Departments of 2 Medicine and 3 Anatomy and Structural Biology and 1 Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Casein kinase 2 (CK2) is a
tetrameric enzyme constitutively expressed in all eukaryotic tissues.
The two known isoforms of the catalytic subunit, CK2 and CK2
',
have been reported to have distinct tissue-dependent subcellular
distributions. We recently described a third isoform of the catalytic
subunit, designated CK2
", which is highly expressed in liver.
Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as
immunofluorescent microscopy revealed that CK2
" was exclusively
localized to the nucleus and preferentially associated with the nuclear
matrix. CK2
and CK2
' were found in nuclear, membrane, and
cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2
" sequence resulted in release of the truncated green fluorescent protein fusion protein from the nuclear matrix and redistribution to both the nucleus and the cytoplasm. Demonstration that the carboxy terminus is necessary but not sufficient for nuclear
retention indicates that the underlying mechanism of CK2
" nuclear
localization is dependent on the secondary structure of the holoenzyme
directed by the carboxy-terminal sequence.
nuclear matrix association; human hepatoma cell line HuH-7; casein kinase 2-green fluorescent protein fusion protein
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INTRODUCTION |
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CASEIN KINASE 2 (CK2;
formerly referred to as casein kinase II) is a highly conserved and
ubiquitously expressed tetrameric enzyme that phosphorylates
serine/threonine residues and is essential for the viability of
eukaryotic cells (41). The tetrameric CK2 holoenzyme
consists of two -subunits, carrying the catalytic activity, and two
-subunits, which have a stabilizing and regulatory function required
for maximal activity and regulation of substrate specificity
(54). Both subunits are constitutively expressed in most
human tissues. To date, more than 150 different proteins in various
tissues and species have been described as potential phosphorylation
targets of CK2 (41). It has become clear that CK2 plays an
important role in regulating physiological and pathological cellular
processes such as cancer development (44) signal
transduction (48), transcriptional control
(15), proliferation (18), and cell cycle
control (28). Although the three-dimensional structure of
CK2 suggests that relatively simple biochemical mechanisms regulate the
substrate specificity of this kinase (14), the mechanisms
that govern its cellular activity have remained largely unknown.
Until recently, eukaryotic cells were thought to contain two isoforms
of the catalytic CK2 subunit, termed CK2 and CK2
', both of which
are widely distributed in different species and tissues. The enzymatic
activities of these two isoforms are equivalent with respect to the
potential tetrameric homo- and heterodimers (
,
'
,
'
'
), and a specific function of the isoforms has not been discovered. A third, novel isoform of CK2
, designated CK2
", which is involved in hepatocellular membrane trafficking, was
recently described (47). The newly cloned CK2
" is
almost identical to the amino acid sequence of CK2
until the
carboxy-terminal 32 amino acids, which appear to be completely
unrelated. The unique CK2
" sequence was previously reported within
genomic CK2
clone RP5-863C7 (gi:5788437) as an intronic repeat
region or Alu sequence (8, 55). The presence of a rarely
translated Alu cassette and the remnants of a poly A tail in the cDNA
suggests that CK2
" is either a CK2
-derived retroposon (4,
33) or the result of alternative splicing, selectively including
an Alu-like exon into the mature mRNA (3, 56).
All three catalytic isoforms exhibit a high degree of identity
(74-90%) with notable differences limited to their
carboxy-terminal regions. The carboxy-terminal 60 amino acids of CK2
are completely unrelated to the carboxy-terminal domain of CK2
' and
differ substantially from CK2
" within the last 35 amino acids. This
indicates that a potential functional difference of the three isoforms
would be very likely related to the carboxy terminus. Experiments with truncated isoforms suggested that the carboxy-terminal ends of CK2
and CK2
' did not considerably influence the enzymatic activity (14) or subcellular localization (39).
Although there appears to be no effect of the CK2
" carboxy terminus
on enzymatic activity, the present study shows that the CK2
"
carboxy-terminal 35 amino acids are involved in the regulation of its
subcellular localization, which might be the key to understanding the
differential function of this isoform (47).
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MATERIALS AND METHODS |
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Cell culture. Cell culture reagents were all obtained from GIBCO-Life Technologies (Rockville, MD), plasticware from Becton Dickinson (Franklin Lakes, NJ), and all other chemicals from Sigma (St. Louis, MO) unless otherwise indicated. HuH-7 cells were cultured in minimal essential medium (MEM) containing 50 µg/ml gentamicin and 10% fetal bovine serum (FBS; Gemini). Primary human hepatocytes were prepared as described previously (62) and maintained in DMEM plus gentamicin, 10% FBS, 10 µg/ml insulin, and 10 µg/ml dexamethasone. The tissues were obtained from ongoing programs at Albert Einstein College of Medicine under approval from the institutional Committee on Clinical Investigations.
Subcellular fractionation by discontinuous sucrose gradient
centrifugation.
Cell fractionation and isolation of intact nuclei via discontinuous
sucrose gradient centrifugation was performed according to standard
protocols (2). Briefly, HuH-7 cells, grown in a 100-mm
culture dish, were scraped into 1 ml of PBS containing 1 mM
dithiothreitol DTT and protease inhibitors (protease inhibitor cocktail, Sigma). Cells were homogenized for 30 s at low speed (Tissue Tearor; Biospec Products, Bartlesville, OK). Successful cell
lysis without disruption of the nuclei was confirmed by phase-contrast microscopy. The homogenate was layered on top of a 25-60% (wt/vol) discontinuous sucrose gradient and centrifuged for 1 h at 35,000 g (SW41 rotor; Beckman, Palo Alto, CA). After
centrifugation, the top layer was removed and centrifuged for 1 h
at 100,000 g. The supernatant after this second
centrifugation was designated as the "cytoplasmic" fraction and
stored at 80°C until further use. The interface between the 25%
and 60% layers was collected as the crude membrane/microsomal
fraction. The nuclei pelleted below the 60% sucrose cushion were
either extracted as described in Preparation of nuclear
extracts or lysed by sonication in 2× SDS sample buffer.
Preparation of nuclear extracts.
Nuclear extracts were prepared essentially as described previously
(7), with the following modifications. HuH-7 or Trf1 cells
were grown in 100-mm tissue culture dishes and scraped into 1 ml of
ice-cold hypotonic lysis buffer [10 mM HEPES, pH 7.4, 10 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1× protease inhibitor cocktail, 0.4% Nonidet P-40
(NP-40)]. Complete lysis, as assessed by light microscopy, was
achieved by pipetting harvested cells up and down five times through a
1-ml pipette tip (USA Scientifics, Ocala, FL). Lysates were centrifuged
at low speed (800 g) for 3 min at 4°C. The supernatant was
ultracentrifuged (100,000 g, 1 h, 4°C), and the
resulting supernatant was considered as the cytosolic fraction. The
nuclear pellet from the initial "low-speed" centrifugation was
washed once with 1 ml of lysis buffer without detergent and centrifuged
again at 800 g for 3 min. The pellet was then resuspended in
40-60 µl of a high-salt extraction buffer (20 mM HEPES, 400 mM
NaCl, 1 mM EDTA, 1 mM DTT, 1× protease inhibitor cocktail) with or
without detergent (1% NP-40) and extracted for 30 min at 4°C with
constant mixing. The samples were centrifuged for 20 min at 20,000 g. The resulting supernatants were designated as the nuclear
extracts and stored at 80°C until further use.
Preparation of nuclear matrix.
Preparation of nuclear matrix (NM) was performed as described
previously with modifications (61). To prepare nuclear
extracts, HuH-7 and Trf1 cells were scraped into 1 ml of hypotonic
lysis buffer consisting of 10 mM HEPES, pH 7.4, 10 mM NaCl, 0.1 mM
EDTA, 1 mM DTT, 1× protease inhibitor cocktail, and 0.4% NP-40. The lysate was centrifuged at 800 g and 4°C for 5 min. The
resulting nuclear pellet was resuspended in 1 ml of NM extraction
buffer (10 mM Tris · HCl, pH 7.4, 1% Tween 40, 0.5% sodium
deoxycholate, 10 mM NaCl, 4 mM vanadyl ribonucleoside complex, and 1×
protease inhibitor cocktail) to separate the matrix from the
nucleoplasm and other non-NM-associated nuclear proteins and incubated
10 min at 4°C with gentle rocking. The samples were centrifuged as above, and the pellet was suspended in NM digestion buffer (10 mM
PIPES, pH 6.8, 0.5% Triton X-100, 50 mM NaCl, 0.3 M sucrose, 3 mM
MgCl2, 1 mM EGTA, 4 mM vanadyl ribonucleoside complex, 1× protease inhibitor cocktail, 100 µg/ml RNase A, and 100 µg/ml DNase
I; Roche Molecular Biochemicals, Mannheim, Germany) and incubated for
60 min at room temperature. The digested sample was centrifuged again
at low speed (600 g and 4°C for 5 min), and the final
pellet was suspended in NM resuspension buffer (50 mM
Tris · HCl, pH 7.9, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1× protease inhibitor cocktail) as the NM and stored at 80°C.
Immunofluorescence.
HuH-7 cells were cultured in 35-mm dishes containing microscope
coverslips that were coated with collagen I (Sigma). At 70-80% confluence, cells were washed twice with ice-cold PBS and fixed for 10 min in PBS containing 4% paraformaldehyde at 4°C. Coverslips were
removed from the dishes, rinsed four times with cold PBS, and incubated
10 min with 0.05% Triton X-100 to permeabilize cell membranes. Cells
were rinsed as before and then blocked by incubation for 2 h in
blocking buffer consisting of PBS, 2% donkey serum, 1% BSA, and 0.1%
Tween 20. Cells were then incubated at 4°C overnight with 1:100
dilutions of antibodies against the different CK2 isoforms. After
incubation with primary antibody, cells were rinsed four times with PBS
and were incubated 1 h at room temperature with 1:100 diluted
Cy3-labeled secondary antibody (donkey anti-rabbit; Jackson
Laboratories, West Grove, PA) in blocking buffer. After being rinsed an
additional four times with PBS cells were mounted on a microscope
slide, and the edges of the coverslips were sealed with clear nail
polish. The subcellular distribution of fluorescent proteins was
determined with an Olympus IX70 fluorescent microscope equipped with a
charge-coupled device (CCD) digital camera.
Western blot analysis.
Western blot analysis of CK2 isoforms was performed by resolving
30-60 µg of cytoplasmic or nuclear protein extracts by
SDS-polyacrylamide gel electrophoresis (PAGE). The gel was blotted to
nitrocellulose membranes (Bio-Rad, Hercules, CA) with a semi-dry
blotting chamber (Bio-Rad). The membrane was blocked overnight in PBST
(PBS + 0.1% Tween 20)-10% nonfat dry milk and then incubated for
90 min with isoform-specific polyclonal rabbit antibodies, each diluted
1:3,000 in PBST plus 2% nonfat dry milk. The antibodies against
CK2
, -
', and -
were a gift from Dr. David W. Litchfield
(University of Manitoba, Winnipeg, MB, Canada; Refs. 27,
28, 39). The antibody against the carboxy
terminus of CK2
" was described recently (47). After
being washed three times for 10 min with PBST, the membrane was
incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit
antibody (Life Technologies) diluted 1:20,000 in PBST-2% nonfat dry
milk. The membrane was washed as before. Signals were visualized by
chemiluminescence (West Pico Kit; Pierce, Rockford, IL).
Construction and analysis of green fluorescent protein fusion
proteins.
Different parts of the open reading frame (ORF) of the CK2" cDNA
without 5'- and 3'-untranslated regions was amplified by "high-fidelity" PCR (High Fidelity PCR Mix; Life Technologies) utilizing a forward primer that contained an EcoRI (Promega,
Madison, WI) and a reverse primer with an ApaI (Promega) cut
site. The PCR reaction (200 µl) was ethanol precipitated, digested
with these enzymes (1 h, 37°C), and subsequently gel purified. The fragment was then cloned in frame downstream of green fluorescent protein (GFP) into the plasmid vector pEGFP-C1 (Clonetech, Palo Alto,
CA). Before the in vitro ligation (T4 ligase; Life Technologies), the
plasmid was linearized with EcoRI/ApaI,
dephosphorylated (alkaline phosphatase; Roche Diagnostics) and gel
purified. Five microliters of the ligation reaction was used to
transform DH5
-competent cells (Life Technologies), which were plated
on LB plates containing 50 µg/ml kanamycin. Clones of the human CK2
and CK2
" were kindly provided by Dr. David W. Litchfield. Plasmid
DNA was prepared with the Qiafilter Midi Prep kit (Qiagen, Valencia,
CA). Plasmid DNA (0.5 µg) was transfected into HuH-7 cells at 60%
confluence with the Lipofectamine Plus reagent (Life Technologies).
After 36-40 h, cells were trypsinized and replated on MatTek
culture dishes containing a 0.17-mm coverslip in the bottom (MatTek,
Ashland, MA). After an additional 24 h of incubation, dishes with
living cells were analyzed for GFP fusion protein expression with an Olympus IX70 fluorescent microscope equipped with a CCD digital camera.
Kinase assay of immunoprecipitated GFP fusion proteins.
For immunoprecipitation of expressed GFP-CK2" fusion proteins (± carboxy-terminal 32 amino acids), HuH-7 cells were lysed with RIPA
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5%
sodium deoxycholate, 1 mM EDTA, and protease inhibitor cocktail; Sigma)
48 h after transient transfection. Anti-GFP antibody (Abcam,
Cambridge, UK) was attached to immobilized protein A/G (Pierce) and
incubated with transfected cell lysates for 1 h with constant
mixing at 4°C. The A/G beads were washed three times with RIPA buffer
and then twice with kinase assay buffer (in mM: 50 Tris-Cl, pH 7.4, 10 MgCl2, and 1 DTT). After the washed immune complexes were
resuspended in kinase buffer, CK2 activity was measured with the
CK2-specific synthetic peptide RRADDSDDDDD (Calbiochem, San Diego, CA)
as substrate. Kinase reactions contained (in mM) 50 Tris-Cl, pH 7.4, 150 NaCl, 10 MgCl2, 0.1 [
-32P]ATP
(specific activity 200-500 cpm/pmol), and 0.1 synthetic peptide.
Reactions were initiated by the addition of immune complex and
terminated after 30 min at 30°C by spotting an aliquot (10 µl) of
the reaction mix on phosphocellulose paper (P81; Whatman) followed by
four wash steps with 1% phosphoric acid as described previously
(13). Less than one-tenth of the kinase activity immunoprecipitated from cells transfected with pEGFP-CK2
" constructs was detected in cells transfected with pEGFP alone, which served as the
negative control.
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RESULTS |
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Subcellular localization of CK2" in hepatocytes is exclusively
nuclear.
To determine the subcellular localization of CK2
", we fractionated
HuH-7 cell homogenates on a discontinuous sucrose gradient. This
protocol resulted in three fractions, a cytosolic fraction, a crude
cellular membrane fraction, and a nuclear fraction. The nuclear pellet
was lysed by sonication in 2× SDS sample buffer, and equal amounts of
cytosolic, membrane, and nuclear proteins were resolved on SDS-PAGE.
The gel was blotted to a nitrocellulose membrane and probed with
antibodies against CK2
", CK2
, or CK2
' (Fig.
1). The immunoblot revealed that in HuH-7
cells, CK2
" exhibits an exclusive nuclear localization. In contrast,
the two other catalytic subunit isoforms, CK2
and CK2
', were
distributed throughout all three of these compartments. This result was
confirmed by an indirect immunofluorescence assay, in which HuH-7 cells
were probed with antibodies against CK2
" and CK2
. As shown in
Fig. 2, anti-CK2
" stained nuclei of
the cells, whereas anti-CK2
stained both the nuclei and the
cytoplasm. Probing with anti-CK2
' showed a staining pattern similar
to that obtained with anti-CK2
(data not shown). To rule out the
possibility that the CK2
" isoform is a HuH-7-specific variant,
nuclear extracts and cytosol were prepared from primary human
hepatocytes. As illustrated in Fig. 3,
CK2
" was detected in the nuclei of isolated human hepatocytes but
not in the cytosol, whereas the CK2
isoform was found in both
compartments. This isoform distribution was equivalent to that seen in
HuH-7 cells.
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CK2" is associated with nuclear matrix.
Because CK2 has been described to be associated with the NM of various
cell types (9), we examined whether CK2
" in HuH-7 cells
was bound to the NM or whether it was preferentially a nucleoplasmic protein. CK2
" as well as CK2
, CK2
', and CK2
were detected in the NM fraction (Fig. 4A).
The selective enrichment of NM protein in this fraction was
demonstrated by the presence of lamin B (Fig. 4B), a widely
accepted marker for in vitro NM protein preparations (1, 36,
63). All the subunits were also detected in nuclear extracts
when prepared in the presence of detergent. Removal of detergent from
the extraction buffer, decreasing the probability of releasing
matrix-bound proteins, led to a complete loss of detectable CK 2
"
but not of CK2
, CK2
', and CK2
. Together, these results
suggested that CK2
" is tightly associated with the NM of HuH-7
cells, whereas the other CK2 subunits also exist in the nucleoplasm as
well as in the cytoplasm of these cells. The nuclear extracts and the
NM of Trf1 cells, an HuH-7-derived mutant cell line that has recently
been described to be CK2
" deficient (47), served as a
negative control and showed no detectable CK 2
" but equivalent
amounts of CK2
. The fact that Trf1 nuclear extracts and matrix
tested negative for CK2
" also demonstrated the specificity of the
antibodies.
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Isoform-specific carboxy terminus of CK2" determines nuclear
localization.
CK2
" and CK2
have a common region that extends over 92% of the
entire ORF. The amino acid identity between the two isoforms in this
common region exceeds 99% (single amino acid mismatch at position 127;
Fig. 5). The sequence difference between
the isoforms is solely due to the last 32 carboxy-terminal amino acids of CK2
" and 38 amino acids of CK2
, respectively. These carboxy termini appear to be unrelated. Therefore, we hypothesized that the
tight association of CK2
" with elements of the NM might be determined by its carboxy terminus. To address this possibility, the
cDNAs encoding CK2
" with or without the last 32 amino acids of the
ORF as well as the CK2
" carboxy-terminal 32 amino acids were cloned
in frame downstream (carboxy terminal) of GFP into the expression
vector pEGFP-C1. Fusion protein expression on transient transfection of
HuH-7 cells was confirmed by immunoblot analysis of cytosolic and
nuclear extracts with anti-GFP antibody (Fig. 6) and recovery of kinase activity in
anti-GFP immunoprecipitates from whole cell lysates. Transfection with
pEGFP alone served as a control, and the expressed GFP was detected in
both cytosol and nuclei as a major band resolving at ~35 kDa on
SDS-PAGE. The fusion protein containing the full-length CK2
" ORF
(EGFP-CK2
"FL) was detected mainly in the nuclear
extracts, whereas the fusion protein with the deleted carboxy terminus
CK2
" ORF (EGFP-CK2
"
32) as well as the fusion
protein containing only the carboxy-terminal 32 amino acids
(EGFP-CK2
"C-term) were evenly distributed in cytosol and
nuclei. These results were confirmed by fluorescent microscopy. GFP
alone showed no particular distribution in HuH-7 cells (Fig.
7A), being detected equally in
cytosol and nuclei. In contrast, EGFP-CK2
"FL was
localized exclusively to the nuclei of HuH-7 cells (Fig. 7B), whereas EGFP-CK2
"
32 as well as
EGFP-CK2
"C-term were found in both cytosol and nuclei
(Fig. 7, C and D), displaying almost the same
distribution as EGFP alone (Fig. 7A). Further insight into
the mechanism leading to the exclusive nuclear localization of CK2
"
was provided by analysis of the NM binding pattern of the different GFP
fusion proteins (Fig. 8). These
experiments revealed that, of the three fusion proteins, only
EGFP-CK2
"FL exhibited significant binding to the NM,
confirming a specific role for the carboxy-terminal sequence in nuclear
retention of the CK2
" isoforms.
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Nuclear localization of CK2" is not dependent on interference
with a nuclear export signal.
To further examine the mechanism by which the isoform-specific carboxy
terminus of CK2
" determines nuclear localization, we investigated
the possibility that the carboxy terminus interferes with a nuclear
export signal (NES), resulting in the prevention of shuttling back to
the cytoplasm. EGFP-CK2
"
32-transfected HuH-7 cells, in
which protein synthesis was blocked for 1 h prior to treatment,
were treated with 20 ng/ml leptomycin B (LMB; a kind gift from Dr. M. Yoshida, Dept. of Biotechnology, University of Tokyo, Tokyo, Japan),
known to strongly inhibit nuclear export mediated by
leucine-rich NES and possibly other export signals
(59). As shown in Fig.
9, there was no difference between
the distribution of EGFP-CK2
"
32 in HuH-7 cells treated
with LMB (Fig. 9B) and in untreated cells (Fig.
9A). This result indicates that the redistribution of the fusion protein lacking the carboxy terminus into the cytosol is not
accomplished by this classic NES. As a control to confirm that LMB was
used in a concentration sufficient to inhibit nuclear export in
HuH-7 cells, we examined the distribution of an expressed EGFP-I
B
construct (gift from Dr. S. Miyamoto, Dept. of Pharmacology, University
of Wisconsin, Madison, WI). I
B, which physiologically inhibits the transcription factor nuclear factor (NF)-
B, is mainly found in the cytoplasm but able to translocate to the nucleus, from
which it shuttles back into the cytoplasm via a LMB-sensitive NES
(20). Figure 8C shows the predominantly
cytoplasmic distribution of I
B, whereas the nucleus shows a weak
signal. On treatment with LMB, I
B accumulated rapidly in the nucleus
of HuH-7 cells (Fig. 9D). This result confirmed the findings
of a previous study (20) and suggested that the LMB
concentration used in the present study was sufficient to inhibit a
leucine-rich NES. In addition to an unchanged
EGFP-CK2
"
32 distribution, there was no effect of LMB
on the subcellular localization of the other isoforms of the catalytic
CK2 subunit (CK2
and CK2
') as indicated by immunoblot analysis
(Fig. 10).
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DISCUSSION |
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CK2 is a messenger-independent serine/threonine protein
kinase that is ubiquitously distributed in all eukaryotic tissues (9, 14). Until recently, two isoforms of the catalytic
subunit were known (CK2 and CK2
') in combination with the
activity-enhancing CK2
subunit proposed to form the tetrameric
holoenzyme (12, 13) composed of two
- and two
-subunits (
2
2,
'
2,
'2
2). The
discovery of a third isoform of the CK2
subunit (CK2
") as the
result of expression cloning a cDNA that complements the phenotype of a
trafficking mutant cell line derived from HuH-7 cells (47) extends the potential composition of the CK2 holoenzyme in these cells.
Furthermore, the expression of this isoform exhibits particular functions in the trafficking of membrane proteins in HuH-7 cells. Because the catalytic activity of CK2
and CK2
" appear to be equivalent, the subcellular localization of CK2
" might be the key to
understanding this functional difference in more detail.
Although CK2 has been shown to phosphorylate many nuclear substrates,
the reports on nuclear localization of the holoenyzme as well as single
subunits have been contradictory. There are groups that have described
CK2 as exclusively localized to the nucleus (14), whereas
others have suggested that CK2 is predominantly cytosolic (6,
45). Recently, direct visualization of a GFP-CK2 fusion
protein in living cells showed a predominant nuclear localization with
a significant cytoplasmic pool (30). Consistent with the various roles proposed for CK2, the distribution of the enzyme between
the nucleus and cytosol appears to be dependent on the tissue type
examined and the state of differentiation or stage of the cell cycle at
which the studies were performed (31). Such differences
extend beyond the holoenzyme in that differential localization of the
CK2
, CK2
', and CK2
subunits was reported (5, 43,
60). Indeed, it has been suggested that the localization and
potential differential function of CK2 within the various organelles of
the cell may be based on their subunit stoichiometry (12). On the basis of this notion, we set out to determine
the subcellular localization of the newly described CK2
" in the
HuH-7 cell line.
Consistent with the presence of a highly conserved cluster of
preferentially basic residues (KKKIKREIK; see Fig. 5) or monopartite nuclear localization signal (NLS; Ref. 22), recently shown
to be sufficient for nuclear localization of a truncated CK2-GFP fusion protein (23, 30, 40), all three members of the
CK2
family were recovered in the nuclear fraction. The crystal
structure of CK2
isolated from Zea mays
(38), which is 75% identical to the human isoform,
suggests that the NLS would be accessible to a nuclear import receptor
and adapter proteins, members of the superfamily of proteins identified
in human tissue known as
-/
-importin (34, 42, 58).
In contrast to CK2
", which was exclusively localized to the nuclear
fraction, CK2
and CK2
' were distributed through all three
fractions that were isolated. The broader distribution for CK2
and
CK2
' was confirmed by indirect immunofluorescence microscopy (Fig.
2; data for CK2
' not shown), and similar findings in primary human
hepatocytes established that the unique nuclear localization of CK2
"
was not a tissue culture artifact seen only in HuH-7 cells.
The nucleus is organized by a fibrillar network, the nuclear lamina and
the nucleolus that together are referred to as the NM
(37). The NM provides the internal scaffolding for nuclear functions related to transcription (35) and cell growth
and proliferation (25, 49, 50), all functions in which CK2
has been implicated to play a significant regulatory role (15,
18, 28, 44, 48). It is well documented that the NM is a
subnuclear site of CK2 association (14), and it has been
suggested that it is this association that provides the physiological
relevance to CK2 nuclear localization (3, 5, 9).
Therefore, we determined whether the newly described CK2" was bound
to the NM or was primarily a nucleoplasmic protein in HuH-7 cells.
Extraction of the nuclear fraction under different conditions suggested
that CK2
" was preferentially associated with the NM (Fig. 4). In
contrast, CK2
, CK2
', and CK2
were equally distributed between
nucleoplasmic and NM fractions. Contrary to other reports in different
cell types (9), this finding suggests that CK2
,
CK2
', and CK2
are not preferentially associated with the NM in
HuH-7 cells. On the basis of this subunit distribution, the potential
composition of the nuclear tetrameric CK2 holoenzyme in HuH-7 cells
would consist of any two of the three catalytic
-subunits in
combination with two of the regulatory
-subunit. However, this
distribution does not eliminate the possibility of a monomeric enzyme
comprised of single catalytic
-subunits in the absence of the
regulatory
-subunit (24). A monomeric configuration
would be less likely if the
-subunit were produced in excess in
HuH-7 as previously shown in COS cells (61).
The most obvious candidate to explain the dissimilar pattern of
subcellular distribution between CK2 and CK2
" is the difference in their respective carboxy-terminal regions. Transient transfection of
HuH-7 cells with the vector encoding the GFP-CK2
"FL
fusion protein confirmed the almost exclusively nuclear localization of
the CK2
" isozyme (Fig. 7). Deletion of the last 32 amino acids of
the carboxy terminus of CK2
" resulted in cytosolic and nuclear localization of the fusion protein, a distribution similar to that of
CK2
and CK2
' as detected by cell fractionation. This finding
clearly supports a positive role for this part of the molecule in
determining the subcellular distribution of CK2
" and potentially of
the holoenzyme containing this isoform, in contrast to a previous study
in which the isoform-specific carboxy terminus was not shown to
influence the subcellular distribution of epitope-tagged CK2
and
CK2
' (39).
Localization of macromolecules in both the nucleus and cytoplasm
implies an active shuttle pathway (17, 51). Although there
is no direct evidence for the bidirectional traffic of CK2, the rapid
translocation of nuclear CK2 to the cytoplasm in response to androgen
withdrawal (11), and its nuclear deposition in response to
chemical inducers of apoptosis (16), suggests that
CK2 is a member of this unique class of proteins. Movement from the
nucleus to the cytoplasm through the nuclear pore complex is often
directed by a leucine-rich NES (34). Inspection of the
amino acid sequences of all three CK2 isoforms failed to identify
this classic NES. This observation was supported by the failure of LMB,
which is widely used to inhibit nuclear export through this classic NES (19, 20, 59), to prevent redistribution of the fusion
protein EGFP-CK2
"
32 (deleted carboxy terminus;
Fig. 9) into the cytosol of HuH-7 cells or shift CK2
and CK2
" to
the nucleus (Fig. 10). However, this finding does not eliminate an
uncommon NES, such as the more recently described nucleocytoplasmic
bidirectional shuttling signals usually reserved for RNA binding
proteins (32), being uncovered by the deletion of the
carboxy-terminal sequence. Alternatively, deletion of the carboxy
terminus region could open cryptic phosphorylation sites, located at
amino acid positions 60-64 and 96-100, which surround the
putative CK2
" NLS (amino acids 74-83). In that case, autophosphorylation might result in cytoplasmic retention due to
interference of NLS recognition (10, 21, 57), thereby reducing the rate of nuclear import and tilting the steady-state distribution of the CK2
" to the cytosol.
It appears that the CK2" carboxy-terminal amino acids, although
necessary, are not sufficient for high-affinity NM recognition and that
the site of NM interaction resides within the common region of the
isoforms. This assumption is supported by the following findings.
1) All three isoforms associate with the NM fraction, even
in the absence of CK2
" (Fig. 4). 2) The isoform-specific carboxy terminus of CK2
" was not capable of altering the subcellular distribution of the EGFP-CK2
"C-term fusion protein (Fig.
7D). 3) Only the EGFP-CK2
"FL
fusion protein was found to bound to the NM, whereas
EGFP-CK2
"
32 and EGFP-CK2
"C-term
showed no NM association (Fig. 8). To date, a number of proteins that interact selectively with CK2
isoforms potentially mediating NM
binding have been identified (21, 26, 46). A reduction in
the association of CK2
" with any one of these or a yet to be defined
CK2-interacting protein could regulate the subcellular distribution of
the isoform.
Presently, it is not clear whether the differential pattern of CK2,
CK2
', and CK2
" NM association reflects an isoform-specific functional difference. Although the restricted nuclear localization of
CK2
" might reflect a distinct physiological role, the exact function
remains speculative. As mentioned earlier, this subunit isoform was
discovered in the course of investigating the molecular basis for the
defect of a previously isolated endocytic trafficking mutant (Trf1)
isolated from HuH-7 cells. The cell line Trf1 is defective in the
distribution of subpopulations of cell surface receptors for
asialoorosomucoid, transferrin, and mannose-terminating glycoproteins
without affecting total receptor expression (52). The
pleiotropic phenotype of Trf1 also includes an increased sensitivity to
Pseudomonas toxin and deficient assembly and function of gap junctions (53). Transfection of Trf1 cells with CK2
"
cDNA was capable of fully restoring the parental phenotype
(47). Connection of the Trf1 phenotype, assumed to be the
consequence of a cytosolic event(s), with the nuclear localization of
the CK2
" isoforms raises two possibilities. One explanation could be
that the absence of a small cytoplasmic pool of CK2
" that remained
undetected in this study is responsible for the Trf1 phenotype. This
would be consistent with the hypophosphorylation of the
asialoglycoprotein receptor's cytoplasmic domain containing a CK2
phosphorylation site in TRf1 cells (47). In this case, the
carboxy terminus of CK2
" would need to confer a unique substrate
specificity to the isozyme, because little difference in total CK2
activity for the synthetic peptide (RRADDSDDDDDD) was evident between
Trf1 and HuH-7 cell lysates (data not shown). A more intriguing
explanation would be that NM-associated CK2
" is an upstream element
of a signal transduction cascade that ultimately extends into the
cytosol as previously suggested for the other CK2 isoforms (16,
29). Consistent with this notion is the finding that even a
modest overexpression of CK2
results in its preferential association with NM (61) and restoration of the parental phenotype to
CK2
-transfected Trf1 cells (47).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Hermeet Malhi and Dr. Sanjeev Gupta for providing
primary human hepatocytes, Dr. M. Yoshida for providing LMB, Dr. D. Litchfield for providing CK2 isoform-specific antibodies and
constructs, and Dr. S. Miyamoto for providing pEGFP-IB. Our thanks
go also to Dr. T. Meier for helpful discussions and critical reading of
the manuscript.
![]() |
FOOTNOTES |
---|
* P. Hilgard and T. Huang contributed equally to this work.
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41918 and DK-17702. P. Hilgard was supported by a grant of Deutsche Forschungsgemeinschaft, Bonn, Germany (HI 735/1-1).
Address for reprint requests and other correspondence: R. Stockert, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: stockert{at}aecom.yu.edu).
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.
April 10, 2002;10.1152/ajpcell.00070.2002
Received 15 February 2002; accepted in final form 8 April 2002.
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