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J. Biol. Chem., Vol. 278, Issue 46, 45611-45619, November 14, 2003

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Scapinin, a Putative Protein Phosphatase-1 Regulatory Subunit Associated with the Nuclear Nonchromatin Structure^*

Junji Sagara¶, Tsukasa Higuchi, Yukiko Hattori, Mie Moriya, Haritha Sarvotham, Hiroshi Shima||, Haruki Shirato||, Kunimi Kikuchi||, and Shunichiro Taniguchi

From the Department of Molecular Oncology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 and the ^||Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan

Received for publication, May 19, 2003 , and in revised form, August 14, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654–1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein phosphatase-1 (PP1)¹ is a major eukaryotic serine/threonine protein phosphatase regulating diverse cellular processes such as muscle contraction, glycogen metabolism, RNA processing, and neuronal signaling (1–3). The catalytic subunit of PP1 binds to regulatory subunits that are critical for substrate specificity and spatial control of PP1 within the cell (4).

It is thought that the nuclear nonchromatin structures, such as the nuclear matrix (NM) and nuclear lamina, are implicated not only in the spatial segregation of chromatins but also in the regulation of various nuclear processes associated with them (5–7). However, the regulatory mechanisms of the nuclear nonchromatin structure are not fully understood. The -catalytic subunit of PP1 is associated with NM during interphase, and PP1 is a mitotic lamin phosphatase (8, 9). Recruitment of PP1 to the nuclear envelope by a PP1-binding protein, protein kinase A-anchoring protein AKAP-149, is a prerequisite for nuclear lamina assembly at the end of mitosis (10). Identification of PP1-binding proteins associated with the nuclear nonchromatin structure will provide insights into the regulatory roles of PP1 in the nuclear structure and gene expression.

Aberrant nuclear structures are hallmarks of neoplastic transformation (11). Nuclear lamins alter during differentiation and transformation (12, 13). Many reports have described striking alterations in the NM proteins induced by transformation (14–18). The mechanisms and physiological importance of this phenomenon are not fully understood, and identification of the alterations may provide more insight into differentiation and transformation.

HL-60 cells have been used as an in vitro model of differentiation because various compounds can cause differentiation of these cells into granulocytes or monocytes (19, 20). In this study, we analyzed differentiation-associated alterations of the chromatin-depleted nuclear structure, referred to as the NM-intermediate filament (IF) scaffold (21), of HL-60 cells. We then identified a novel protein, scapinin, which was tightly attached to the NM-IF scaffold of the nucleus and down-regulated by differentiation in HL-60 cells. Furthermore, we identified PP1 as a scapinin-binding protein by the yeast two-hybrid assay.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—HL-60 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. To induce differentiation, HL-60 cells (2 x 10⁵/ml) were treated with 100 nM all-trans-retinoic acid (RA). Maximum reduction of nitro blue tetrazolium (a differentiation marker) occurred after treating HL-60 cells with RA for 5 days. HL-60 cells (5 x 10⁵/ml) were also treated with 10 nM phorbol 12-myristate-13-acetate or 1.2% dimethyl sulfoxide. U937, K562, and Jurkat cells were cultured in RPMI 1640, and HeLa, WM35, SKNSH, and GOTO cells were cultured in Dulbecco's modified minimal essential medium. All media were supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin.

Preparation of the NM-IF Scaffold and IF Fraction of HL-60 Cells—The NM-IF scaffold was prepared as described previously (21). Briefly, the cells were suspended in 0.5% Triton X-100 and cytoskeleton buffer (10 mM PIPES, pH 6.8, 100 mM KCl, 300 mM sucrose, 3 mM MgCl₂,1mM EGTA, 4 mM vanadyl riboside complex) on ice for 5 min before separating the particulate (P1) and soluble (Sol) fractions by centrifugation at 600 x g for 5 min. The P1 fraction was incubated with 0.15 mg/ml DNase I at 23 °C for 30 min. Ammonium sulfate (1 M) was added to obtain a final concentration of 0.25 M, and the chromatin-depleted fraction (P2) was then collected by centrifugation. The P2 fraction was further extracted with a 2 M NaCl solution. Fey et al. (21) referred to this final particulate fraction as the NM-IF scaffold. The NM-IF scaffold was separated into the NM and IF fractions as described previously (23). The NM-IF scaffold was dissolved in an 8 M urea solution containing 20 mM MES, pH 6.6, 0.1 mM MgCl₂, 1 mM EGTA, and 1% 2-mercaptoethanol. After ultracentrifugation at 100,000 x g for 1 h, the supernatant was dialyzed against a buffer containing 25 mM imidazole, pH 7.1, 150 mM KCl, 5 mM MgCl₂ and 0.125 mM EGTA. The reassembled (IF fraction) and soluble (NM) proteins were separated by centrifugation at 10,000 x g for 15 min. To release RNA-containing matrix, the NM-IF scaffold was digested with RNase A in cytoskeleton buffer containing protease inhibitors; NaCl was then added to a final concentration of 2 M from a stock solution of 4 M NaCl in cytoskeleton buffer. 1 mM dithiothreitol was included in all solutions used in preparation of the NM-IF scaffold and RNase A digestion. RNase A (Sigma) was boiled for 20 min to destroy DNase and protease contaminants. The efficiency of RNase A treatment was monitored by the loss of heterogeneous nuclear ribonucleoprotein (hnRNP)-U. Anti-hnRNP-U antibody was purchased from Santa Cruz Biotechnology.

Development of a Monoclonal Antibody—The NM-IF scaffold proteins from proliferating or RA-treated HL-60 cells were mixed with complete Freund's adjuvant and immunized to BALB/c mice (intraperitoneal, 0.4 mg of protein/mouse). After 3 weeks, mice were boosted with 0.2 mg of the NM-IF scaffold proteins mixed with incomplete Freund's adjuvant (intraperitoneal). At 3 days after the boost, the spleen cells were collected, fused with myeloma cells, and selected in HAT medium (22). The NM-IF scaffold proteins from proliferating or RA-treated HL-60 cells were suspended in phosphate-buffered saline by sonication at a final concentration of 10 µg/ml, and each suspension was added to 96-well enzyme-linked immunosorbent assay plates at 0.1 ml/well. The anti-scapinin antibody exhibited a positive reaction in an enzyme-linked immunosorbent assay plate coated with the NM-IF scaffold proteins of proliferating cells but was negative in that of RA-treated cells.

Cloning of Scapinin cDNAs—A gt11 cDNA library was constructed from poly(A)⁺ RNA of HL-60 cells and immunoscreened as described (22). Positive clones were amplified by PCR with gt11 forward primer 5'-GGTGGCGACGACTCCTGGAGCCCG-3' and gt11 reverse primer 5'-TTGACACCAGACCAACTGGTAATG-3'. The amplified DNA fragment was subcloned into the pBluescript SK^- vector (Stratagene) and subjected to DNA sequencing. The 5'-region of the DNA fragment was cloned using a 5'/3'-RACE kit (Roche Applied Science) according to the manufacturer's instructions. The 3'-region of the DNA fragment was cloned by the 3'-RACE system (Invitrogen). The amplified fragment was ligated into the pGEM-T easy vector (Promega) and sequenced. The coding regions of scapinin-S and scapinin-L were obtained by RT-PCR using a RT primer 5'-AATCTCTATGGCCTGTGGAA-3', a reverse primer 5'-TCTCTATGGCCTGTGGAATCT-3', a forward primer 5'-CTGGATGAGATGGACCAAACG-3', a template poly(A)⁺ RNA of HL-60 cells, and a high fidelity RNA PCR kit (Takara, Tokyo, Japan). The RT-PCR products of 1.35 and 1.55 kb were separated by 1% agarose gel electrophoresis, integrated into pBluescript SK^- (SmaI site), and sequenced.

Western Blotting and Immunofluorescence Analyses—Western blotting was performed as described previously (22). Immunofluorescence was performed as below. Whole HL-60 cells and the NM-IF scaffold of HL-60 cells were spread on glass slides using Cytospin II (Shandon Southern, Sewickly, PA), fixed with cold methanol for 15 min, and soaked with TBS containing 1% bovine serum albumin and 2% fetal bovine serum at room temperature for 15 min. The cells were incubated for 1 h at room temperature with culture medium from an antibody-producing hybridoma. After rinsing several times with TBS, the cells were incubated with fluorescein isothiocyanate (FITC)- or rhodamine-conjugated goat anti-mouse IgG antibody for 30 min. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) at 1 µg/ml for 5 min. To visualize the morphology of the NM-IF scaffold, specimens were incubated for 10 min with a FITC-labeled anti-lamin B antibody at 1 µg/ml. The anti-lamin B antibody was labeled using a Fluoro-Tag^TM FITC conjugation kit (Sigma). After rinsing with TBS, the specimens were observed using a fluorescent microscope (Axiovert S100, Carl Zeiss, Germany). HL-60 cells were also immunostained with rabbit polyclonal antibodies (Santa Cruz Biotechnology) against the splicing factor SC35, promyelocytic leukemia protein, or proliferating cell nuclear antigen.

Northern Blotting Analysis—Total RNAs of tumor cells were separated by electrophoresis in 1% agarose gels containing formaldehyde, blotted onto Hybond^TM-N (Amersham Biosciences) membranes, and then cross-linked with 7000 J/cm² ultraviolet light. A 3'-untranslated region of scapinin cDNA (0.5 kb), amplified by PCR using the forward primer 5'-TGATACCAACACTGAACATT-3' and reverse primer 5'-AACAGGAATCAAAATGTTGCT-3', was used as the probe. The probe was labeled using the Multiprime DNA labeling system (Amersham Biosciences) and [³²P]dCTP and hybridized with the blots in 5 x SSC overnight at 68 °C. Human multiple-tissue RNA blots (Clontech) were also examined.

Expression of Scapinin—cDNAs for both scapinin-S and scapinin-L were integrated into pcDNA3 (EcoRV site) and transfected to COS-7 cells using the transfection reagent FuGENE 6 (Roche Applied Science). The coding regions of scapinin-S and scapinin-L were amplified by PCR using the following primers to introduce restriction enzyme sites: 5'-gggaattcCCAGCTCTGGATGAGATGGAC-3' (forward primer) and 5'-gggctcgagATCTCCAACCACTTTCCAT-3' (reverse primer). The PCR products were then inserted into pEGFP-C2 (Clontech). The pEGFP-C2-scapinin constructs were transfected into cells grown on glass coverslips.

Yeast Two-hybrid Assay—Protein-protein interactions were analyzed with the MATCHMAKER^TM two-hybrid system (Clontech) according to the manufacturer's instructions. The full-length and fragmented cDNAs of scapinin-L were inserted into the EcoRI-PstI sites of pGBT9 in-frame with the GAL4 binding domain. The yeast strain HF7c was transformed by the bait plasmid and sequentially by pACT2 plasmid containing the human placenta MATCHMAKER^TM cDNA library (Clontech). Cotransformants grown up in tryptophan-, leucine-, and histidine-deprived SD media were analyzed for -galactosidase activity. -Galactosidase activities were measured according to the Yeast Protocols Handbook (Clontech). Recombinant yeast strains were incubated at 30 °C overnight in 5 ml of the appropriate SD medium. Then, 2 ml of the overnight culture was added to 8 ml of YPD medium and incubated at 30 °C until the A₆₀₀ > 0.5. After washing with Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄), 0.5 ml of the second culture was resuspended in 0.1 ml of Z buffer. Cells were lysed by repeated freeze/thawing and diluted with 0.7 ml of Z buffer containing -mercaptoethanol (100:0.27). The reaction was then initiated by the addition of 160 µl of 4 mg/ml o-nitrophenyl -D-galactopyranoside (Sigma) and incubated at 30 °C. The A₄₂₀ was measured after stopping the reaction with 0.4 ml of 1 M Na₂CO₃. -Galactosidase activity was determined from the values of A₄₂₀ and A₆₀₀ using Equation 1

(Eq. 1)

where t is the elapsed time of the reaction (min). U > 2. 0 was recognized as high (++), U = 0.2–2.0 as low (+), and U < 0.2 as negative (-).

Coimmunoprecipitation Assay—The -catalytic subunit of PP1 was expressed as a HA-tagged protein using the pcDNA3 expression vector as described (24). 5 x 10⁶ COS-7 cells were cotransfected with pcDNA3-scapinin (S or L) and pcDNA3-HA-PP1c. 1.25 µg of DNA of each plasmid was used for the transfection. For the control experiments, empty pcDNA3 plasmid was added to adjust the total DNA to 2.5 µg. Transfected cells were lysed in immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 10% glycerol, 1% Triton X-100, and protease inhibitors) with the aid of sonication, and cell debris was removed by centrifugation at 15,000 x g for 20 min. The cell lysate was divided into two aliquots and incubated with either anti-scapinin antibody-conjugated Sepharose 4B or anti-HA antibody-conjugated agarose beads (Roche Applied Science) for 3 h. Immune complexes were collected by centrifugation (1,500 x g, 1 min), washed three times, and eluted in SDS-sample buffer. Immunoprecipitates were analyzed by Western blotting using either anti-scapinin or anti-HA (Roche Applied Science) antibodies.

PP1 Binding Assay by Far-Western Analysis—Scapinin was purified from HL-60 cells. The NM-IF scaffold was dissolved in RIPA buffer (0.2% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, and 50 mM Tris-HCl, pH 8.0), and the clarified lysate was incubated with anti-scapinin antibody-conjugated Sepharose 4B for 3 h at 4 °C. After washing three times with RIPA buffer (SDS concentration was reduced to 0.1%), scapinin was eluted in SDS-sample buffer. Purified scapinin was separated by SDS-PAGE and then blotted on nitrocellulose membrane (Schleicher & Schuell). Far-Western analysis was performed as described (25). Briefly, blots were incubated with or without 0.2 µg/ml recombinant PP1c- (Sigma) in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) containing 0.5 mM MnCl₂ for 4 h at 4 °C. Bound PP1c was detected by incubation with rabbit anti-PP1c antibody (Santa Cruz Biotechnology) for 1 h followed by incubation with goat horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody for 30 min.

PP1 Inhibition Assay—Scapinin deletion mutants were expressed as fusion proteins with GST. The cDNA fragment of scapinin was amplified by PCR using a specific primer set, to which EcoRI (forward primer) or SalI (reverse primer) recognition sequences were added. After digestion with EcoRI and SalI, the PCR product was ligated into pGEX-4T-1 (Amersham Biosciences) and cleaved with EcoRI and SalI. GST fusion proteins were produced in BL21 bacteria and purified with glutathione-Sepharose 4B (Amersham Biosciences). The catalytic subunit of PP1 was purified from rabbit skeletal muscles, and PP1 activity was measured as described (25). Reaction mixtures (30 µl) contained 50 mM Tris-HCl, 0.15 mM EDTA, 15 mM 2-mercaptoethanol, 0.01% (w/v) Brij 35, 0.3 mg/ml bovine serum albumin, 5 mM caffeine, 10 µM [³²P]phosphorylase a, and 100 ng/ml PP1 with various concentrations of GST or GST-scapinin fusion proteins.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Association of Scapinin with the Nuclear Scaffold in HL-60 Cells—We developed monoclonal antibodies against NM-IF scaffold proteins of HL-60. We obtained a monoclonal antibody, an anti-scapinin antibody, which recognized two polypeptides, with apparent molecular masses of 60 and 75 kDa; both polypeptides were concomitantly concentrated in the NM-IF scaffold (Fig. 1A). The NM-IF scaffold was separated further into the NM and IF fractions by solubilization in an 8 M urea solution before reassembly in a physiological solution as described previously (23). Reassembled proteins (IF fraction) were separated from soluble proteins (NM fraction) by centrifugation (Fig. 1B). The two polypeptides were mostly recovered in the IF fraction.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Association of scapinin with the NM-IF scaffold in proliferating HL-60 cells. A, HL-60 cells were extracted sequentially as described by Fey et al. (21). Cultured suspension cells were collected, suspended in a 0.5% Triton X-100 and cytoskeleton buffer, and separated into soluble (Sol) and particulate (P1) fractions. The chromatin DNA was released from the P1 fraction by digestion with DNase I followed by the addition of 1 M ammonium sulfate to a final concentration of 0.25 M. The chromatin DNA-depleted fraction (P2) was collected and further extracted with 2 M NaCl. The resultant insoluble fraction was referred to as the NM-IF scaffold. 40 µg of each protein fraction was separated by SDS-PAGE and subjected to Coomassie Brilliant Blue staining (CBB) or Western blotting (WB) with the anti-scapinin antibody. Histones (arrows) are depleted from the NM-IF scaffold. B, the NM-IF scaffold was dissolved in an 8 M urea solution, and insoluble materials were discarded by ultracentrifugation. The solubilized proteins were dialyzed against an IF-polymerizing buffer, and the resultant reassembled (IF) and soluble (NM) proteins were separated by centrifugation. Aliquots of each fraction were analyzed as above. C, aliquots of the NM-IF scaffold of HL-60 cells were digested with indicated concentrations of RNase A (boiled) in cytoskeleton buffer containing protease inhibitors at 20 °C for 20 min. The RNase-A resistant insoluble fraction was collected and subjected to Western blotting with anti-scapinin antibody (upper panel) or anti-hnRNP-U antibody (lower panel). Recovery was expressed as a percent against mock treatment (0 µg/ml RNase A).

The anti-scapinin antibody strongly stained the nuclei of HL-60 cells (Fig. 2A). Immunostaining also showed association of the scapinin antigen with the NM-IF scaffold of the nucleus (Fig. 2B). These results demonstrate that the scapinin antigen is a component of the nuclear scaffold of HL-60 cells.

View larger version (35K): [in this window] [in a new window]   FIG. 2. Localization of scapinin in the nucleus. A, proliferating HL-60 cells were collected by centrifugation, fixed in cold methanol (-20 °C), and immunostained with the anti-scapinin antibody. FITC-conjugated goat anti-mouse Ig antibody was used as the secondary antibody. The nuclei were stained with DAPI. B, the NM-IF scaffold was prepared as in Fig. 1, fixed in cold methanol, and immunostained with the anti-scapinin antibody and rhodamine-labeled anti-mouse Ig goat serum. The nuclei were stained with FITC-conjugated anti-lamin B antibody. Bars, 10 µm.

Cloning of Two Splicing Variants of Scapinin—We obtained a cDNA fragment (1.0 kb) by immunoscreening of a gt11 cDNA library and then cloned a 2.3-kb cDNA by 3'- and 5'-RACE. The 2.3-kb cDNA encoded a novel polypeptide of 518 amino acids (Fig. 3A). Prediction of the protein sorting signals and localization sites (PSORT; www.psort.ims.u-tokyo.ac.jp) of scapinin indicated nuclear localization at 94.1% reliability. To prepare the coding region, we performed RT-PCR using primer sets that were designed outside the start and stop codons. As a result, we obtained two RT-PCR products that encoded two splicing variant polypeptides of 448 (scapinin-S) and 518 (scapinin-L) amino acids, respectively (Fig. 3A). A search of the human genome databases (www.ncbi.nlm.nih.gov/genome) demonstrated that the scapinin gene was located in chromosome 20q13.32 (130 kb) and composed of 13 exons. Scapinin-S lacks exon 5 encoding 70 amino acids (shaded in Fig. 3A). Expression experiments revealed that scapinin-S and scapinin-L cDNAs encoded 60- and 75-kDa polypeptides, respectively, which comigrated with the authentic polypeptides of HL-60 cells (Fig. 3B).

View larger version (38K): [in this window] [in a new window]   FIG. 3. Two splicing variants of scapinin. A, two cDNAs encoding the splicing variant polypeptides of scapinin were obtained by immunoscreening a gt11 cDNA library with the anti-scapinin antibody, 5'/3'-RACE, and RT-PCR as described under "Experimental Procedures." The long form (L) of scapinin, which is composed of 518 amino acids, is presented in this figure. The short form with 468 amino acids (scapinin-S) lacks 70 amino acids (shaded) containing a proline-rich sequence (bold underline). Thin underlines represent putative nuclear localization signals. The anti-scapinin antibody used in this study recognizes the polypeptide from amino acids 131 to 350. B, scapinin-S and scapinin-L cDNAs were expressed in COS-7 cells using the pcDNA3 expression vector, and the cell lysates were subjected to Western blotting. The expressed scapinin-S and scapinin-L comigrate with the 60-kDa and 70-kDa polypeptides of endogenous scapinin-S and scapinin-L of HL-60 cells, respectively.

Scapinin Expression in Human Tumor Cell Lines and Normal Tissues—Northern blotting analyses showed that scapinin was expressed in HL-60 (leukemia), U937 (leukemia), and GOTO (neuroblastoma) cells, but not in K562 (leukemia), Jurkat (lymphoma), HeLa (uterine carcinoma), WM35 (melanoma), or SKNSH (neuroblastoma) cells (Fig. 4A). In normal human tissues, scapinin transcripts were abundant in the brain (Fig. 4B). When the same blot was exposed for longer, scapinin transcripts were detectable in the thymus, spleen, and placenta (data not shown). The high expression of scapinin in the brain was not anticipated. We then analyzed human EST clones. As shown in Table I, 21 of a total of 58 scapinin EST clones were derived from brain cDNA libraries. The other scapinin EST clones were derived from pooled tissues (mixtures of normal tissues or whole embryo) or tumor tissues.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Expression of scapinin in human cell lines and tissues. A, 20 µg of total RNAs from tumor-derived cell lines was subjected to Northern blotting analysis. A scapinin cDNA fragment (0.5 kb) in the 3'-untranslated region was used as a probe. HL-60 (leukemia), U937 (leukemia), K562 (leukemia), Jurkat (lymphoma), HeLa (epitheloid carcinoma), WM35 (melanoma), SKNSH (neuroblastoma), and GOTO (neuroblastoma) cell lines were examined. B, an RNA blot of normal human tissues (Clontech) was hybridized with the scapinin cDNA probe as above. Each blot was rehybridized with a glyceraldehyde-3-phosphate dehydrogenase probe (GAPDH) as a control.

View this table: [in this window] [in a new window]   TABLE I Human EST clone analysis of scapinin Scapinin ESTs were obtained by BLAST searches of the human EST data base (www.ncbi.nlm.nih.gov) using scapinin-L cDNA (2.3 kb) as a query sequence.

Down-regulation of Scapinin in HL-60 Cells by Induction of Differentiation—Western and Northern blotting showed that scapinin was decreased by RA treatment of HL-60 cells (Fig. 5A). Furthermore, down-regulation of scapinin was induced by incubation with other differentiation-inducing agents, phorbol 12-myristate-13-acetate and dimethyl sulfoxide (Fig. 5B). Immunostaining showed that the positive signals for scapinin antigen in the nucleus were reduced in HL-60 cells treated with differentiation-inducing chemical compounds (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 5. Down-regulation of scapinin in HL-60 cells during differentiation induced by various chemical compounds. Scapinin was measured by Western blotting (WB) and Northern blotting (NB) as described in Figs. 1 and 4, respectively. HL-60 cells were cultured with (A) 100 nM RA for 3 days, or (B) 10 nM phorbol 12-myristate-13-acetate for 1 day (PMA) or 1.2% dimethyl sulfoxide for 1 day (DM). HL-60 cells were also cultured without chemical compounds (C) as a control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (35K): [in this window] [in a new window]   FIG. 6. Association of expressed scapinin-S and scapinin-L with the NM-IF scaffold in the cells. A, pcDNA3 expression plasmids encoding scapinin-S or scapinin-L were transfected to 2 x 106 COS-7 cells. After 48 h of incubation, the NM-IF scaffold was prepared as in Fig. 1. 20 µg of protein of the whole cell lysate (Whole), soluble fraction (Sol), and NM-IF scaffold were subjected to Western blotting analysis with the anti-scapinin antibody. B, aliquots of the NM-IF scaffold of scapinin-expressing cells were digested with the indicated concentrations of RNase A (boiled) in cytoskeleton buffer as in Fig. 1C. The RNase-A resistant insoluble fraction was collected and subjected to Western blotting with anti-scapinin antibody (top and middle panels) or anti-hnRNP-U antibody (bottom panel). Recovery of each polypeptide was expressed as a percent against mock treatment (0 µg/ml RNase A).

View larger version (58K): [in this window] [in a new window]   FIG. 7. Subcellular localization of expressed scapinin-S and scapinin-L in the cells. HeLa cells were grown on glass coverslips and transfected with pEGFP-c2 expression plasmids encoding GFP alone, GFP-scapinin-S, or GFP-scapinin-L. After 24 h of incubation, the distributions of GFP and GFP-scapinin fusion proteins were observed. GFP-scapinin-L was localized in the nucleus and some cytoplasmic extensions. An arrow in the right panel (GFP-scapinin-L) points to cellular extensions, which are enlarged in the inset picture of GFP-scapinin-L staining. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   FIG. 8. Binding of PP1c to scapinin and inhibition of PP1 activity by scapinin. A, coimmunoprecipitation assay. pcDNA3 expression plasmids encoding scapinin-S, scapinin-L, or HA-tagged -catalytic subunit of PP1 (HA-PP1c) were transfected to 5 x 106 COS-7 cells as indicated. After 48 h of incubation, cells were lysed in immunoprecipitation buffer by sonication. Cleared cell lysates were subjected to coimmunoprecipitation experiments using either anti-HA or anti-scapinin antibodies. The resultant immunoprecipitates (IP) were analyzed by Western blotting (WB) using either anti-scapinin or anti-HA antibodies (upper two panels). Cell lysates were also analyzed by Western blotting (lower two panels). B, far-Western blotting analysis (Far-WB). Scapinin was affinity purified from HL-60 cells using anti-scapinin antibody-coupled Sepharose 4B and blotted onto nitrocellulose membrane. The blots were incubated without (-) or with (+) recombinant PP1c, and bound PP1c was detected with rabbit anti-PP1c antibody (first antibody) and goat horseradish peroxidase-anti-rabbit immunoglobulin antibody (second antibody). Coomassie Brilliant Blue staining (CBB) of purified scapinin is presented. C, assignment of a PP1 binding region by a yeast two-hybrid assay. The catalytic subunit cDNA of PP1 and each scapinin cDNA fragment were inserted into a vector set of the two-hybrid system and introduced into yeast HF7c. Selection marker-positive yeast colonies were subjected to an in vitro -galactosidase assay. -Galactosidase activity was expressed in arbitrary units: ++ (high), + (low), and - (negative) (see "Experimental Procedures"). D, inhibition of PP1 activity by scapinin in vitro. Each scapinin fragment was produced as a fusion protein with GST and subjected to a PP1 inhibition assay in which phosphorylated phosphorylase a was used as the substrate (24). Inhibitor-2 was used as a positive control for PP1 inhibition.

To determine the binding region of scapinin with PP1c, deletion mutants of scapinin were subjected to the yeast two-hybrid binding assay. This experiment revealed that the COOH-terminal region (about 70 amino acids) of scapinin was the PP1 binding region (Fig. 8C). In this region, a short binding sequence (RVXF motif), which is present in many PP1-binding proteins, is absent.

It is known that many PP1-binding proteins inhibit PP1 activity. Deletion mutants of scapinin were expressed as GST fusion proteins and subjected to PP1 inhibition assays (Fig. 8D). GST-scapinin (438–518) inhibited PP1 activity, whereas GST-scapinin (465–518) and GST did not. Importantly, a deletion mutant lacking 10 amino acids from the COOH terminus, GST-scapinin (438–508), moderately inhibited PP1 activity. These results indicate that scapinin binds to and inhibits PP1 and that its COOH-terminal region is responsible for PP1 binding and PP1 inhibiting activities.

Scapinin Family—BLAST searches demonstrated the presence of scapinin homologs in worms (Caenorhabditis elegans), insects (Drosophila melanogaster), mice, and humans, although their functions were unknown. In particular, the NH₂-terminal and COOH-terminal regions were conserved among them (Fig. 9). Searches of the conserved domain data base showed the presence of an RPEL repeat (pfam0257, smart0707) in the COOH-terminal conserved region (Fig. 9B). The RPEL repeat is named after the four conserved amino acids (arginine, proline, glutamic acid, and leucine) that it contains. The function of the RPEL repeat is unknown. Furthermore, all of these proteins have a proline-rich sequence, except for scapinin-S, which lacks the proline-rich region after RNA alternative splicing (Fig. 9C). These results suggest that scapinin and its homologs are members of a novel family that are structurally conserved from worms to humans. We therefore termed this family the scapinin family.

View larger version (65K): [in this window] [in a new window]   FIG. 9. Alignment and structure of scapinin family proteins in humans (scapinin, KIAA0680, AK025436 [GenBank] , KIAA1733, and dj227A7.2), D. melanogaster (AE003479 [GenBank] ), and C. elegans (F26H9.2). The highly conserved regions that reside in the NH2 terminus (A) or COOH terminus (B) of scapinin are aligned. Protein BLAST searches of the conserved domain data base in NCBI reveals RPEL repeats (shaded) in the highly conserved regions. The RPEL repeat (smart00707) is named after the four conserved amino acids it contains; however, the function is unknown. A putative PP1 binding region in scapinin is indicated (top line)in B. C, schematic structures of the scapinin family. Scapinin family proteins share conserved NH2-terminal and COOH-terminal regions, although there are several exceptions. A long glutamine repeat exists in the NH2-terminal region of the D. melanogaster protein. Scapinin family proteins have proline-rich sequences. A putative PP1 binding region in scapinin is also indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The NM-IF scaffold includes a nuclear filament network composed of RNA and RNPs, which is released from the nuclear insoluble scaffold by RNase digestion (21). About half of the scapinin was released from the NM-IF scaffold by RNase digestion, but the balance was resistant to extensive RNase A digestion (Figs. 1C and 6B). By contrast, hnRNP-U was mostly released by such treatment. These results suggest that a scapinin-containing structure is stabilized by interaction with the RNA-based nuclear network and is impaired and partially released by the destruction of RNA-base nuclear network. Alternatively, some of the scapinin is directly associated with the RNA-based nuclear network. Scapinin has no RNA binding motifs existing in RNPs and other RNA-binding proteins. Further studies are needed to clarify the relationships between scapinin and the RNA-based nuclear network.

Most of the regulatory subunits bind to PP1 through a short conserved sequence, the RVXF motif. However, some putative regulatory subunits do not possess the RVXF motif but bind tightly to PP1 by combination of weak interactions at multiple sites (4, 29). Scapinin does not possess an RVXF motif. PP1 binding and inhibition assays demonstrated that the COOH-terminal segment of 70 amino acids is needed for the full PP1 binding and inhibitory activities (Fig. 8). The deletion experiment demonstrated that a critical region for PP1 binding resides around amino acids 449–465 (Fig. 8C). In addition, deletion of 10 amino acids from the COOH terminus reduced the PP1 inhibiting activity (Fig. 8C). These results suggest that multiple sites in the COOH-terminal region of scapinin are responsible for the full PP1 binding activity.

PP1 plays crucial roles in the regulation of various cellular processes in both the nucleus and the cytoplasm. It is thought that the nuclear nonchromatin structures, such as the NM and nuclear lamina, are implicated not only in the spatial segregation of chromatins but also in the regulation of various nuclear processes associated with them. However, the regulatory mechanisms of the nuclear scaffold are not fully understood. Scapinin is a putative PP1 regulatory subunit associated with a nuclear nonchromatin structure, suggesting a regulatory role of scapinin in the nucleus. Therefore, further studies on scapinin may provide an insight into the regulatory roles of the nuclear scaffold system.

Human promyelocytic leukemia HL-60 cells, which proliferate continuously in suspension cultures, can be induced to differentiate into morphologically and functionally mature granulocytes or monocytes by incubation with a wide variety of compounds (19, 20). Scapinin was down-regulated in HL-60 cells during differentiation induced by RA, dimethyl sulfoxide, and phorbol 12-myristate-13-acetate. Expression of PP1c- was not reduced in HL-60 cells during differentiation. The down-regulation of scapinin occurred at the transcriptional level (Fig. 5). Northern blotting and EST clone analyses suggest that scapinin expression is restricted to some types of tumors and that it is not a housekeeping gene product (Fig. 4 and Table I). Therefore, scapinin may be related to transformed or immature phenotypes of some types of tumor cells. In normal adult tissues, scapinin is abundant in the brain, suggesting a role in the brain.

Expression experiments showed that scapinin-S was localized in the nucleus, but scapinin-L was also localized in some cytoplasmic extensions in addition to the nucleus (Fig. 7). The result of the expression experiments for scapinin-L (Fig. 7) seemed to be inconsistent with the immunostaining studies on HL-60 cells (Fig. 2). Western blotting analyses showed that scapinin-L was the minor component compared with scapinin-S in HL-60 cells (Fig. 2). Thus, a possible explanation for this discrepancy is that the immunostaining only reflects the distribution of scapinin-S. Immunostaining studies on GOTO and U937 cells, in which scapinin-L was the minor component as in HL-60 cells, also showed nuclear localization of scapinin (data not shown). Further studies are needed to determine the mechanism responsible for retaining scapinin-L in the cytoplasmic extensions and the significance of this phenomenon.

Scapinin-S lacks 70 amino acids containing a proline-rich sequence (Fig. 3A). It is known that some proline-rich sequences act as protein-protein interaction domains, which are implicated in various cellular processes such as cell motility and gene expression (30, 31). Scapinin family proteins also contain proline-rich sequences that match or resemble those proline-rich motifs involved in protein-protein interactions (Fig. 9C). Thus, identification of proteins binding to the prolinerich sequence may reveal functional differences and, possibly, differential distribution between scapinin-S and scapinin-L.

Scapinin is a member of a novel protein family present from C. elegans to Homo sapiens. These scapinin family proteins exhibit homology to each other as a whole and, with the exception of KIAA1733, they share a well conserved COOH-terminal sequence composed of 150 amino acids (Fig. 9B). This region includes several RPEL repeats. The RPEL repeat is a conserved domain present in transcription factors (myocardin and MKL1) and an oncogene of OTT-MAL (32–34). These RPEL-containing proteins have a SAP motif that is a putative DNA binding domain predicted to be involved in chromosome organization. The COOH-terminal region composed of 70 amino acids, to which the putative PP1 binding domain of scapinin was assigned, is highly conserved among scapinin family proteins (Fig. 9, B and C). This fact indicates the possibility that the scapinin family represents a novel PP1-binding protein family. Further studies are needed to assess this prediction.

	FOOTNOTES

 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB098521 [GenBank] (scapinin-L) and AB098522 [GenBank] (scapinin-S).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this paper.

^¶ To whom correspondence should be addressed. Tel.: 81-263-37-2723; Fax: 81-263-37-2724; E-mail: sagara{at}sch.md.shinshu-u.ac.jp.

¹ The abbreviations used are: PP1, protein phosphatase-1; DAPI, 4',6-diamidino-2-phenylindole; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; hnRNP, heterogeneous nuclear ribonucleoprotein; IF, intermediate filament; MES, 4-morpholineethanesulfonic acid; NM, nuclear matrix; P1, particulate; P2, chromatin-depleted fraction; PIPES, 1,4-piperazinediethanesulfonic acid; PP1c, -catalytic subunit of PP1; RA, all-trans-retinoic acid; RACE, rapid amplification of cDNA ends; RT, reverse transcription; Sol, soluble; TBS, Tris-buffered saline.

	ACKNOWLEDGMENTS

 
We are extremely grateful to Dr. K. Ayukawa (National Institute of Neuroscience, Japan) for efforts in the early days of this study and to Drs. T. Miyamoto and K. Hashizume (Shinshu University) for valuable advice on the yeast two-hybrid assay.

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