A Novel Casein Kinase 2 alpha -Subunit Regulates Membrane Protein Traffic in the Human Hepatoma Cell Line HuH-7*

Xiaoying ShiDagger , Barry Potvin§, Tianmin HuangDagger , Philip HilgardDagger , David C. Spray, Sylvia O. Suadicani||, Allan W. Wolkoff**Dagger Dagger , Pamela Stanley§, and Richard J. StockertDagger Dagger §§

From the Dagger  Marion Bessin Liver Research Center and the Departments of § Cell Biology,  Neuroscience, ** Anatomy and Structural Biology, and Dagger Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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), CK2alpha ". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7×HuH-7 and HuH-7×TRF1 expressed equivalent amounts of total CK2alpha . Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha ' and the novel CK2alpha " isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha ', the mutant did not express CK2alpha ". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha " apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha " 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 CK2alpha 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

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 alpha 2-macroglobin receptors (6).

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 CK2alpha ", is 91% identical to the CK2alpha isoform. Transfection and overexpression of either the novel CK2alpha " isoform or the previously described CK2alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -MEM without serum. Cells were fused by exposure for 1 min to a 44% solution of polyethylene glycol 1000 in serum-free alpha -MEM. The monolayers were then washed four times with 1.5 ml of serum-free alpha -MEM and once with 1.5 ml of alpha -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 alpha -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

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 alpha -subunit of human CK2. As there already exists a CK2alpha ' isoform (8), we termed this new isoform CK2alpha ".

Transfection of CK2alpha " and Human CK2alpha cDNAs into TRF1 Cells-- The polymerase chain reaction-recovered cDNA encoding CK2alpha " was cloned back into the expression vector pBK-CMV, and the fidelity of the resulting construct (pBK-CMV-CK2alpha ") was established by DNA sequencing. Clones of the human CK2alpha 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-CK2alpha " and pRC-CMV-HA-CK2alpha were transfected into TRF1 cells using a LipofectAMINE Plus kit. The G418-resistant colonies were isolated and expanded.

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 CK2alpha " Subunit-- A peptide containing 16 amino acids near the carboxyl terminus from amino acids 366 to 381 of CK2alpha " (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.

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-CK2alpha subunit antibody (Upstate Biotechnology, Inc.) diluted to 1 µg/ml, CK2alpha and CK2alpha ' isoform-specific antibody diluted 1:5000 (kindly provided by Dr. David W. Litchfield), or CK2alpha " 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-CK2alpha " antibody, a mixture of 1 µg of CK2alpha " peptide, 3 µl of anti-CK2alpha " 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.

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 -70 °C.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).



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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.

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.



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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.

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).



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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.

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 CK2alpha subunit. The new CK2alpha isoform obtained from HuH-7 cells, termed CK2alpha ", has 1507 nucleotides with an open reading frame encoding 385 amino acids (Fig. 4). The cDNA sequence of CK2alpha " is 65.2% identical to CK2alpha cDNA.



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Fig. 4.   Amino acid sequences of CK2alpha and CK2alpha ". The amino acid sequence of CK2alpha (GenBankTM/EBI accession number X70251) is aligned with the deduced sequence of the newly cloned CK2alpha " 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 CK2alpha and CK2alpha " are totally different.

The differences between CK2alpha and CK2alpha " 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 CK2alpha " and CK2alpha are totally different. Despite this difference, when CK2alpha " was expressed in Escherichia coli, cell lysates exhibited a kinase activity equal to that of bacteria transformed with the CK2alpha cDNA using a synthetic peptide (RRKDLHDDEENDAMSITA) as substrate (13). The unique CK2alpha " sequence has previously been reported within clone RP5-863C7 (gi:5788437) as an intronic repeat region (14, 15). Although the genomic structure of CK2alpha " 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 CK2alpha " is either a CK2alpha -derived retroposon (16, 17) or the result of alternative splicing, selectively including an Alu-like exon into the mature mRNA (18, 19).

Expression of CK2alpha Isoforms by HuH-7, TRF1, and Transfected TRF1 Cells-- Western blot analysis using antibody against the common region of CK2alpha and CK2alpha " (amino acids 70-91 of the CK2alpha subunit) indicated that TRF1 cells expressed 40.6 ± 2.7% of the total CK2alpha 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 CK2alpha and CK2alpha " (Fig. 5B). Analysis using antibodies against peptides unique to CK2alpha and CK2alpha ' indicated that TRF1 cells expressed amounts of CK2alpha and CK2alpha ' comparable to the parental HuH-7 cell line (Fig. 6). The predicted molecular mass of CK2alpha " was equivalent to that of the CK2alpha subunit and could not be by resolved from CK2alpha on SDS-PAGE. To determine the expression of CK2alpha " in HuH-7 and TRF1 cells, a rabbit polyclonal antibody directed against a specific region of the C terminus of CK2alpha " was prepared. CK2alpha " expression by HuH-7, TRF1, and CK2alpha "-transfected TRF1 cells was determined by immunoblot analysis. As illustrated in Fig. 6, only HuH-7 and TRF1 cells transfected with CK2alpha " expressed a 44-kDa protein, consistent with the predicted molecular mass of CK2alpha ", whereas TRF1 cells expressed no such protein.



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Fig. 5.   A, expression of CK2alpha and CK2alpha " 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 CK2alpha (TRF1-alpha ), CK2alpha " (TRF1-alpha "), 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 CK2alpha and CK2alpha ". 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 CK2alpha and CK2alpha " 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 CK2alpha isoforms in HuH-7, TRF1, and CK2alpha "-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 CK2alpha "-transfected TRF1 (TRF1-alpha ") 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 CK2alpha isoforms. Antibody deposition was detected by chemiluminescence.

Restoration of Pseudomonas Toxin Resistance-- Transfection of TRF1 cells with the cDNA recovered by expression cloning confirmed that the cloned CK2alpha " 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 CK2alpha or CK2alpha " 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 CK2alpha , resulting in overexpression of the isoform, or with CK2alpha " 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 CK2alpha (TRF1-alpha ), CK2alpha " (TRF1-alpha "), 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 CK2alpha ", CK2alpha , 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.

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 CK2alpha " 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 CK2alpha " 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 (approx 20 µm from the injected cell; n = 12 fields) and to the second cell tier of cells (approx 40 µm from the injected cell) was increased by 70 and 90%, respectively, in the TRF1 cells transfected with CK2alpha ", 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 CK2alpha " 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 CK2alpha " 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 CK2alpha " 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 CK2alpha " transfectant, whereas the conduction velocity remained low in these cells (C).

The reversion to a parental-like gap junction-mediated coupling between TRF1 cells induced by transfection with CK2alpha " 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 CK2alpha " 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 CK2alpha " 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 CK2alpha " isozyme, altering the mobilization of Ca2+, as opposed to a direct effect on gap junction formation.

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 CK2alpha " 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 CK2alpha " 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 CK2alpha " expression. Confluent HuH-7, TRF1, and CK2alpha "-transfected TRF1 (TRF1-alpha ") 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

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 CK2alpha ", is 91% identical in amino acid sequence and 65.2% identical in nucleotide sequence to the previously described CK2alpha (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, CK2alpha and CK2alpha ', have been identified (8). They are the products of distinct genes localized to different chromosomes (27, 28) The newly cloned CK2alpha " represents a new isoform of the CK2alpha family. It is almost identical to CK2alpha 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 CK2alpha " and that TRF1 cells have no detectable CK2alpha ". Transfection of TRF1 cells with a cDNA encoding either CK2alpha or the newly described CK2alpha " restored the parental phenotype to TRF1 cells. Whether reversion of the TRF1 phenotype is due to the absolute level of CK2alpha kinase activity or to a distinct localization of CK2alpha ", which can be compensated for by overexpressing CK2alpha , remains to be resolved.

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 CK2alpha ". 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 CK2alpha ". 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).

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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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