0.3% Triton 100), 1 µl of [-32P]ATP (Amersham
Corp., 6000 Ci/mmol), and 10 ng of purified CKII (Life Technologies,
Inc.) (39, 40). Reactions were terminated by adding 3 µl of loading
buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl, pH 6.8, 0.1% bromphenol blue) and boiling for 5 min. Proteins were then
resolved on a 10% polyacrylamide gel followed by autoradiography. In
selected experiments, CKII was immunoprecipitated using 300 µg of
total NIH 3T3 cell extracts and a mixture of polyclonal antibodies
directed against the
and
subunit of CKII (39). These extracts
were then incubated for 15 min at 37 °C in the presence of GST or
GST/CR3HD (3 µg) in a 50-µl volume containing 5 µl of reaction
buffer (250 mM Tris-HCl, pH 7.5, 100 mM
MgCl2, 50 µM ATP) and 0.5 µl of
[
-32P]ATP (Amersham Corp., 6000 Ci/mmol). In selected
experiments, Cut proteins were immunoprecipitated together with CKII
using 1 mg of total cell extract and a mixture of different monoclonal antibodies directed against Cut. As we have observed that Cut proteins
are dephosphorylated at the end of
G1,2 extracts
were obtained from cells that had been maintained for 24 h in DMEM
plus 0.4% calf serum and then incubated in DMEM plus 10% fetal bovine
serum (FBS) for 3 h. The immunoprecipitates were then incubated in
the same conditions as described above with 0.5 µl of
[
-32P]ATP. Proteins were then resolved on a 10%
polyacrylamide gel followed by autoradiography.
Plasmid Constructions and Site-directed Mutagenesis-- The GST/CR1 and GST/CR3HD vectors have been described previously (23, 24). The GST/CR1 and GST/CR3HD mutants were created by substituting the codon for Ser400 and Ser972 with codons for Ala by site-directed mutagenesis according to the method of Deng and Nickoloff (41). The cDNA sequence for the murine Cut protein, Cux, can be obtained from GenBankTM, accession no. X75013 (3). The nucleotide and amino acid numbers used in the text are taken from this cDNA sequence and its deduced amino acid sequence. The mCut-expressing vector was prepared by inserting a fragment of Cux cDNA, from nucleotides 318 to 4575, into the mammalian expression vector pXM139 from the Genetic Institute. This cDNA fragment includes the entire coding sequence of Cux. The integrity of the cDNA sequence was confirmed by DNA sequencing. The mCut mutant, mCutmut, was created by substituting the codon for Ser400, Ser789, and Ser972 with codons for Ala. Mutations were confirmed by DNA sequencing. The CKII expressing vectors have been described elsewhere (42).
Transient Transfections and Preparation of Total Cell
Extracts--
NIH 3T3 cells were grown in DMEM medium supplemented
with 10% FBS. Cells were plated at a density of 3 × 105 cells per 100-mm plate 24 h prior to transfection.
All transfection experiments were repeated at least three times.
Transient transfection in NIH 3T3 were performed using the calcium
phosphate precipitation method. 5 µg of each plasmid were used per
plate, and the total amount of transfected DNA was kept constant by
addition of appropriate quantities of the parental expression vector.
Following transfection, cells were serum-starved for 24 h in DMEM
medium supplemented with 0.4% FBS. Cells were harvested with 1 ml of
TEN buffer (40 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl), and total extracts were prepared by resuspending
cell pellets in 30 µl of 10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM dithiothreitol. Cells were then
incubated for 10 min on ice, and total extracts were recovered by
centrifugation at 12,000 rpm for 10 min and retrieval of the
supernatant. Extracts were either used immediately or quick-frozen in a
dry ice-ethanol bath and stored at 80 °C.
Electrophoretic Mobility Shift Assays (EMSA)--
Bacterially
expressed proteins (50 ng) were preincubated for 5 min at room
temperature with 50 ng of poly(dI-dC) in 25 mM NaCl, 10 mM Tris, pH 7.5, 1 mM MgCl2, 5 mM EDTA, pH 8, 5% glycerol, and 1 mM
dithiothreitol. Total extracts prepared as described above were
incubated in the same buffer with a nonspecific competitor, either 1.5 µg of poly(dI-dC) for transfected cells or 0.5 µg of a polymerase
chain reaction-amplified random oligonucleotide for nontransfected
cells. In selected experiments, extracts were preincubated for 30 min
at room temperature with 2 units of calf intestinal phosphatase, and a
nonspecific competitor was added for the last 5 min of reaction. A
double-stranded nucleotide containing a Cut-consensus binding site
(upper strand, 5-AAAAGAAGCTTATCGATACCGT-3
) was end-labeled using the
Klenow polymerase and 10 pg of the probe (20,000 cpm) was then added to
the protein mixture for 15 min. Samples were then loaded on a 5%
polyacrylamide gel (30:1) and separated by electrophoresis at 8 V/cm
for 2 h in 50 mM Tris, 0.38 M glycine, 1 mM EDTA, pH 8.5. Gels were then dried and visualized by
autoradiography.
CAT Assays--
Transfected cells were harvested with 1 ml of
TEN buffer as described above, centrifuged, and resuspended in 100 µl
of 0.25 M Tris, pH 7.5. Cells were then subjected to three
cycles of freeze-thaw. Following centrifugation, cellular extracts were
recovered and used directly in CAT assays or stored frozen at
80 °C. CAT assays were performed as described previously (43) and
visualized by autoradiography.
Western Blot Analysis-- Protein extracts were recovered as described above and centrifuged, and 20 µg were resuspended in 40 µl of Laemli buffer. Proteins were then boiled for 5 mn and loaded onto 6% SDS-polyacrylamide gel. The gel was soaked for 10 min in a solution of 0.1 M Tris, 0.192 M glycine, 20% (v/v) methanol, and proteins were electrotransferred to nylon membranes for at least 6 h at 4 °C. Blots were then washed five times with TBS (10 mM Tris, pH 8, 150 mM NaCl) supplemented with 0.1% Tween (TBS, Tween 0.1%), and incubated overnight at 4 °C in TBS containing 5% milk and 2% bovine serum albumin to prevent nonspecific binding of the antibody. Following five washings, blots were then incubated with a polyclonal antibody directed against Cut diluted in TBS buffer (5% milk, 0.5% bovine serum albumin). After washings, membranes were incubated with a second antibody conjugated to horseradish peroxidase for 40 min at room temperature and washed five times in TBS, Tween 0.5%, one time in TBS, Tween 0.1% and one time in TBS. Proteins were then visualized using the ECL system of Amersham Corp. according to the instructions of the manufacturer.
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RESULTS |
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Bacterially Expressed Cut Repeats Are Phosphorylated in Vitro by CKII-- The evolutionarily conserved Cut repeats were previously shown to function as specific DNA binding domains either alone or in conjunction with the Cut homeodomain. The sequence alignment between Cut repeats of the murine and Drosophila melanogaster Cut proteins is presented in Fig. 1. It should be noted that, except for one amino acid, Cut repeat sequences are identical between human and mouse. Sequence analysis revealed the presence, in each of the Cut repeats, of consensus phosphorylation sites for CKII (Fig. 1). As a first step to investigate whether CKII can modulate Cut repeat activity, we verified if CKII can phosphorylate GST fusion proteins containing either Cut repeat 1 (GST/CR1) or Cut repeat 3 and homeodomain (GST/CR3HD). When incubated for 30 min in the presence of CKII and radiolabeled ATP, both GST/CR1 and GST/CR3HD were phosphorylated (Fig. 2, lanes 3 and 5). By comparison, GST alone was not phosphorylated by this enzyme (Fig. 2, lane 2). To confirm the identity of the phosphorylation sites, we prepared mutated GST/Cut repeat fusion proteins in which serines 400 and 972 within CR1 and CR3, respectively, were substituted with alanines, GST/CR1S400A and GST/CR3HDS972A. Following incubation with CKII, no phosphorylation of the mutated proteins was observed (Fig. 2, compare lanes 3 and 5 with lanes 4 and 6, respectively), confirming that Ser400 and Ser972 are the sites phosphorylated by CKII in vitro.
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In Vitro Phosphorylation by CKII Decreases DNA Binding by Cut-- The addition of a negative charge within Cut repeat DNA binding domains would be predicted to cause a reduction in DNA binding. To test this hypothesis, GST/Cut repeat fusion proteins were first incubated in the presence of CKII, with or without and cold ATP, and then tested in an EMSA using oligonucleotides encoding a Cut binding site. CKII by itself (without ATP) had no effect on DNA binding (Fig. 3, lanes 1 and 5, and data not shown). Phosphorylation of GST/CR1 and GST/CR3HD by CKII significantly reduced the DNA binding activity of the Cut proteins (Fig. 3, compare lanes 1 and 5 with lanes 2 and 6, respectively). By contrast, no reduction in DNA binding was observed when the mutated proteins GST/CR1S400A and GST/CR3HDS972A were tested (Fig. 3, compare lanes 3 and 4 and lanes 7 and 8). We conclude that CKII-mediated phosphorylation of Ser400 and Ser972 in Cut repeats 1 and 3, respectively, leads to a reduction in DNA binding.
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Cut Is Phosphorylated in Vivo-- In preliminary experiments, Cut DNA binding activity was found to be very weak in cells synchronized in early G1, although the protein itself was detected by Western blot analysis (data not shown). We hypothesized that Cut DNA binding in these cells could be inhibited by phosphorylation. To verify this hypothesis, total cell extracts were treated with calf intestinal phosphatase and then tested in EMSA. An increase in Cut DNA binding activity was observed following phosphatase treatment (Fig. 4A, compare lane 1 with lane 2). The retarded complex was specific for Cut since it was inhibited in the presence of monoclonal antibodies directed against Cut proteins. A similar increase in Cut DNA binding following phosphatase treatment was also observed using extracts from exponentially growing cells, however, the increase was the most substantial with cells maintained in low serum for few days essentially because basal Cut DNA binding was the lowest in these cells (data not shown). Interestingly, Cut DNA binding activity of dephosphorylated extract was decreased following a further treatment with purified protein kinase CKII (data not shown). We therefore performed additional experiments to investigate whether CKII could be one of the kinases responsible for Cut phosphorylation. CKII was immunoprecipitated from cell extracts and incubated for 15 min in the presence of GST/CR3HD and radiolabeled ATP. GST/CR3HD, but not GST alone, was efficiently phosphorylated (Fig. 4B, compare lanes 1 and 2). We next verified whether CKII could phosphorylate the endogenous Cut protein. CKII and Cut proteins were immunoprecipitated from total cell extracts and then incubated for 15 min in the presence of radiolabeled ATP. As seen in Fig. 4C, the endogenous Cut protein was phosphorylated in these conditions.
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Forced Expression of CKII Causes a Decrease in Cut DNA
Binding--
We investigated whether overexpression of CKII would
affect DNA binding by Cut. NIH 3T3 cells were cotransfected with
vectors encoding the CKII and
subunits and a plasmid expressing
either the entire murine Cut protein (mCut) or only the CR3HD region. The next day, the medium was changed for DMEM containing 0.4% calf
serum. Twenty-four hours later, total cell extracts were prepared and
tested in EMSA using oligonucleotides encoding a Cut-binding site. In
this system, a retarded complex was generated by the exogenously
expressed Cut proteins. This was demonstrated by the following facts.
No such complex was observed in untransfected cells (Fig. 4 and data
not shown). The retarded complex did not appear when the reaction took
place in the presence of a specific anti-Cut antibody, while an
unrelated antibody had no effect on this complex (Fig.
5, lanes 3 and 4).
Cotransfection of CKII expressing vectors significantly reduced DNA
binding activity by mCut or the CR3HD protein (compare lane
5 with 6, and lane 7 with 8). In
contrast, DNA binding was not affected by CKII overexpression when we
tested the mutant CR3HDS972A, in which Ser972
was replaced with alanine (Fig. 5, lanes 9 and
10). Similarly, CKII overexpression did not affect DNA
binding by the mutant mCutmut, in which Ser400,
Ser789, and Ser972 were replaced with alanine
residues (Fig. 5, lanes 1 and 2). Western blots
were performed on extracts from transfected cells expressing mCut and
mCutmut. The wild-type and mutated proteins were expressed
at comparable levels in the presence or absence of CKII expressing
vectors, suggesting that the varying levels of DNA binding were not a
result of differences in protein stabilities, but were most likely due to post-translational modifications (Fig. 5B).
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CKII Activation Inhibits Cut Repression Activity in Vivo-- Mammalian Cut proteins have been found to function as transcriptional repressors (2, 3, 16, 18, 19, 25). We have shown that the murine and human Cut proteins can repress the herpes simplex minimal thymidine kinase (HSV-tk) promoter by occupying the Sp1 and CCAAT sites upstream of the TATA box (25). Since CKII overexpression caused a reduction in mCut DNA binding, we hypothesized that it should also interfere with the repression function of mCut. To verify this, NIH 3T3 cells were transfected with a tkCAT reporter plasmid in the presence or absence of vectors expressing CKII and mCut proteins. Expression of tkCAT was repressed by mCut as previously reported, whereas CKII by itself had no effect (Fig. 6). When both mCut and CKII were co-expressed, no repression was observed (Fig. 6). In contrast, CKII had no effect on repression mediated by the mutant mCutmut (Fig. 6). As before, Western blots were performed on extracts from transfected cells to confirm that mCut and mCutmut were expressing at similar levels in the presence or absence of CKII vectors. Altogether, these results suggest that CKII phosphorylate Cut repeats causing a reduction in DNA binding and repression activity.
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DISCUSSION |
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In the present study, we have investigated the functional significance of putative CKII phosphorylation sites in Cut repeat DNA binding domains. The consensus phosphorylation site for CKII is (S/T)XX(D/E). Each of the three Cut repeats in Cut proteins from human, mouse, rat, dog, and D. melanogaster contain the sequence (S/T)VS(D/E). Moreover, in human a novel transcription factor, hepatocyte nuclear factor 6, has been found to contain a single Cut repeat and a Cut-type homeodomain (44). Hepatocyte nuclear factor 6 contains the sequence TLSD at a similar position within its single Cut repeat. The importance of these sites for modulation of Cut repeat activity is supported by the fact they are conserved in all Cut repeats of the Cut family.
We have shown that CKII in vitro can phosphorylate GST/CR1
and GST/CR3HD fusion proteins and that replacement of the first serine
for alanine in the sequence SVSD/E abolished the ability of
CKII to phosphorylate Cut repeats 1 and 3. Phosphorylation at this
position introduces a negative charge within the Cut repeat DNA binding
domain, and this would be expected to reduce electrostatic interactions
with DNA. Indeed, DNA binding by GST/CR1 and GST/CR3HD was decreased
following phosphorylation with CKII. Our results also indicate that DNA
binding by the murine Cut protein is inhibited by phosphorylation since
DNA binding was increased following treatment of nuclear extracts with
calf intestinal phosphatase. In cotransfection studies, overexpression
of CKII subunits and
reduced DNA binding by wild type mCut but
not by the mutant mCutmut, in which Ser400,
Ser789, and Ser987 were replaced with alanine
residues. These results suggested that CKII can phosphorylate mCut in
cells and that the phosphorylation sites correspond to those predicted
from sequence analysis. However, it is difficult to definitively
establish whether a site is indeed phosphorylated by a specific kinase
in vivo. Our mutagenesis and cotransfection experiments
suggest that CKII is likely to phosphorylate Cut repeats, but it is
also formally possible that another kinase acts on these sites in
vivo.
We have not been able to test the effect of CKII-mediated phosphorylation on a GST/Cut repeat 2 (GST/CR2) fusion protein. Although other groups have reported that GST/Cut repeat 2 can bind to DNA, we have not been able to reproduce these results despite many attempts with several independent constructs (21, 22). The reason for this discrepancy is currently unknown. We have been able, however, to phosphorylate GST/CR2 with CKII in vitro (data not shown).
Several reports have implicated growth factors in the activation of
CKII (45-48). However, in some recent studies, alterations in CKII
activity are not routinely observed following growth factor stimulation
(49, 50). We have compared Cut DNA binding in G0 and early
G1 to verify whether there is a decrease in DNA binding that could be ascribed to CKII activation at the start of
G1. To our surprise, our results indicated that Cut DNA
binding is almost undetectable in G0 and early
G1, although the protein is clearly expressed (Fig. 4 and
data not shown). We have detected an increase in Cut DNA binding later
in the cell cycle, at a time that corresponds to entry into S phase.
Using antibodies specific for CKII and
subunits, we have
immunoprecipitated CKII from cells in different phases of the cell
cycle and tested its ability to phosphorylate GST/Cut repeat fusion
proteins in
vitro.3 Since we did not
observe any significant difference in CKII activity, we hypothesize
that CKII sites in Cut repeats are constitutively phosphorylated and
that increase in Cut DNA binding activity is in fact the result of a
change in the activity of a phosphatase.
Previous studies have established that Cut repeats can function as specific DNA binding domains, either independently (CR1 and CR2) or in cooperation with the Cut homeodomain (CR3HD) (21-24). Other homeodomain proteins, the Paired, Pou, and LIM domain proteins, have been found to contain a bipartite DNA binding domain (51-56). However, Cut proteins represent an unusual class of transcription factors in that they contain multiple DNA binding domains, CR1, CR2, and the bipartite Cut repeat 3/homeodomain (CR3HD). How native Cut proteins bind to DNA is not yet entirely understood, but the available data permit few predictions to be made. Individual Cut repeats were found to bind to DNA when expressed as part of a dimeric fusion protein (GST/CR) but not as monomers (maltose binding protein (MBP)/CR) (19, 21, 23, 24). Since GST fusion proteins exist as dimers and MBP as monomers, we can assume that only dimers of Cut repeat 1 or 2 can bind to DNA with high affinity. In contrast, a monomer of CR3HD, linked to either the MBP or a histidine tag, was able to bind to DNA. Moreover, the Cut homeodomain on its own did not exhibit DNA binding specificity, but it was capable of specific and high affinity DNA binding when acting in conjunction with CR3 (24). Thus, it is likely that in the native Cut protein, Cut repeat 3 forms a bipartite DNA binding domain together with the Cut homeodomain. This composite domain would drive the first interaction with DNA and cooperative DNA binding would then allow Cut repeat 1 and 2 to make a stable interaction with other sites. It is also possible that Cut repeats 1 and 2 interact together to form another bipartite DNA binding domain. Yet another possibility is that not only CR3 but also CR1 and CR2 may have the potential to interact with the Cut homeodomain to form a bipartite DNA binding domain. Comparison of the properties of the three Cut repeats revealed that individual Cut repeats exhibit overlapping, yet distinct, DNA binding specificities. Thus, depending on the specific modes of DNA binding that prevail in various situations, Cut proteins may bind to different targets. How would phosphorylation by CKII affect these various modes of interaction with DNA? Since CKII phosphorylation sites are present within each of the three Cut repeats, we can reasonably assume that interaction with DNA is going to be inhibited no matter what mode of interaction is chosen. Moreover, we would consider it unlikely that phosphorylation by CKII would favor one mode DNA binding over another. Clearly, additional studies are necessary to fully understand how Cut proteins bind to DNA. Yet, results of the present studies strongly suggest that CKII is an important modulator of Cut DNA binding.
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ACKNOWLEDGEMENTS |
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We are grateful to Bruno Luckow for the gift of the BL5CAT (tkCAT) plasmid and Dr. J. Fred Mushinski for the gift of NIH 3T3 cells.
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FOOTNOTES |
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* This research was supported in part by Medical Research Council of Canada Grant MT-11590 and National Cancer Institute of Canada Grant 3497 (to A. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from The Royal Victoria Hospital Department of Medicine and Research Institute.
Research Scientist of the National Cancer Institute of
Canada.
¶¶
Recipient of a scholarship from the Fonds de la
Recherche en Santé du Québec. To whom correspondence should
be addressed: Molecular Oncology Group, Room H-5.08, McGill
University, 687 Pine Ave. West, Montreal, Quebec, Canada, H3A 1A1.
Tel.: 514-842-1231 (ext. 5832); Fax: 514-843-1478; E-mail:
alain{at}lan1.molonc.mcgill.ca.
1 The abbreviations used are: hCut, human Cut; mCut, murine Cut; CR, Cut repeat; HD, homeodomain; CKII, casein kinase II; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase; MBP, maltose-binding protein.
2 O. Coqueret, G. Bérubé, and A. Nepveu, submitted for publication.
3 O. Coqueret and A. Nepveu, unpublished observations.
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REFERENCES |
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