Nuclear localization of an o-glycosylated protein phosphotyrosine phosphatase from human cells

Histochemical staining of immunoprecipitates of p65, a component of human M phase-promoting factor, identified the molecule as having phosphatase activity. The enzyme, purified 3400-fold from mitotic cell extracts by (NH^SCL precipitation, DEAE chromatography, and immunoaffinity chromatography on immobilized anti-p65 IgG, was inhibited by Zn²⁺and Na₃VO₄but not NaF or β-glycerophosphate;³²P-labeled poly(Glu, Tyr) was more efficiently dephosphorylated than phosphorylated histone or phosphorylase a. Indirect immunofluorescence showed most of the phosphatase to be localized in the nucleus of interphase cells, with a fine, granular distribution unaltered by detergent extraction; in mitotic cells, p65 was localized on chromosomes. ELISA of subcellular fractions confirmed this localization. Immunoreactive p65 was recovered from immobilized wheat germ agglutinin (WGA) upon elution with N-acetylglucosamine; similarly, WGA recognized immunoaffinity-purified p65 on blots. Alkaline hydrolysis of blotted protein prevented WGA binding, indicating that phosphatase p65, like a small group of other nuclear proteins, contains O-linked carbohydrate terminating in N-acetylgluco-samine.

Examination of the histone kinase profiles of human cells, generated by overlaying native gels of cell extracts with substrate, led to the identification of several distinct species whose activities were maximal at mitosis (Halleck et al. 1987). Antisera prepared against two of them each recognized the same 65000 M_r polypeptide, designated p65. While these anti-p65 antisera revealed the molecule to be present throughout the cell cycle, and to have no detectable protein kinase activity itself, at mitosis it associates with the p34^cdc2 kinase complex (Meikrantz et al. 1990), which probably accounts for the mitosis-specific histone kinase activity seen in native gels. Because protein phosphatases, like protein kinases, play an essential role in regulating mitosis (reviewed by Cyert and Thomer, 1989), including activation of p34^cdc2 (reviewed by Nurse, 1990), p65 was examined for phosphatase activity. This report demonstrates that p65 is in fact a protein phosphotyrosine phosphatase that is unique in its nuclear localization and O-linked glycosylation.

Human D98/AH-2 cells (ATCC CCL 18.3) were cultured as described (Fabisz-Kijowska et al. 1987). Mitotic cells were collected by mechanical shake-off after 16–18h in 40 ng ml⁻¹ nocodazole and homogenized (Meikrantz et al. 1990). Cytoplasmic (Meikrantz et al. 1990) and chromosomal and nuclear fractions (Halleck et al. 1987; Fabisz-Kjjowska et al. 1987) were prepared as described.

Following reduction and alkylation of samples (Dwyer and Blobel, 1976) SDS–PAGE was carried out in 0.5mm 7.5% gels (Laemmli, 1970), and protein blotted onto Immobilon-P membranes in 7.5 mw Tris, 1.2 mM borate (pH9.2) for Ih at 7.5 W. Blots were probed with anti-p65 IgG as previously described (Meikrantz et al. 1990) or with biotinylated wheat germ agglutinin (WGA) in 50mM Tris, 1.0mNbCI, 0.05% Tween 20 (pH 7.4) and detected with [^12eI]streptavidin. β-Elimination of O-linked glycosides was carried out by incubation of blots overnight in 0.1mNbOH (Spiro, 1972) prior to WGA probing.

p65 immunoprecipitates were prepared exactly as described (Meikrantz et al. 1990). In one experiment (Fig. 1, lane 2), mitotic extract was boiled for 10 min in 2% SDS, diluted 20-fold with the immunoprecipitation buffer previously described and brought to 0.5% Triton X-100 prior to immunoprecipitation.

(NH₄)SO₄ (20–60%) precipitates of mitotic cell extract were applied to DEAE-Sepharose in TE (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH 8.0 at 4 °C) containing ImM phenylmethylsulfonyl fluoride (PMSF) and 1 μg ml⁻¹ leupeptin. After washing with TE/0.1m NaCl, the column was eluted with 10×TE and the eluent applied directly to an anti-p65 IgG immunoaffinity column (Meikrantz et al. 1990). Following binding for 1h at room temperature and washing with TE/1.0mNbCI, the column was eluted with 0.2 m citrate, pH 3.0, to remove p65-binding proteins (Meikrantz et al. 1990). p65 was then eluted with 50% ethylene glycol, pH 8.0 (Meikrantz et al. 1990). Alternatively, (NH₄)₂SO₄-fractionated extract was applied to WGA-agarose (Sigma) and washed with TE/1.0MNaCl/0.05% (v/v) Tween 20. Bound protein was eluted with TE/0.1m TV-acetylglucosamine containing 0.5 him PMSF and 0.1 mM dithiothreitol (DTT) and detected by biotinylation and [¹²⁵I]streptavidin overlay following SDS–PAGE and transfer to Immobilon, as previously described (Meikrantz et al. 1990).

Protein blotted following SDS–PAGE was renatured in situ by overnight incubation at 4°C in 100 mM Hepes, 0.2% Chaps, 10 mM MgCl₂, 50 mM KC1 (pH 7.4). Phosphatase activity was assayed in situ by incubation in 0.1mTtis-HCI (pH 9.5) containing 50 μg ml⁻¹ bromochloroindolyl phosphate and lmgml^-1 nitroblue tétrazolium for 1 h with gentle agitation.

Samples (50 μl) of column fractions were assayed in disposable cuvette tubes in a 1ml volume containing Im diethanolamine, 4him MgCl₂ and l mg ml⁻¹ p-nitrophenyl phosphate, pH9.5. Following incubation for 30 min at room temperature, reactions were stopped by the addition of 0.5 ml of 1.5 m NaOH and the amount of nitrophenol produced was calculated from the absorbance at 405 nm.

[³²P]phosphorylase a was prepared by incubating 1000 i.u. of phosphorylase kinase (Sigma) in 1ml of labeling buffer (TO um ATP at 35 μ Ci ml^{− 1}, 5mM DTT, 50mM Tris-HCl, 50mM KC1, 5mM MgCl₂,1 mM CaCl₂, pH 7.4) for 20 min at 37 °C; contaminating phosphorylase b (∼ 2%) was phosphorylated on serine by the kinase. Histone labeled with ³²P on serine and threonine was prepared by incubating 1000 i.u. of cyclic AMP-dependent protein kinase catalytic subunit (Sigma) with 5 mg of mixed histones (Type II-A, Sigma) for 30 min at 37 °C in 1 ml of labeling buffer without CaCl₂. Poly(Glu, Tyr) (4:1, Glu:Tyr; 20 000–50 000 M_r, Sigma) labeled with ³²P on tyrosine was prepared by incubating 10 mg of the synthetic copolymer with 100 μl (120 μg) of mitotic cell extract in 1 ml total volume of labeling buffer with 10 mM MnCl₂ replacing the MgCl₂. Under these conditions, incorporation of ³²P is ∼10000-fold greater than when mitotic extract is incubated alone. Reactions were terminated by addition of 0.5 ml of 100% trichloroacetic acid (TCA). TCA pellets were washed five times with absolute ethanol and resuspended in 1ml of 10×TE before exchanging via Sephadex G25 into reaction buffer (0.1m triethanolamine, 100 mM NaCl, 4mM MgCl₂, 0.1 mM DTT, lmgml^-1 BSA, pH7.2). Phosphatase reactions were initiated by the addition of 0.2 pg of immunoaffinity-purified p65 to 100 μl of reaction volumes containing an excess of labeled substrate: either 200 μg of phosphorylase a (10 000 cts min⁻¹ mg⁻¹), 80 μg of histone (25 000 cts min⁻¹ mg⁻¹) or 100 μg of poly(Glu, Tyr) (20000 cts min⁻¹mg⁻¹). After 30min at room temperature, reactions were terminated by adding 100 μl of 2 m perchloric acid. Acid-soluble ³²PP_i in triplicate samples was corrected for P_i release in the absence of added p65.

Cells grown to 50–70% confluence on glass coverslips were fixed with 5% formaldehyde (EM grade, Polysciences) in PBS for 20 min (this and all other steps carried out at room temperature), incubated with 0.13 m glycine for 30 min to eliminate remaining reactive aldehyde groups, then permeabilized with freshly prepared methanol :acetone (1:1, v/v) for 2 min. Following incubation with PBS/0.1% BSA for 1 h, coverslips were incubated with anti-p65 (1:100 in PBS/0.1% BSA/0.05% Tween 20) for 15 min, rinsed twice with PBS/0.05% Tween 20 and once with PBS/0.1% BSA, followed by incubation for 15 min with rhodamine-conjugated goat anti-rabbit IgG (1:100, Organon-Teknika/Cappel) and washing as above. Similar results were obtained using 0.5% glutaraldehyde or cold methanol as fixative, or using 0.5% Triton X-100 as the permeabilizing agent. Double staining was performed by incubating anti-p65-stained coverslips for 15 min with 4,6-diamidino-2-phenylindole (DAPI, Sigma) at 0.25 pg ml^-1 in PBS before mounting in PBS/50% glycerol.

Mitotic extracts were immunoprecipitated with antibodies to p65. When immunoprecipitated proteins were separated by SDS–PAGE and transferred to Immobilon, renatured in situ and stained for phosphatase activity, a band at M_r 65 000 was seen (Fig. 1, lane 1). The band persisted even when mitotic extract was boiled in SDS prior to immunoprecipitation to eliminate co-precipitation of proteins other than p65 (lane 2), indicating that the phosphatase activity is inherent in the p65 polypeptide itself. No activity was precipitated by pre-immune antisera (lane 3) or when rabbit antibody was omitted from the immunoprecipitation reaction (lane 4).

Purification of p65 from crude homogenates by (NH₄)₂SO₄ precipitation, DEAE chromatography and immunoaffinity chromatography results in a final fraction containing only a single 65 000 M_r polypeptide (Fig. 2, lane 1; detected by biotinylation of blotted protein followed by [¹²⁵I]streptavidin overlay, since p65 stains poorly with Coomassie Blue or silver), which is recognized by anti-p65 antibodies (lane 2). As shown in a representative experiment in Table 1, the phosphatase activity of the final fraction is enriched 3400-fold with respect to the crude extract. In other experiments specific activities from 1100 to 4800 i.u. mg⁻¹, corresponding to enrichments of 1000-to 4400-fold, have been obtained. The purified phosphatase was sensitive to Na₃VO₄ and ZnCl₂, but was largely unaffected by NaF or β-glycerophosphate (Table 2), a pattern of response distinctive of protein phosphotyrosine phosphatases (reviewed by Ballou and Fischer, 1987).

Purified p65 was assayed for its ability to dephosphorylate ³²P-labeled proteins: phosphorylase a, phosphorylated on serine by phosphorylase kinase; histones, phosphorylated on serine and threonine by cyclic AMP-dependent protein kinase; and the synthetic copolymer poly(Glu, Tyr), phosphorylated on tyrosine by crude mitotic extract. As shown in Table 3, poly(Glu,Tyr) was the preferred substrate, hydrolyzed at a specific activity comparable to those reported for other protein phosphotyrosine phosphatases purified to homogeneity (Ballou and Fischer, 1987; Tonks et al. 1988a; Jones et al. 1989). This preference for phosphotyrosine is consistent with the enzyme’s robust hydrolysis of p-nitrophenylphosphate, which is structurally similar to phosphotyrosine, as well as its sensitivity to specific inhibitors of tyrosine phosphatases. In contrast, histone was a considerably poorer substrate, while déphosphorylation of phosphorylase a was barely detectable above background.

Subcellular localization of p65 was determined by indirect immunofluorescence. In interphase cells, anti-p65 staining was most intense in the nucleus, which exhibited a fine, granular distribution of fluorescence, although fainter staining of the cytoplasm was also seen (Fig. 3A). Nuclear staining persisted if cells were extracted with nonionic detergent prior to fixation (Schliwa and van Blerkom, 1982), although cytoplasmic staining appeared to be reduced. In mitotic cells, the chromosomes, identified by DAPI staining (Fig. 3C), were intensely stained by anti-p65 (Fig. 3D). This localization was confirmed by ELISA of subcellular fractions of interphase and mitotic cells. As shown in Table 4, the immunoreactivity of both the nuclear compartment of interphase cells and the chromosomal compartment of mitotic cells was 30-fold greater than their cytosolic counterparts. Thus p65 appears to be associated primarily with detergentinsoluble nuclear structures (i.e. chromosomes or nuclear matrix) throughout the cell cycle.

The resistance of p65 to staining with Coomassie Blue or silver suggested that it might be a glycoprotein (Andrews, 1986). To test this possibility, blots of immunoaffinity-purified p65 were probed with wheat germ agglutinin (WGA). As shown in Fig. 4 (lane 1), WGA bound to p65.

Similarly, when mitotic extract was adsorbed with WGA–agarose, a 65 000 M_r protein retained on the column and eluted with N-acetylglucosamine was recognized by anti-p65 antibodies (Fig. 4, lane 2). O-linked sugars can be distinguished from N-linked sugars by their susceptibility to β-elimination at alkaline pH (Spiro, 1972). Incubating blots of immunoaffinity-purified p65 overnight in 0.1m NaOH abolished WGA binding to p65 (Fig. 4, lane 3); binding of anti-p65 IgG was not affected (lane 4). Thus p65 is a glycoprotein containing O-linked carbohydrate terminating in N -acetylglucosamine.

p65, a constituent of the p34^cdc2 histone kinase complex, is a protein phosphatase: anti-p65 antibodies immunoprecipitate a phosphatase of Af_r 65 000, detected histo-chemically on blots following renaturation of protein; purification of p65 to homogeneity results in a 3400-fold purification of phosphatase activity, which is able to remove phosphate from several protein substrates. The marked preference for phosphotyrosine over phosphoserine/phosphothreonine, along with dramatic inhibition of activity by Zn²⁺ and

, identifies p65 as a protein phosphotyrosine phosphatase. Its molecular weight, nuclear localization and O-linked glycosylation make it unlike any phosphotyrosine phosphatase previously described. p65 is also unique among purified phosphotyrosine phosphatases in having significant activity against phosphoserine/phosphothreonine; and unlike phosphotyrosine phosphatases 1A and IB, the principal activities in human placenta (Tonks et al. 1990a,6), p65 is insensitive to both polycations and polyanions, and is inhibited, rather than stimulated, by DTT (Table 2).

Recently, a new class of intracellular glycoproteins with O-glycosidic bonds has been described, which seem to be particularly abundant in nuclear and chromatin fractions, and include a variety of transcription factors, viral proteins, and structural components of the nuclear pore–matrix–lamina fraction (Hart et al. 1989). The susceptibility of WGA labeling of p65 to β-elimination at alkaline pH shows that p65 is an O-linked glycoprotein. Among the O-linked glycoproteins in Drosophila is a polypeptide, abundant on chromosomes, of M_r 66 000 (Kelly and Hart, 1989). Interestingly, the banding patterns on Drosophila chromosomes produced by histochemical staining for phosphatase (Danielli and Catcheside, 1945) are similar to those obtained when the M_r 65000 protein is localized by staining with FITC-WGA (Kelly and Hart, 1989).

The ability of anti-p34^cdc2 antibodies to co-precipitate p65 from mitotic extracts (Meikrantz et al. 1990) indicates that both protein phosphatase (p65) and protein kinase (p34^cdc2) activities are present in a single complex. Such stable associations of protein phosphatases with protein kinases are not uncommon. For example, myosin lightchain kinase from rabbit skeletal muscle copurifies with myosin light-chain phosphatase (Morgan et al. 1976). In neurons, calcineurin (phosphatase 2B) is found in association with cyclic AMP-dependent protein kinase II (reviewed by Nestler and Greengard, 1982). And, in polyoma-infected cells, the cellular tyrosine kinase pp60^c’”^re and cellular protein phosphatase 2A are both complexed with the viral middle T antigen (Pallas et al. 1990). In each of these cases, the other subunits of the complexes, including the kinases, are suspected to be substrates for the associated phosphatases. In the case of p34^cdc2, dephosphorylation on tyrosine (and perhaps threonine) is required at mitosis for activation of the molecule’s latent histone Hl kinase activity (evidence summarized by Nurse, 1990). Since phosphatase p65 associates with p34^cdc2 specifically at mitosis, it will be of considerable interest to determine whether p65 is responsible for this activation.