From the Marion Bessin Liver Research Center and the
Departments of § Cell Biology, ¶ Neuroscience,
** Anatomy and Structural Biology, and
Medicine, Albert Einstein College of
Medicine, Bronx, New York 10461 and the
University Sao Judas
Tadeu, Sao Paulo 03166-000, Brazil
Received for publication, September 19, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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A previously isolated endocytic trafficking
mutant (TRF1) isolated from HuH-7 cells is defective in the
distribution of subpopulations of cell-surface receptors for
asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin,
and mannose-terminating glycoproteins. The pleiotropic phenotype of
TRF1 also includes an increased sensitivity to Pseudomonas
toxin and deficient assembly and function of gap junctions. HuH-7×TRF1
hybrids exhibited a normal subcellular distribution of ASGR, consistent
with the TRF1 mutation being recessive. A cDNA
expression library derived from HuH-7 mRNA was transfected into
TRF1 cells, which were subsequently selected for resistance to
Pseudomonas toxin. Sequence analysis of a recovered
cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2 Receptor-mediated endocytosis, a universal mechanism for the
uptake of macromolecules by cells, is initiated by the binding of
ligand to specific cell-surface receptors, followed by a complex series
of intracellular vesicular transfers (1). The asialoglycoprotein receptor (ASGR)1 is a
prototype of the class of receptors that constitutively enter cells via
clathrin-coated pits and traffic the endocytic pathway, recycling
between early endosomal compartments and the cell surface (2). Two
subpopulations of ASGR, States 1 and 2, were originally identified
based on the kinetics of ligand transport and the identification of
parallel endocytic pathways (3). Changes in both the phosphorylation
(4) and acylation (5) status of the receptor have been suggested to
affect the transition between these states. The existence of receptor
subpopulations has also been suggested for other class II recycling
receptors, including the low density lipoprotein, mannose 6-phosphate,
and A mutant affecting endocytic traffic (TRF1) was previously isolated
from HuH-7 cells using a dual selection protocol (7). To avoid the
selection of receptor-minus mutants, selection pressure was applied
against two independent cell-surface receptors. Gelonin, an inhibitor
of protein synthesis, was covalently coupled to two glycoproteins with
oligosaccharide chains terminating in either galactose
(asialoorosomucoid (ASOR)) or mannose (ovalbumin) and simultaneously
delivered to mutagenized HuH-7 cells. The mutation expressed by TRF1
cells reduces the surface expression of several unrelated membrane
proteins and provides genetic evidence for the existence of receptor
subpopulations. Due to a selective redistribution of State 2 receptors
in TRF1 cells, the surface binding of ASOR was reduced by 50% and that
of transferrin by 30% compared with parental HuH-7 cells. Anterograde
steps of intracellular endocytic processing of ASOR, including
internalization, endosomal acidification, and ligand degradation, were
not significantly altered by the TRF1 mutation.
The pleiotropic phenotype of TRF1 cells also results in a
marked increase in sensitivity to Pseudomonas toxin (7). In
this study, we took advantage of this sensitivity to expression-clone a
cDNA that complements the TRF1 mutation. The cloned gene,
designated CK2 Somatic Cell Hybridization--
The origin of the HuH-7 and TRF1
human hepatoma cell lines was described previously (7). The HuH-7
neomycin-resistant cell line was isolated following transfection with
pRc/RSV (Invitrogen) DNA using LipofectAMINE (Life Technologies,
Inc.) according to the manufacturer's protocol. HuH-7
Zeocin-resistant and TRF1 neomycin-resistant cell lines were isolated
following electroporation of ~2 × 106 cells with 10 µg of pZeoSV2(+) (Invitrogen) or pRc/RSV DNA in a Bio-Rad Gene-Pulser
II with a capacitance setting of 975 microfarads and a voltage setting
of 0.3 kV.
To generate somatic cell hybrids, ~1 × 106 cells of
each line were plated in a 35-mm tissue culture dish in 2 ml of Determination of Ploidy by Propidium Iodide Staining--
Hybrid
colonies were removed from flasks or dishes by washing with 5 ml of PBS
without divalent cations, followed by treatment with 2 ml of 5 mM EDTA in PBS without divalent cations. Clumps of cells
were disaggregated by four passages through a syringe fitted with a
20-gauge needle. Approximately 5 × 105 cells were
transferred to centrifuge tubes and pelleted at 1200 rpm for 10 min in
an IEC HN-SII centrifuge. The cell pellet was washed once with 5 ml of Earle's balanced salt solution and resuspended in 1 ml of 0.1%
sodium citrate-phosphate (pH 7.8) containing 50 µg/ml propidium
iodide (Sigma). After a 10-min incubation on ice, ploidy was estimated
by determining the DNA content of single cells by FACS analysis using a
Becton-Dickinson FACScan cytometer.
Library Construction--
HuH-7 mRNA was isolated using a
QIAGEN mRNA isolation kit, and an HuH-7 expression library was
prepared in the pBK-CMV vector system by Stratagene. To isolate plasmid
DNA for transfection, the cDNA library was amplified by
transformation of competent cells (XLI, Stratagene) and selection on
LB/kanamycin plates. After incubation at 37 °C overnight, the cells
were harvested, and plasmid was prepared using a QIAGEN maxiprep kit.
Sensitivity of Cells to Pseudomonas Toxin--
The sensitivity
of cells to toxins was determined essentially as described previously
(7). Briefly, the cells were removed from nearly confluent T-75 flasks
by treatment with PBS containing 5 mM EDTA, resuspended in
RPMI 1640 medium with 10% FBS, and diluted to 8 × 104 cells/ml. A range of Pseudomonas toxin
(Sigma) concentrations was prepared in the same medium, and 0.1 ml was
added to the wells of a 96-well plate, followed by 0.1 ml of cells
(8 × 103). Cells were incubated at 37 °C until the
control wells (without toxins) reached confluence. The concentration of
each toxin required to kill 90% of the cells (D10 value)
was determined by a methylthiazolyltetrazolium assay.
Expression Cloning of a cDNA That Complements the TRF1
Mutation--
Ten 100-mm plates of TRF1 cells grown to 80% confluence
in RPMI 1640 medium containing 10% FBS were transfected with the HuH-7 library (4 µg/plate) using a LipofectAMINE Plus kit (Life
Technologies, Inc.). After 48 h, G418 (200 µg/ml) was added, and
the cells were cultured for 2 weeks. The surviving colonies were
cultured for another 2 weeks in medium containing
Pseudomonas toxin (0.2 ng/ml) and G418 (200 µg/ml) until
the cells transfected with the pBK-CMV vector alone died.
Pseudomonas toxin-resistant colonies were isolated and
expanded. Genomic DNA was isolated using a QIAGEN QIAamp kit, and the
integrated plasmid cDNA was retrieved by polymerase chain reaction
using T3/T7 primers. The DNA Sequencing Facility of the Albert Einstein
College of Medicine performed sequence analysis of the cDNAs. The
two most resistant clones to Pseudomonas toxin had identical
cDNA sequences. Comparison with the NCBI Database showed the
sequence to be 91% identical to the Transfection of CK2 Preparation of Post-nuclear Supernatants and Cell Lysates--
A
post-nuclear supernatant fraction was prepared by the method of Tyc and
Steitz (10). Briefly, cells (1 × 107) were scraped
into 10 ml of ice-cold PBS and centrifuged for 5 min at 1000 × g. After resuspension in homogenization buffer (10 mM HEPES/KOH (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 1 mM
dithiothreitol, 30 µl/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride) and incubation on ice for 15 min, the
cells were disrupted by 15 strokes with a tight-fitting pestle. The
cell homogenate was centrifuged for 10 min at 1000 × g, and the protein concentration was determined in the
supernatant fraction by bicinchoninic acid protein assay reagent
(Pierce). Cell lysates were prepared by direct suspension of cells in
lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1% Nonidet P-40, 30 µl/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride) and passage 20 times through a 21-gauge
needle. After centrifugation at 15,000 × g for 10 min, the supernatants were used for protein assay and Western blotting.
Preparation of Rabbit Polyclonal Antibody to the Human CK2 Western Blotting--
Forty µg of protein from post-nuclear
supernatants or cell lysates was resolved by 10% SDS-PAGE and
transferred to a PVDF membrane. The PVDF membrane was blocked for
1 h at room temperature in TBS/Tween buffer (150 mM
NaCl, 50 mM Tris-HCl (pH 7.8), and 0.1% Tween 20)
containing 10% dry milk. The membrane was incubated for 1 h at
room temperature in one of the following rabbit anti-human CK2 antibody
preparations: anti-CK2 Cell-surface Binding of 125I-ASOR--
Cells grown
to confluence in 60-mm dishes were washed two times with binding buffer
(135 mM NaCl, 1.2 mM MgCl2, 0.81 mM MgSO4, 27.8 mM glucose, 2.5 mM CaCl2, and 25 mM HEPES (pH
7.2)). Following washing, the cells were incubated at 37 °C for
1 h. To assay surface binding, cells were chilled to 4 °C and
incubated for 1 h at 4 °C with 1 µg/ml 125I-ASOR
in 1.5 ml of binding buffer. Nonspecific binding was determined from
dishes that also contained 100 µg of unlabeled ASOR. Unbound ligand
was removed by three washes with binding buffer, and surface-bound 125I-ASOR was released by incubation for 10 min at 4 °C
in 1 ml of 20 mM EGTA in TBS (7, 11).
Evaluation of Gap Junctional Coupling--
The presence of
functional gap junction channels was directly assessed by both
cell-to-cell transfer of Lucifer yellow, a small molecular mass
fluorescent dye (<1 kDa), and communication of mechanically induced
intercellular Ca2+ waves.
Dye Transfer--
Cells were injected with Lucifer yellow (5%
in 150 mM LiCl) until they glowed brightly through
microelectrodes using hyperpolarizing pulses or brief overcompensation
of the negative capacitance control on a World Precision Instruments
electrometer as described previously (12). Cells were photographed 1-2
min after dye injection using a Nikon Diaphot microscope equipped with
a xenon arc lamp configured for epifluorescence using barrier and
excitation filters optimized for detection of fluorescein emission.
Exposure times using TMAX 200 film were usually 20-30 s. Dye transfer
was evaluated as the number of contacting cells receiving dye from the
injected cell.
Intercellular Ca2+ Wave
Propagation--
Intracellular free Ca2+ levels were
measured in cells grown to confluence on glass-bottom microwells (Model
15, Mattek Corp., Ashland, MA) and loaded at 37 °C for 45 min with 5 µM Indo-1/AM (Molecular Probes, Inc., Eugene, OR), a
radiometric Ca2+ indicator. After rinsing with PBS, Indo-1
fluorescence was imaged using an argon ion laser with excitation at 351 nm and emission at 400 and 480 nm. Images were acquired using the Nikon
real-time confocal microscope (RCM8000) and analyzed as described
previously (12). The intercellular propagation of Ca2+
waves was initiated by brief, gentle focal mechanical stimulation of
one cell in the confocal field using a pulled-glass micropipette (1-2-µm outer diameter). Indo-1 fluorescence ratio values were plotted as a function of time, and graphs were fit with least-squares polynomial regression as described previously (12). Two parameters of
Ca2+ wave propagation were used for comparison of the
Ca2+ signaling between cells: efficacy (the number of
responding cells in the field) and conduction velocity (the time
required for half-maximal Ca2+ increase divided by the
distance from the stimulated cell).
Phosphate Labeling of ASGR--
Cells (1 × 106) preincubated at 37 °C in phosphate-free MEM
supplemented with 10% dialyzed FBS for 1 h were labeled with
[32P]orthophosphate (250 µCi/ml) for 3 h in the
same medium. Following labeling, cells were washed three times with
ice-cold PBS, harvested by scraping, and centrifuged at 300 × g for 10 min. The cell pellet was resuspended in 0.5 ml of
water to which 2× lysis buffer (100 mM Tris (pH 7.4), 300 mM NaCl, and 2% Nonidet P-40 containing protease inhibitor
mixture (Sigma) and 4 µM okadaic acid) was added and
maintained at 4 °C for 30 min with constant mixing, followed by
centrifugation at 14,000 × g for 10 min. Antiserum to
human ASGR was added to supernatants containing equal amounts of
32P-labeled proteins as determined by trichloroacetic acid
precipitation. Following a 1-h incubation at 4 °C, immobilized
protein A/G (Pierce) was added, and incubation was continued for an
additional 1 h. Immobilized protein A/G recovered by
centrifugation at 10,000 × g was washed three times
with radioimmune precipitation assay buffer and than a final wash with
PBS. Antigen released by heating to 95 °C for 3 min was resolved by
10% SDS-PAGE, and the gel was stained and fixed. The dried gels were
exposed to Biomax film with an intensifying screen (Eastman Kodak Co.)
at Expression Cloning of a Gene That Complements the TRF1
Mutation--
Prior to expression cloning, it was determined whether
the TRF1 mutation is expressed as a dominant or recessive
phenotype. HuH-7 and TRF1 cells were stably transfected with plasmids
encoding genes for either neomycin or Zeocin antibiotic resistance.
Cell lines resistant to each antibiotic were isolated and fused by exposure to polyethylene glycol 1000. Hybrid cells (HuH-7×HuH-7 or
HuH-7×TRF1) were selected for resistance to both antibiotics. After
subculturing, cells were stained with propidium iodide and analyzed by
FACS to determine DNA content. As would be expected, hybrid cells
contained approximately twice as much DNA as parental cell lines (Fig.
1).
The hallmark of the mutation expressed by TRF1 cells is the reduction
in cell-surface expression of several unrelated membrane proteins,
including ASGR (7). Restoration of cell-surface ASGR binding activity
in the HuH-7×TRF1 hybrid would be consistent with a normal receptor
distribution and suggest that the TRF1 mutation is
recessive. The binding of 125I-ASOR was used to estimate
the number of bioactive ASGRs at the cell surface. Both hybrid cell
lines exhibited somewhat greater cell-surface 125I-ASOR
binding activity than TRF1 cells or the antibiotic-resistant parental
lines (Fig. 2). Based on this result, the
TRF1 mutation was presumed recessive, and a cDNA library
was derived from HuH-7 mRNA for expression cloning a complementary
cDNA that corrected the TRF1 phenotype.
Our previous observation that TRF1 cells are 10 times more sensitive to
Pseudomonas toxin than the parental HuH-7 cell line (7)
suggested that complementation of the TRF1 mutation could be
selected for by resistance to Pseudomonas toxin. From
preliminary experiments, it was found that, although the difference in
sensitivity to Pseudomonas toxin was constant, the absolute
concentration required to kill >90% of the cells varied depending on
the source and age of the Pseudomonas toxin preparation. To
determine the amount of Pseudomonas toxin to use for
selection, a sensitivity assay was preformed on TRF1 and HuH-7 cells.
Cells were plated in 96-well dishes, exposed to increasing
concentrations of Pseudomonas toxin, and cultured until
control cells reached confluence. The concentration at which ~90% of
the cells were killed was determined by a methylthiazolyltetrazolium
assay as described previously (7). For this particular preparation of
Pseudomonas toxin obtained from Sigma, 0.2 ng/ml killed 90%
of the TRF1 cells, whereas 90% of the HuH-7 cells survived (Fig.
3).
An HuH-7 expression cDNA library constructed in pBK-CMV was
prepared by Stratagene using mRNA isolated from HuH-7 cells. Stable transfectants (1 × 105/plate) were obtained from 10 plates of TRF1 cells (1 × 107/plate) transfected with
4 µg of plasmid DNA from the HuH-7 library. The surviving colonies on
each plate were pooled and subjected to selection with 0.2 ng/ml
Pseudomonas toxin. After 14 days of selection,
G418-resistant colonies from pBK-CMV vector-only-transfected cells were
dead, whereas 15 clones survived from the library-transfected cells.
Identification of the Transfected Gene--
Of the surviving
clones, two were as resistant to Pseudomonas toxin as the
parental HuH-7 cell line (>50% cell viability at 0.5 ng/ml
Pseudomonas toxin). The other 13 clones exhibited
intermediate levels of toxin resistance (<50% cell viability at 0.3 ng/ml Pseudomonas toxin). Transfected cDNA was retrieved
from the two clones by polymerase chain reaction with T3/T7 sequence
primers using genomic DNA as template. Sequence analysis indicated that
the two transfectants harbored the same cDNA encoding a protein
with 91.2% amino acid identity to the human CK2
The differences between CK2 Expression of CK2 Restoration of Pseudomonas Toxin Resistance--
Transfection of
TRF1 cells with the cDNA recovered by expression cloning confirmed
that the cloned CK2 Restoration of Gap Junctions--
Gap junctional communication
between TRF1 cells is significantly reduced compared with that observed
for the parental human hepatoma cell line HuH-7 (12). Dye transfer
between TRF1 cells compared with TRF1 cells transfected with CK2
The reversion to a parental-like gap junction-mediated coupling between
TRF1 cells induced by transfection with CK2 ASGR Phosphorylation Status--
Previous studies of ASGR
phosphorylation showed that phosphate incorporation into the H2 subunit
occurs in serine residues and requires a 57-nucleotide sequence
encoding a 19-amino acid peptide cis to the transmembrane
domain (20). Since the TRF1 mutation appears to alter the
distribution of ASGR without affecting the absolute concentration of
receptor (7), it was determined whether the reduction of CK2 To identify the molecular basis of the TRF1 mutation,
an expression cloning strategy was adopted to isolate cDNA that
complemented the TRF1 phenotype. First, it was shown that the
TRF1 mutation behaves recessively in HuH-7×TRF1 hybrids.
This is somewhat unusual, as cells defective in various stages of
membrane protein traffic described by several laboratories have proven
to be dominant or to exhibit a conditional phenotype (21-24).
Nevertheless, a cDNA obtained from a parental HuH-7 library
corrected the TRF1 phenotype.
The gene that complemented TRF1 cells, designated as CK2 The disruption of trafficking of several membrane proteins by the
TRF1 mutation suggests that the TRF1 phenotype is likely to
result from a defect at a common point affecting protein sorting. Kinase and phosphatase activities have been shown to control both general and cargo-specific trafficking (29). In particular, CK2
phosphorylates proteins that traffic between the Golgi and the plasma
membrane. For example, CK2-mediated phosphorylation of the cytoplasmic
tail of furin is required for its localization to the
trans-Golgi network (30, 31). A CK2 phosphorylation site
(e.g. ESEER) on the cytoplasmic domain of the
cation-dependent mannose receptor and furin has been shown
to determine the high affinity interaction of activator protein-1 Golgi
assembly proteins with phosphofurin acidic cluster-sorting
protein-1, meditating retrieval to the trans-Golgi
network (32, 33). This interaction has been suggested to act as a
dominant determinant controlling receptor sorting.
The proteins affected by the TRF1 mutation, including
ASGR (2), the transferrin (34) and mannose (32) receptors, and furin (31),2 possess a CK2
consensus motif in their predicted cytoplasmic tail, supporting the
biochemical basis of the pleiotropic TRF1 phenotype as reduced
expression of CK2 Previous studies of ASGR phosphorylation showed that phosphate
incorporation into the H2 subunit was detected only in serine residues
and required a 57-nucleotide sequence encoding a 19-amino acid peptide
cis to the transmembrane domain (20). Based on surface
labeling, it was suggested that only H2 protein isoforms expressing the
57-nucleotide encoded peptide trafficked efficiently to the cell
surface. Although the 57-nucleotide insert does not encode a serine
residue, its deletion could allosterically alter the putative CK2
recognition motif located just two amino acids upstream. Failure to
phosphorylate the H1 or H2 subunit of ASGR may alter interaction with
sorting determinants, as demonstrated for other membrane proteins (29),
thereby affecting the subsequent distribution of the State 2 ASGR
subpopulation as previously suggested (3) and disrupting one of the
parallel trafficking pathways proposed for ASGR. Taken together, these
findings point to a significant role for CK2-mediated phosphorylation
in maintaining the normal subcellular distribution of diverse membrane
proteins affected by the TRF1 mutation.
".
Western blot analysis of TRF1 proteins revealed a 60% reduction in
total CK2
expression. Consistent with this finding, the hybrids
HuH-7×HuH-7 and HuH-7×TRF1 expressed equivalent amounts of total
CK2
. Immunoblots using antibodies against peptides unique to the
previously described CK2 isoforms CK2
and CK2
' and the novel
CK2
" isoform showed that, although TRF1 and parental HuH-7 cells
expressed comparable amounts of CK2
and CK2
', the mutant did not
express CK2
". Based on the genomic DNA sequence, RNA transcripts
encoding CK2
" apparently originate from alternative splicing of a
primary transcript. Protein overexpression following transfection of
TRF1 cells with cDNAs encoding either CK2
or the newly cloned
CK2
" restored the parental HuH-7 phenotype, including
Pseudomonas toxin resistance, cell-surface ASGR binding
activity, phosphorylation, and the assembly of gap junctions. This
study suggests that HuH-7 cells express at least three CK2
isoforms
and that the pleiotropic TRF1 phenotype is a consequence of a reduction
in total CK2 expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-macroglobin receptors (6).
", is 91% identical to the CK2
isoform.
Transfection and overexpression of either the novel CK2
" isoform or
the previously described CK2
isoform restore the parental phenotype
to TRF1 cells. This study provides the first evidence that CK2 plays a significant role in ASGR trafficking between an intracellular compartment and the plasma membrane.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MEM
(Life Technologies, Inc.) containing 10% FBS. The following day, the medium was removed, and the plates were washed once with 1.5 ml of
-MEM without serum. Cells were fused by exposure for 1 min to a 44%
solution of polyethylene glycol 1000 in serum-free
-MEM. The
monolayers were then washed four times with 1.5 ml of serum-free
-MEM and once with 1.5 ml of
-MEM containing 10% FBS. Two ml of
medium with serum was then added to each plate. After incubation overnight at 37 °C, cells were removed by treatment with 5 mM EDTA in PBS without divalent cations, and 2 × 105 cells were plated in 100-mm tissue culture dishes in 10 ml of
-MEM containing 10% FBS. After an additional 48 h, the
medium was replaced with medium containing 500 µg/ml G418 and 400 µg/ml Zeocin. Plates were returned to a 37 °C incubator and
examined for the presence of colonies weekly. Small colonies, generally observed within 3 weeks, were subcloned at 8-10 weeks, and the cultures were gradually expanded. Hybrids formed between the HuH-7 neomycin-resistant and HuH-7 Zeocin-resistant cell lines at a frequency
of ~5 × 10
3. In the case of the TRF1
neomycin-resistant and HuH-7 Zeocin-resistant fusion partners, a lower
frequency of 3 × 10
4 was observed. Few,
if any, colonies were observed in the absence of polyethylene glycol
treatment
-subunit of human CK2. As there
already exists a CK2
' isoform (8), we termed this new isoform
CK2
".
" and Human CK2
cDNAs into TRF1
Cells--
The polymerase chain reaction-recovered cDNA encoding
CK2
" was cloned back into the expression vector pBK-CMV, and the
fidelity of the resulting construct (pBK-CMV-CK2
") was
established by DNA sequencing. Clones of the human CK2
subunit were
kindly provided by Dr. David W. Litchfield (University of Manitoba,
Winnipeg, Manitoba, Canada). The cDNA was cloned in the pRC-CMV
expression vector with a hemagglutinin tag (9). The plasmids
pBK-CMV-CK2
" and pRC-CMV-HA-CK2
were transfected into TRF1 cells
using a LipofectAMINE Plus kit. The G418-resistant colonies were
isolated and expanded.
"
Subunit--
A peptide containing 16 amino acids near the carboxyl
terminus from amino acids 366 to 381 of CK2
"
(LLSSTVYPPWPPKVL) was synthesized with a cysteine residue at the
carboxyl terminus as a linker. The Laboratory of Macromolecular
Analysis at the Albert Einstein College of Medicine performed peptide
synthesis and verification. This cysteine-terminating peptide was
linked to maleimide-activated keyhole limpet hemocyanin (Pierce)
according to the manufacturer's directions. Covance Research Products,
Inc. raised antibody to the peptide in rabbits.
subunit antibody (Upstate Biotechnology,
Inc.) diluted to 1 µg/ml, CK2
and CK2
' isoform-specific antibody diluted 1:5000 (kindly provided by Dr. David W. Litchfield), or CK2
" isoform-specific antibody diluted 1:3300 in TBS/Tween containing 2% dry milk. The membrane was washed five times for 5 min
each time and incubated for 30 min in horseradish peroxidase-conjugated goat anti-rabbit IgG antiserum diluted 1:5000 in TBS/Tween containing 1% dry milk. After washing five times for 5 min each time with TBS/Tween, the membrane was incubated for 20 s in
chemiluminescence reagents (Super Signal-Pierce) and exposed to
Fuji film. To determine the specificity of rabbit anti-CK2
"
antibody, a mixture of 1 µg of CK2
" peptide, 3 µl of
anti-CK2
" antibody, 200 µl of PBS was rotated overnight at 4 °C
and incubated for 1 h at room temperature in 10 ml of TBS/Tween
containing 2% nonfat dry milk before use. Band intensities were
determined by scanning films used for chemiluminescent detection.
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA content of hybrid cells. HuH-7 cells
transfected with the G418 resistance gene (HuH-7neo) or the
Zeocin resistance gene (HuH-7zeo), TRF1 cells transfected
with the G418 resistance gene (TRF1neo), and the somatic
cell hybrids HuH-7neo×HuH-7zeo and
HuH-7zeo×TRF1neo were stained with propidium
iodine, and ploidy was estimated by determining the DNA content of
single cells by FACS analysis.
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Fig. 2.
The TRF1 mutation behaves
recessively in hybrids. HuH-7, TRF1, HuH-7zeo,
TRF1neo, and HuH-7 hybridized with HuH-7
(H×H) or with TRF1 (H×T)
were grown to confluence (~2 × 106 cells/60-mm
dish) to assure maximum binding activity. Cells were washed three times
with binding buffer and preincubated for 1 h in the same buffer.
Cells were chilled to 4 °C and incubated for 1 h in 1.5 ml of
binding buffer containing 125I-ASOR (1 µg) with or
without 100 µg of unlabeled ASOR. Surface-bound 125I-ASOR
(ng/mg of protein; mean ± S.D.) was determined from triplicate
dishes in three independent experiments by 20 mM
EGTA-released radioactivity.
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Fig. 3.
Pseudomonas toxin sensitivity of
TRF1 cells. HuH-7 and TRF1 cells were incubated in increasing
concentrations of Pseudomonas toxin until control cells
grown in the absence of toxin were confluent. The mean number of viable
cells determined from triplicate dishes by a methylthiazolyltetrazolium
assay as described previously (7) is expressed as a percentage of
control.
subunit. The new
CK2
isoform obtained from HuH-7 cells, termed CK2
", has 1507 nucleotides with an open reading frame encoding 385 amino acids (Fig.
4). The cDNA sequence of CK2
" is
65.2% identical to CK2
cDNA.
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Fig. 4.
Amino acid sequences of
CK2 and CK2
".
The amino acid sequence of CK2
(GenBankTM/EBI accession
number X70251) is aligned with the deduced sequence of the newly cloned
CK2
" using BLAST 2 (NCBI Database). From amino acids 1 to 353, there
is only one amino acid difference in the derived protein sequence (Thr
to Ala at position 127). After amino acid 353, the predicted protein
sequences of CK2
and CK2
" are totally different.
and CK2
" are mainly localized to the
C terminus. From amino acids 1 to 353, there is only one amino acid
substitution in the derived protein sequence (Thr to Ala at position
127, due to a single nucleotide difference, A to G). After amino acid
353, the predicted protein sequences of CK2
" and CK2
are totally
different. Despite this difference, when CK2
" was expressed in
Escherichia coli, cell lysates exhibited a kinase activity
equal to that of bacteria transformed with the CK2
cDNA using a
synthetic peptide (RRKDLHDDEENDAMSITA) as substrate (13). The unique
CK2
" sequence has previously been reported within clone RP5-863C7
(gi:5788437) as an intronic repeat region (14, 15). Although the
genomic structure of CK2
" has not been established, the presence of
a translated commonly dispersed repeat or Alu cassette and the
remnants of a poly(A) tail in the cDNA recovered from the original
revertants suggest that CK2
" is either a CK2
-derived retroposon
(16, 17) or the result of alternative splicing, selectively including
an Alu-like exon into the mature mRNA (18, 19).
Isoforms by HuH-7, TRF1, and Transfected TRF1
Cells--
Western blot analysis using antibody against the common
region of CK2
and CK2
" (amino acids 70-91 of the CK2
subunit)
indicated that TRF1 cells expressed 40.6 ± 2.7% of the total
CK2
isoforms expressed by HuH-7 cells (Fig.
5A). Consistent with these
findings, the hybrid cell lines HuH-7×HuH-7 and HuH-7×TRF1, which
exhibited a similar level of cell-surface ASOR binding activity as the
parental HuH-7 cell line (Fig. 2), expressed equivalent levels of
CK2
and CK2
" (Fig. 5B). Analysis using antibodies
against peptides unique to CK2
and CK2
' indicated that TRF1 cells
expressed amounts of CK2
and CK2
' comparable to the parental
HuH-7 cell line (Fig. 6). The predicted
molecular mass of CK2
" was equivalent to that of the CK2
subunit
and could not be by resolved from CK2
on SDS-PAGE. To determine the
expression of CK2
" in HuH-7 and TRF1 cells, a rabbit polyclonal
antibody directed against a specific region of the C terminus of
CK2
" was prepared. CK2
" expression by HuH-7, TRF1, and
CK2
"-transfected TRF1 cells was determined by immunoblot analysis.
As illustrated in Fig. 6, only HuH-7 and TRF1 cells transfected with
CK2
" expressed a 44-kDa protein, consistent with the predicted
molecular mass of CK2
", whereas TRF1 cells expressed no such
protein.
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Fig. 5.
A, expression of CK2 and CK2
" in
HuH-7, TRF1, and transfected TRF1 cells. A post-nuclear supernatant was
prepared as described under "Materials and Methods." Cell proteins
(40 µg) isolated from HuH-7, TRF1, and TRF1 transfected with CK2
(TRF1-
), CK2
" (TRF1-
"), or
the pBK-CMV vector only (pBK) were resolved by 10% SDS-PAGE
and transferred to PVDF membrane. The membrane was stained with Ponceau
S to confirm equal loading prior to probing with polyclonal antibody
against a region common to both CK2
and CK2
". Antibody deposition
was detected by chemiluminescence, and data from four independent
immunoblots were quantified by densitometric scanning (UltroScan XL,
Amersham Pharmacia Biotech). B, expression of CK2
and
CK2
" in hybrid cells. Cell proteins (40 µg) isolated from HuH-7
hybridized with HuH-7 or with TRF1 were resolved by 10% SDS-PAGE,
transferred to PVDF membrane, and processed as described for
A.
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Fig. 6.
Expression of CK2
isoforms in HuH-7, TRF1, and
CK2
"-transfected TRF1 cells. A
post-nuclear supernatant was prepared as described under "Materials
and Methods." Cell proteins (40 µg) isolated from HuH-7, TRF1, and
CK2
"-transfected TRF1 (TRF1-
") cells were
resolved by 10% SDS-PAGE and transferred to PVDF membrane. The
membrane was stained with Ponceau S to confirm equal loading prior to
probing with polyclonal antibody raised against peptides to unique
regions of the various CK2
isoforms. Antibody deposition was
detected by chemiluminescence.
" cDNA was capable of restoring a
Pseudomonas toxin-resistant phenotype to TRF1 cells (Fig.
7A). Whereas TRF1 and
vector-transfected TRF1 cells were 10 times more sensitive to
Pseudomonas toxin than the parental HuH-7 cell line,
transfection of the TRF1 mutant with either CK2
or CK2
" fully
restored Pseudomonas toxin resistance to the HuH-7 level.
Cell-surface ASOR binding activity in TRF1 cells is reduced on the
order of 50% due to the altered trafficking of State 2 receptors (7).
Transfection of TRF1 cells either with CK2
, resulting in
overexpression of the isoform, or with CK2
" fully restored
cell-surface ASOR binding to the parental level, suggesting that normal
ASGR subcellular distribution had been reestablished (Fig.
7B). As was the case for Pseudomonas toxin
sensitivity, transfection with vector alone had no significant effect
on the level of cell-surface ASOR binding activity.
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Fig. 7.
A, reversion of Pseudomonas
toxin sensitivity. HuH-7, TRF1, and TRF1 transfected with CK2
(TRF1-
), CK2
" (TRF1-
"), or
the pBK-CMV vector only (pBK) were incubated in increasing
concentrations of Pseudomonas toxin until control HuH-7
cells (in the absence of toxin) were confluent. The D10
values (concentration of Pseudomonas toxin causing 90% cell
death; mean ± S.D.) were determined from triplicate dishes in
three independent experiments by a methylthiazolyltetrazolium assay as
described previously (7). B, reversion of ASGR cell-surface
distribution. Cell-surface ASOR binding was determined in confluent
HuH-7, TRF1, and TRF1 transfected with CK2
", CK2
, and pBK-CMV
vector only. Cells were chilled to 4 °C and incubated for 1 h
in 1.5 ml of binding buffer containing 125I-ASOR (1 µg)
with or without of 100 µg of unlabeled ASOR. Surface-bound
125I-ASOR (ng/mg of protein; mean ± S.D.) was
determined from the radioactivity released by 20 mM EGTA
and is expressed as a percentage of the parental HuH-7 cell-surface
receptor binding activity.
"
revealed a reversion of the transfectants to the parental phenotype.
Their ability to communicate via gap junction channels was rescued.
After 1-2 min of Lucifer yellow injection in a single TRF1 CK2
"
transfectant, the overall dye spread to the neighboring cells was twice
that observed in TRF1 mutants (p < 0.05) and was not
significantly different from the spread observed in the parental HuH-7
cells (Fig. 8A). Lucifer
yellow transfer to the first cell tier of TRF1 cells (
20 µm from
the injected cell; n = 12 fields) and to the second
cell tier of cells (
40 µm from the injected cell) was increased by
70 and 90%, respectively, in the TRF1 cells transfected with CK2
",
approaching values previously reported for HuH-7 cells (12).
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Fig. 8.
Reversion of gap junction function.
Dye transfer and Ca2+ wave spread between HuH-7,
TRF1, and TRF1 cells transfected with CK2 " are shown. Dye transfer
following microinjection of Lucifer yellow was evaluated as the number
of contacting cells receiving dye from the injected cell
(A); in the TRF1 CK2
" transfectant, dye spread was not
significantly different from that in the parental HuH-7 cells.
Ca2+ wave was mechanically induced by gentle stimulation of
the TRF1 CK2
" transfectant (B, cell A), and
responses in adjacent cells were recorded at 2-s intervals thereafter
(D). The efficacy of the Ca2+ wave transmission
was largely restored in the TRF1 CK2
" transfectant, whereas the
conduction velocity remained low in these cells (C).
" was also observed in
Ca2+ imaging experiments. The ability of TRF1 transfectants
to communicate Ca2+ signals generated by focal mechanical
stimulation of a single cell was markedly enhanced. Whereas the
efficacy of Ca2+ signal transmission (number of responding
cells in the field) in TRF1 mutants was found to be 62% lower than
that observed in HuH-7 cells (12), in TRF1 CK2
" transfectants, it
was only 33% lower, accounting for an almost 75% improvement in TRF1
intercellular communication (Fig. 8C). Although the number
of cells recruited in Ca2+ signaling studies was not
significantly different (Fig. 8, B-D), the conduction
velocity of the Ca2+ signal in TRF1 CK2
" transfectants
remained similar to that in the TRF1 mutants (Fig. 8C). The
failure to revert to the normal rate of Ca2+ flux may be a
result of overexpression of the CK2
" isozyme, altering the
mobilization of Ca2+, as opposed to a direct effect on gap
junction formation.
"
expression in TRF1 cells had any effect on the overall ASGR
phosphorylation status. Following labeling of HuH-7 and TRF1 cells to
steady state with [32P]orthophosphate, equal amounts of
32P-labeled cell proteins were immunoprecipitated with
anti-ASGR antiserum. Based on the results, it became evident that ASGR
expressed by TRF1 cells was hypophosphorylated compared with ASGR in
the parental HuH-7 cell line. Transfection of the TRF1
mutant with CK2
" cDNA restored the HuH-7 level of
32P incorporation into ASGR to that observed in HuH-7 cells
(Fig. 9).
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Fig. 9.
Phosphorylation of ASGR in response to
CK2 " expression. Confluent HuH-7, TRF1,
and CK2
"-transfected TRF1 (TRF1-
") cells
were preincubated in phosphate-free MEM supplemented with 10% dialyzed
FBS for 1 h. Cell lysates were prepared following labeling with
[32P]orthophosphate (250 µCi/ml) for 3 h in the
same medium. Radiolabeled ASGR was immunoprecipitated from cell lysates
containing equal amounts of 32P-labeled proteins determined
by trichloroacetic acid precipitation. Recovered ASGR was resolved by
10% SDS-PAGE, and the fixed gel was prepared for autoradiographic
analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
", is 91%
identical in amino acid sequence and 65.2% identical in nucleotide
sequence to the previously described CK2
(25). CK2 is a ubiquitously
expressed eukaryotic Ser/Thr protein kinase present in the nucleus and
cytoplasm (26). In mammals, two isoforms of the catalytic subunit of
CK2, CK2
and CK2
', have been identified (8). They are the
products of distinct genes localized to different chromosomes (27, 28)
The newly cloned CK2
" represents a new isoform of the CK2
family.
It is almost identical to CK2
in amino acid sequence until the last
32 amino acids, which are completely unique. A peptide antibody to this
C-terminal sequence showed that HuH-7 cells express CK2
" and that
TRF1 cells have no detectable CK2
". Transfection of TRF1 cells with
a cDNA encoding either CK2
or the newly described CK2
"
restored the parental phenotype to TRF1 cells. Whether reversion of the
TRF1 phenotype is due to the absolute level of CK2
kinase activity
or to a distinct localization of CK2
", which can be compensated for
by overexpressing CK2
, remains to be resolved.
". It is not so apparent, however, why connexin-43
(12), a multi-transmembrane protein (35) that differs in several
additional respects from other proteins affected by the TRF1
mutation, would be altered by the loss of CK2
". Gap junctions
localize exclusively to the lateral cell surface. Connexins are not
glycoproteins, and they are not thought to play a role in the endocytic
pathway. Since connexin-43 lacks an obvious CK2 phosphorylation motif,
the failure to form functional gap junctions between TRF1 cells may
reflect altered phosphorylation of an accessory protein necessary
for appropriate connexin-43 trafficking (36).
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-41918 and DK-32972.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.
§§ To whom correspondence should be addressed: Liver Research Center, Albert Einstein College of Medicine, Ullmann 517, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3644; Fax: 718-430-8975; E-mail: stockert@aecom.yu.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M008583200
2 C. Harley, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: ASGR, asialoglycoprotein receptor; ASOR, asialoorosomucoid; CK2, casein kinase 2; MEM, minimal essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline.
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