Translated Alu sequence determines nuclear localization of a novel catalytic subunit of casein kinase 2

Philip Hilgard1,*, Tianmin Huang1,*, Allan W. Wolkoff1,2,3, and Richard J. Stockert1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Casein kinase 2 (CK2) is a tetrameric enzyme constitutively expressed in all eukaryotic tissues. The two known isoforms of the catalytic subunit, CK2alpha and CK2alpha ', have been reported to have distinct tissue-dependent subcellular distributions. We recently described a third isoform of the catalytic subunit, designated CK2alpha ", which is highly expressed in liver. Immunoblot analysis of HuH-7 human hepatoma cell fractions as well as immunofluorescent microscopy revealed that CK2alpha " was exclusively localized to the nucleus and preferentially associated with the nuclear matrix. CK2alpha and CK2alpha ' were found in nuclear, membrane, and cytosolic compartments. Deletion of the carboxy-terminal 32 amino acids from the CK2alpha " 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 CK2alpha " 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunits, carrying the catalytic activity, and two beta -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 CK2alpha and CK2alpha ', 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 (alpha alpha beta beta , alpha alpha 'beta beta , alpha 'alpha 'beta beta ), and a specific function of the isoforms has not been discovered. A third, novel isoform of CK2alpha , designated CK2alpha ", which is involved in hepatocellular membrane trafficking, was recently described (47). The newly cloned CK2alpha " is almost identical to the amino acid sequence of CK2alpha until the carboxy-terminal 32 amino acids, which appear to be completely unrelated. The unique CK2alpha " sequence was previously reported within genomic CK2alpha 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 CK2alpha " is either a CK2alpha -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 CK2alpha are completely unrelated to the carboxy-terminal domain of CK2alpha ' and differ substantially from CK2alpha " 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 CK2alpha and CK2alpha ' did not considerably influence the enzymatic activity (14) or subcellular localization (39). Although there appears to be no effect of the CK2alpha " carboxy terminus on enzymatic activity, the present study shows that the CK2alpha " 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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 CK2alpha 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 CK2alpha 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 CK2alpha , -alpha ', and -beta 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 CK2alpha " 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 CK2alpha " 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 DH5alpha -competent cells (Life Technologies), which were plated on LB plates containing 50 µg/ml kanamycin. Clones of the human CK2 and CK2alpha " 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-CK2alpha " 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 [gamma -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-CK2alpha " constructs was detected in cells transfected with pEGFP alone, which served as the negative control.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Subcellular localization of CK2alpha " in hepatocytes is exclusively nuclear. To determine the subcellular localization of CK2alpha ", 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 CK2alpha ", CK2alpha , or CK2alpha ' (Fig. 1). The immunoblot revealed that in HuH-7 cells, CK2alpha " exhibits an exclusive nuclear localization. In contrast, the two other catalytic subunit isoforms, CK2alpha and CK2alpha ', 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 CK2alpha " and CK2alpha . As shown in Fig. 2, anti-CK2alpha " stained nuclei of the cells, whereas anti-CK2alpha stained both the nuclei and the cytoplasm. Probing with anti-CK2alpha ' showed a staining pattern similar to that obtained with anti-CK2alpha (data not shown). To rule out the possibility that the CK2alpha " isoform is a HuH-7-specific variant, nuclear extracts and cytosol were prepared from primary human hepatocytes. As illustrated in Fig. 3, CK2alpha " was detected in the nuclei of isolated human hepatocytes but not in the cytosol, whereas the CK2alpha isoform was found in both compartments. This isoform distribution was equivalent to that seen in HuH-7 cells.


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Fig. 1.   Detection of the three casein kinase 2 (CK2)alpha isoforms in different fractions of HuH-7 cells by immunoblot. HuH-7 cells were fractionated by sucrose density gradient centrifugation as described in MATERIALS AND METHODS. Nuclei were lysed by boiling in SDS-sample buffer and sonication. Equivalent amounts of cytosolic, membrane, and nuclear proteins (50 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to a nitrocellulose membrane. The same membrane was sequentially probed with the primary antibodies against CK2alpha ", CK2alpha , and CK2alpha ' after the membrane was stripped with 0.1 M NaOH for 5 min at room temperature. The signals were visualized by horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescence.



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Fig. 2.   Subcellular localization of CK2alpha " and CK2alpha in HuH-7 cells by indirect immunofluorescence microscopy. HuH-7 cells were grown on collagenized coverslips, fixed, permeabilized, and incubated with primary antibodies against CK2alpha " and CK2alpha . Bound primary antibodies were detected by Cy3-labeled secondary anti-rabbit antibody. Fluorescent (A) and corresponding phase-contrast (B) images were superimposed (C). Control experiments without primary antibody incubation showed no signal.



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Fig. 3.   Distribution of the CK2alpha isoforms in primary human hepatocytes. Cytosol and nuclear extracts of primary human hepatocytes were prepared as described in MATERIALS AND METHODS. Equal amounts of cytosolic and nuclear proteins (50 µg) were resolved by SDS-PAGE. An equivalent amount of HuH-7 nuclear extracts served as a positive control. The proteins were transferred to a nitrocellulose membrane. The membrane was probed with anti-CK2alpha " as the primary antibody and HRP-conjugated anti-rabbit IgG as the second antibody. Signals were visualized by chemiluminescence. The membrane was stripped with 0.1 M NaOH for 5 min and reprobed with anti-CK2alpha as the primary antibody after complete stripping of the signal had been confirmed by exposure to X-ray film for 5 min.

CK2alpha " is associated with nuclear matrix. Because CK2 has been described to be associated with the NM of various cell types (9), we examined whether CK2alpha " in HuH-7 cells was bound to the NM or whether it was preferentially a nucleoplasmic protein. CK2alpha " as well as CK2alpha , CK2alpha ', and CK2beta 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 2alpha " but not of CK2alpha , CK2alpha ', and CK2beta . Together, these results suggested that CK2alpha " 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 CK2alpha " deficient (47), served as a negative control and showed no detectable CK 2alpha " but equivalent amounts of CK2alpha . The fact that Trf1 nuclear extracts and matrix tested negative for CK2alpha " also demonstrated the specificity of the antibodies.


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Fig. 4.   Subnuclear distribution of CK2 subunits in HuH-7 cells. A: HuH-7 and Trf1 cell lysates (two 150-mm plates) were divided into 3 aliquots and centrifuged at 800 g for 3 min. Two of the three resulting nuclear pellets were used for extraction in 60 µl of hypertonic extraction buffer with or without detergent [extract 1 = no detergent, extract 2 = 1% Nonidet P-40 (NP-40)]. The third nuclear pellet was used for preparation of nuclear matrix as described in MATERIALS AND METHODS and finally resuspended in the same amount of buffer as used for extraction (60 µl). Equivalent volumes from the two nuclear extracts and the matrix preparation, containing 38 (extract 1), 62 (extract 2), and 84 (matrix) µg of total protein, were resolved by 10% SDS-PAGE. After transfer, the nitrocellulose membrane was sequentially probed with anti-CK2alpha ", anti-CK2alpha , and anti-CK2alpha ' as described in Fig. 3. A separate membrane was probed with anti-CK2beta . B: equal amounts of protein (50 µg) from nuclear extracts with and without detergent as well as from the nuclear matrix preparation were resolved by 10% SDS-PAGE. After transfer to nitrocellulose, the membrane was probed for the presence of the known nuclear matrix protein lamin B. Anti-lamin B antibody was a gift from Dr. T. Meier, Albert Einstein College of Medicine.

Isoform-specific carboxy terminus of CK2alpha " determines nuclear localization. CK2alpha " and CK2alpha 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 CK2alpha " and 38 amino acids of CK2alpha , respectively. These carboxy termini appear to be unrelated. Therefore, we hypothesized that the tight association of CK2alpha " with elements of the NM might be determined by its carboxy terminus. To address this possibility, the cDNAs encoding CK2alpha " with or without the last 32 amino acids of the ORF as well as the CK2alpha " 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 CK2alpha " ORF (EGFP-CK2alpha "FL) was detected mainly in the nuclear extracts, whereas the fusion protein with the deleted carboxy terminus CK2alpha " ORF (EGFP-CK2alpha "-32) as well as the fusion protein containing only the carboxy-terminal 32 amino acids (EGFP-CK2alpha "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-CK2alpha "FL was localized exclusively to the nuclei of HuH-7 cells (Fig. 7B), whereas EGFP-CK2alpha "-32 as well as EGFP-CK2alpha "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 CK2alpha " 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-CK2alpha "FL exhibited significant binding to the NM, confirming a specific role for the carboxy-terminal sequence in nuclear retention of the CK2alpha " isoforms.


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Fig. 5.   Alignment of the amino acid sequences of CK2alpha and CK2alpha ". The amino acid sequence of CK2alpha " was compared with CK2alpha (GenBank accession no. X70251). Sequence differences between the 2 isoforms, including the unique carboxy terminus, are highlighted with gray boxes. The predicted nuclear localization signal (NLS) of CK2alpha and CK2alpha " is located within the similar region of the isoforms at position 70-81 (italics). The NLS is surrounded by 2 potential autophosphorylation sites (underlined).



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Fig. 6.   Expression of green fluorescent protein (GFP)-CK2alpha " fusion proteins in HuH-7 cells. HuH-7 were transiently transfected with pEGFP vector alone or with pEGFP containing the CK2alpha " open reading frame (ORF) with (EGFP-CK2alpha "FL) or without (EGFP-CK2alpha "-32) the carboxy-terminal 32 amino acids or containing only this carboxy terminus (EGFP-CK2alpha "C-term). After transfection (48 h), cells were harvested and cytosolic as well as nuclear extracts were prepared. Because transfection efficiencies were consistently higher for the pEGFP vector alone and pEGFP-CK2alpha "-32, the amount of cytosolic and nuclear protein resolved by SDS-PAGE and transferred to nitrocellulose was normalized to the corresponding number of GFP-positive transfected cells for each construct. The membrane was probed with a monoclonal antibody against GFP (Clonetech), and the signal was visualized by chemiluminescence after incubation with a HRP-conjugated anti-mouse secondary antibody. nontransfected, Cytosolic and nuclear proteins from untransfected HuH-7 cells (negative control); EGFP, cytosolic and nuclear proteins from HuH-7 cells transfected with pEGFP alone; EGFP-CK2alpha "FL, cytosolic and nuclear proteins from HuH-7 cells transfected with pEGFP containing the full-length CK2alpha " ORF; EGFP-CK2alpha "-32, cytosolic and nuclear proteins from HuH-7 cells transfected with pEGFP containing the CK2alpha " ORF without the carboxy-terminal last 32 amino acids; EGFP-CK2alpha "C-term, cytosolic and nuclear proteins from HuH-7 cells transfected with pEGFP containing only the carboxy-terminal last 32 amino acids of the CK2alpha " ORF.



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Fig. 7.   Distribution of GFP-CK2alpha " fusion proteins in HuH-7 cells. HuH-7 were transiently transfected with the pEGFP constructs described in Fig. 6. Twenty-four hours after transfection, cells were trypsinized and replated on 35-mm MatTek dishes. After an additional 24 h, the distribution of GFP was determined in viable cells by fluorescent microscopy (I). The corresponding phase-contrast images (II) were superimposed (III). A: HuH-7 cells expressing EGFP alone. B: HuH-7 cells expressing an EGFP-CK2alpha " fusion protein with the full-length ORF of CK2alpha " (EGFP-CK2alpha "FL). C: HuH-7 cells expressing EGFP-CK2alpha " fusion protein without the last 32 carboxy-terminal amino acids of CK2alpha " (EGFP-CK2alpha "-32). D: HuH-7 cells expressing an EGFP-CK2alpha " fusion protein containing solely the last 32 carboxy-terminal amino acids of CK2alpha ", without the entire rest of the molecule (EGFP-CK2alpha "C-term).



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Fig. 8.   Association of the EGFP-CK2alpha " fusion proteins with the nuclear matrix. HuH-7 cells were transfected with the 3 different EGFP-CK2alpha " fusion proteins described in Fig. 6. Cells expressing EGFP alone served as a control. Forty-eight hours after transfection, cells were harvested and the nuclear matrix was prepared. Equivalent amounts of matrix proteins (25 µg) were solubilized in SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose. The membrane was probed with a monoclonal antibody against GFP (Clonetech), and the signal was visualized by chemiluminescence after incubation with an HRP-conjugated anti-mouse secondary antibody.

Transfection efficiencies, as indicated by the number of GFP-positive cells in fluorescent microscopy, were consistently higher for the pEGFP vector alone followed by EGFP-CK2alpha "-32, whereas a much lower number of cells expressed EGFP-CK2alpha "FL and EGFP-CK2alpha "C-term. This difference in transfection efficiency between the EGFP-CK2alpha "-32- and EGFP-CK2alpha "FL-containing vectors was also reflected by a twofold level of CK2 activity in anti-GFP-fusion protein immunoprecipitates from lysates of HuH-7 cells transfected with the pEGFP-CK2alpha "-32 vector compared with cells transfected with the pEGFP-CK2alpha FL vector (data not shown). This difference in activity is not inherent to the CK2alpha fusion proteins, because Escherichia coli lysates displayed an equivalent kinase activity when the bacteria were transformed with a prokaryotic expression vector containing CK2alpha , CK2alpha ", or CK2alpha "-32 cDNA.

Nuclear localization of CK2alpha " is not dependent on interference with a nuclear export signal. To further examine the mechanism by which the isoform-specific carboxy terminus of CK2alpha " 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-CK2alpha "-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-CK2alpha "-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-Ikappa B construct (gift from Dr. S. Miyamoto, Dept. of Pharmacology, University of Wisconsin, Madison, WI). Ikappa B, which physiologically inhibits the transcription factor nuclear factor (NF)-kappa 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 Ikappa B, whereas the nucleus shows a weak signal. On treatment with LMB, Ikappa 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-CK2alpha "-32 distribution, there was no effect of LMB on the subcellular localization of the other isoforms of the catalytic CK2 subunit (CK2alpha and CK2alpha ') as indicated by immunoblot analysis (Fig. 10).


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Fig. 9.   Effect of leptomycin B (LMB) on the subcellular distribution of GFP-CK2alpha "-32 and GFP-Ikappa B. HuH-7 were transiently transfected with pEGFP-CK2alpha "-32 or pEGFP-Ikappa B and plated as described in Fig. 7. Before fluorescent microscopy, cells were treated with cycloheximide (50 µg/ml) for 1 h and subsequently with LMB (20 ng/ml) for 30 min. Controls were treated with cycloheximide alone. The distribution of expressed GFP was determined by fluorescent microscopy (I). The corresponding phase-contrast images (II) were superimposed (III). A: HuH-7 expressing EGFP-CK2alpha "-32 without LMB treatment. B: HuH-7 expressing EGFP-CK2alpha "-32 after 30-min treatment with LMB. C: HuH-7 expressing EGFP-Ikappa B without LMB treatment. D: HuH-7 cells expressing EGFP-Ikappa B after 30-min treatment with LMB.



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Fig. 10.   Effect of LMB on the subcellular distribution of CK2alpha and CK2alpha '. Confluent HuH-7 monolayers were treated with cycloheximide (50 µg/ml) for 1 h and subsequently with LMB (20 ng/ml) for 60 min. Controls remained untreated. Cells were harvested, and cytosolic as well as nuclear extracts were prepared. Equivalent amounts of proteins (50 µg) were resolved by SDS-PAGE and transferred to nitrocellulose. The membranes were probed with the polyclonal antibodies against CK2alpha and CK2alpha ', and the signal was visualized by chemiluminescence after incubation with a HRP-conjugated anti-rabbit secondary antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (CK2alpha and CK2alpha ') in combination with the activity-enhancing CK2beta subunit proposed to form the tetrameric holoenzyme (12, 13) composed of two alpha - and two beta -subunits (alpha 2beta 2, alpha alpha 'beta 2, alpha '2beta 2). The discovery of a third isoform of the CK2alpha subunit (CK2alpha ") 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 CK2alpha and CK2alpha " appear to be equivalent, the subcellular localization of CK2alpha " 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-CK2alpha 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 CK2alpha , CK2alpha ', and CK2beta 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 CK2alpha " 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 CK2alpha -GFP fusion protein (23, 30, 40), all three members of the CK2alpha family were recovered in the nuclear fraction. The crystal structure of CK2alpha 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 alpha -/beta -importin (34, 42, 58). In contrast to CK2alpha ", which was exclusively localized to the nuclear fraction, CK2alpha and CK2alpha ' were distributed through all three fractions that were isolated. The broader distribution for CK2alpha and CK2alpha ' was confirmed by indirect immunofluorescence microscopy (Fig. 2; data for CK2alpha ' not shown), and similar findings in primary human hepatocytes established that the unique nuclear localization of CK2alpha " 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 CK2alpha " 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 CK2alpha " was preferentially associated with the NM (Fig. 4). In contrast, CK2alpha , CK2alpha ', and CK2beta were equally distributed between nucleoplasmic and NM fractions. Contrary to other reports in different cell types (9), this finding suggests that CK2alpha , CK2alpha ', and CK2beta 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 alpha -subunits in combination with two of the regulatory beta -subunit. However, this distribution does not eliminate the possibility of a monomeric enzyme comprised of single catalytic alpha -subunits in the absence of the regulatory beta -subunit (24). A monomeric configuration would be less likely if the beta -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 CK2alpha and CK2alpha " is the difference in their respective carboxy-terminal regions. Transient transfection of HuH-7 cells with the vector encoding the GFP-CK2alpha "FL fusion protein confirmed the almost exclusively nuclear localization of the CK2alpha " isozyme (Fig. 7). Deletion of the last 32 amino acids of the carboxy terminus of CK2alpha " resulted in cytosolic and nuclear localization of the fusion protein, a distribution similar to that of CK2alpha and CK2alpha ' as detected by cell fractionation. This finding clearly supports a positive role for this part of the molecule in determining the subcellular distribution of CK2alpha " 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 CK2alpha and CK2alpha ' (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 CK2alpha 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-CK2alpha "-32 (deleted carboxy terminus; Fig. 9) into the cytosol of HuH-7 cells or shift CK2alpha and CK2alpha " 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 CK2alpha " 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 CK2alpha " to the cytosol.

It appears that the CK2alpha " 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 CK2alpha " (Fig. 4). 2) The isoform-specific carboxy terminus of CK2alpha " was not capable of altering the subcellular distribution of the EGFP-CK2alpha "C-term fusion protein (Fig. 7D). 3) Only the EGFP-CK2alpha "FL fusion protein was found to bound to the NM, whereas EGFP-CK2alpha "-32 and EGFP-CK2alpha "C-term showed no NM association (Fig. 8). To date, a number of proteins that interact selectively with CK2alpha isoforms potentially mediating NM binding have been identified (21, 26, 46). A reduction in the association of CK2alpha " 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 CK2alpha , CK2alpha ', and CK2alpha " NM association reflects an isoform-specific functional difference. Although the restricted nuclear localization of CK2alpha " 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 CK2alpha " 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 CK2alpha " isoforms raises two possibilities. One explanation could be that the absence of a small cytoplasmic pool of CK2alpha " 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 CK2alpha " 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 CK2alpha " 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 CK2alpha results in its preferential association with NM (61) and restoration of the parental phenotype to CK2alpha -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-Ikappa B. 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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