©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Myeloid Leukemia-associated Protein SET Is a Potent Inhibitor of Protein Phosphatase 2A (*)

(Received for publication, February 29, 1996; and in revised form, March 19, 1996)

Mei Li Anthony Makkinje (§) Zahi Damuni (¶)

From the Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I(1) and I(2) have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z.(1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I(2) was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.


INTRODUCTION

A number of defined chromosomal translocations occur in specific subtypes of myeloid leukemia indicating that these translocations play an important role in the process of leukemogenesis(1, 2) . As a result of translocation, nearby oncogenes and other genes involved in the control of proliferation or differentiation can be activated through alterations in regulatory DNA sequences that leave the encoded protein intact (e.g. Myc) or through formation of fusion genes that encode chimeric proteins (e.g. Bcr-Abl, E2A-Pbx, and Pml-RAPalpha)(1, 2) . However, although several of the chromosomal translocations and the resulting fusion genes that occur in leukemia have been identified, often the function of the individual proteins encoded by the fusion transcripts has not been determined(1, 2) .

Protein phosphatase 2A (PP2A) (^1)is a major mammalian protein serine threonine phosphatase that regulates diverse cellular processes(3, 4, 5, 6) . In cells, PP2A is thought to exist as heterotrimeric forms termed PP2A(1) and PP2A(0) and composed of a catalytic C subunit and A and B (B, B`, or B") subunits (3, 4, 5, 6) . A dimeric form of PP2A, termed PP2A(2), has also been isolated from numerous sources and is composed of the A and C subunits (3, 4, 5, 6) . However, this enzyme is thought not to exist in vivo because the missing B subunit may have been lost during the isolation procedures(3, 4, 5, 6) . Two forms of the A and C subunits exhibiting apparent M(r) values of 65,000 and 36,000 and 86% (7, 8) and 97% identity (9, 10, 11, 12, 13) in their predicted amino acid sequences, respectively, have been identified by molecular cloning methods. In addition, several distinct B subunits of apparent M(r) 54,000, 55,000, 56,000, 72,000, and 130,000 have also been identified (e.g. see Refs. 7 and 14-17). Although the significance of the different A, B, and C subunits is not well understood, the distinct substrate specificities of the various trimeric PP2A forms appear to be conferred, at least in part, by the variable B subunit(3, 4, 5, 6) .

However, despite progress on the structure of PP2A, little information is available on the regulation of this enzyme. Nonetheless, evidence has emerged that PP2A is inactivated by phosphorylation (18, 19) and activated by methylation (20) of its C subunit. In addition, we recently purified to apparent homogeneity from extracts of bovine kidney two heat-stable and PP2A-specific inhibitor proteins designated I(1) and I(2)(21) . These inhibitor proteins act noncompetitively and exhibit apparent K values in the nanomolar range(21) . The inhibitor proteins appear to be PP2A-specific because, in contrast to PP2A, they do not affect the activities of PP1(C), PP2B, and PP2C(21) , the other major mammalian protein serine threonine phosphatases(3, 4, 5, 6) . Furthermore, I(1) and I(2) did not affect the activities of 11 different protein kinases(21) . Because the purified preparations exhibited distinct peptide patterns following cleavage with Staphylococcus aureus V8 protease, I(1) and I(2) may be the products of distinct genes(21) . However, direct evidence for this possibility was not provided.

This study was undertaken to determine the identity of I(2) on a firm basis. In this communication, we show that the purified bovine kidney preparations of I(2) represent a truncated form of SET (22) , a largely nuclear protein also termed PHAP-II (putative class II human histocompatibility leukocyte-associated protein II) (23) and TAF (template-activating factor)(24) . In acute non-lymphocytic myeloid leukemia, the SET gene, which is located on chromosome 9q34 centromeric to c-abl and nup214, is fused to nup214 (also termed can) apparently as a result of translocation(22) . This SET-Nup214 fusion gene encodes a 5-kilobase transcript that contains a single open reading frame predicting a chimeric SET-Nup214 protein of apparent M(r) 150,000(2, 22) . The results suggest that fusion of SET with Nup214 in acute myeloid leukemia may impair the normal regulation of PP2A and contribute to leukemogenesis.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain MBP(25) , PP1(C)(26) , and PP2B (26) and bovine kidney I(1)(21) , I(2)(21) , PP2A(1)(26) , PP2A(2)(26) , PP2A(C)(26) , PP2C(26) , and protamine kinase (27) were purified to apparent homogeneity as described. Human kidney polyadenylated RNA was from Clontech. Synthetic oligonucleotides were synthesized at Life Technologies, Inc. Other materials are given elsewhere(27, 28, 29, 30, 31) .

P-Labeled MBP was prepared by incubation with the protamine kinase (27) exactly as described(21) . Assay of PP2A(1), PP2A(2), PP2A(C), PP1(C), PP2B, and PP2C was performed as reported(21, 26) . One unit of phosphatase activity was defined as the amount of enzyme that catalyzed the release of 1 nmol of phosphoryl groups/min from P-labeled MBP. To ensure linearity, the extent of phosphoryl group release was limited to <10%. Inhibitor protein activity was measured as described(21) . One unit of inhibitor protein activity was defined as the amount of protein that inhibited 1 unit of PP2A(2) by 50% in the standard assay.

SDS-PAGE was performed in slab gels (12% acrylamide) with 0.1% SDS and Tris/glycine buffer, pH 8.3(32) . Protein bands were detected by staining with Coomassie Blue. Protein was determined as described (33) .

Generation, Purification, and Sequencing of Tryptic Peptides

Aliquots (2 µg) of purified bovine kidney I(2) were subjected to SDS-PAGE, followed by electrophoretic transfer onto Immobilon P membranes. After staining with Ponceau S, bands corresponding to I(2) were cut out and subjected to overnight incubation at 37 °C with N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (0.5 mg) as described(34) . Tryptic peptides were then resolved by high pressure liquid chromatography reversed-phase chromatography on an Aquapore RP-300 column (1 times 250 mm) equilibrated with 0.1% (v/v) trifluoroacetic acid. After washing with this solution, the column was developed, at a flow rate of 0.15 ml/min, with a linear gradient from 0.1% trifluoroacetic acid to 0.08% trifluoroacetic acid containing 38.5% (v/v) acetonitrile in 30 min, followed by a linear gradient from 0.08% trifluoroacetic acid containing 38.5% acetonitrile (v/v) to 0.08% trifluoroacetic acid containing 59.5% (v/v) acetonitrile in 10 min. Fractions (0.045 ml) were collected, and the absorbance at 214 nm was determined. The amino acid sequences of peptides eluting at about 38% (peptide I) and 48% (peptide II) acetonitrile and the N terminus of I(2) were determined using standard reagents for gas phase chemistry on an automated protein sequencer (Applied Biosystems) equipped with an on-line UV detector to identify phenylthiohydantoin derivatives.

Generation of SET cDNA

First strand SET cDNA was generated at 42 °C for 30 min in 0.02 ml of 10 mM Tris-HCl, pH 8.3, containing 50 mM KCl, 5 mM MgCl(2), 20 units of RNase inhibitor, 1 mM of each dNTP, 0.5 µg of human kidney polyadenylated RNA (Clontech), 50 pmol of the 3` SET-based oligonucleotide (GGATCCTAGTCATCTTCTCCTTC), and 2.5 units of murine leukemia virus reverse transcriptase (Perkin-Elmer). After heat denaturation at 99 °C for 5 min, a 0.005-ml aliquot of the mixture was subjected to amplification by polymerase chain reaction in 0.1 ml of 20 mM Tris-HCl, pH 8.3, containing 10 mM KCl, 10 mM (NH(4))(2)SO(4), 0.1% Triton X-100, 2 mM MgCl(2), 0.1 mg/ml bovine serum albumin, 100 pmol of the 3` and 5` (CCGCGGAGCAGCCATATGTCGGC) SET-based oligonucleotides, 0.2 mM of each dNTP, and 5 units of recombinant pfu DNA polymerase (Stratagene). After heat denaturation for 5 min at 94 °C, polymerase chain reaction amplification for 30 cycles was performed as follows: 30 s at 94 °C, 30 s at 55 °C, and 2.5 min at 72 °C except that, in the last cycle, extension was carried out for 7 min. The amplified cDNA (854 base pairs) was subcloned into the SmaI site of pUC18 and used to transform Escherichia coli ONE SHOT as recommended by the manufacturer (Invitrogen). After growth at 37 °C in Luria-Bertani medium (35) containing 100 µg/ml ampicillin, the pUC18 containing SET cDNA was purified from the cells by the alkaline lysis method(35) . The identity of the purified cDNA with the coding region of SET (22) (GenBank accession no. M93651) was confirmed by sequencing both strands using M13 sequencing primers and the dideoxynucleotide chain termination method (U. S. Biochemical Corp. TAquence sequencing kit and DuPont NEN [alpha-S] dATP).

Expression and Purification of SET

SET cDNA was excised from pUC18 by incubation with BamHI and NdeI and then isolated with GLASSMILK (Geneclean II, BIO 101, Inc.) from low melting agarose following gel electrophoresis. pET-21a DNA (50 ng) (Novagen) was linearized with BamHI and NdeI and dephosphorylated with calf intestinal alkaline phosphatase. This DNA was then incubated overnight at 16 °C with 10 ng of the purified cDNA in 0.01 ml of 20 mM Tris-HCl, pH 7.6, containing 1 mM ATP, 5 mM MgCl(2), 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, and 4 units of T4 DNA ligase (Invitrogen) as recommended (35) . The mixture was then used to transform E. coli BL21(DE3) (Novagen). A 5-ml overnight culture of these cells was then inoculated into 500 ml of Terrific Broth containing 100 µg/ml ampicillin. After growth at 37 °C to log phase, IPTG was added (final concentration, 0.5 mM), and the cells were grown for an additional 2 h. Unless indicated otherwise, all subsequent operations were performed at 4 °C. After centrifugation for 15 min at 3,000 times g in a Beckman JA-10 rotor, cells were resuspended in buffer A (25 mM Tris-HCl, pH 7.4, containing 10% glycerol, 4 mM EDTA, 1 mM benzamidine, 0.1 mM phenylmethanesulfonyl fluoride, and 14 mM beta-mercaptoethanol) and lysed at 1200 p.s.i. with a French press. After centrifugation at 39,000 times g for 30 min in a Beckman JA-20 rotor, pellets were discarded, and to the supernatant was added 30 volumes of buffer B (buffer A containing 1 mM instead of 4 mM EDTA). The mixture was applied onto a column (2.5 times 8.5 cm) of poly-L-lysine-agarose equilibrated with buffer B. After washing with 500 ml of buffer B, the column was developed with a 600-ml linear gradient from 0 to 1.0 M NaCl. Inhibitor activity was recovered at about 0.65 M NaCl. Active fractions were pooled and mixed at room temperature with 2 volumes of a 99% ethanol solution. After centrifugation at 30,000 times g for 10 min in a Beckman JA-14 rotor, pellets were resuspended in buffer B and subjected to gel permeation chromatography on a calibrated column (2.5 times 95 cm) of Sephacryl S-200 equilibrated with buffer B containing 0.1 M NaCl and 0.01% Brij 35. A major peak of inhibitor protein activity emerged at V(0) (220 ml) from Sephacryl S-200. This activity peak was not detected following chromatography on Sephacryl S-200 of extracts from mock-transformed or transformed cells that had been incubated in the absence of IPTG. Fractions containing this inhibitory activity were pooled, and 20 g/liter trichloroacetic acid was added with stirring for 10 min. After centrifugation, the supernatant was discarded, and pellets were resuspended in a solution of 70% ethanol. The mixture was centrifuged and the supernatant was discarded. This procedure was repeated three times. The final pellets were resuspended in buffer C (buffer A containing 0.1 mM instead of 4 mM EDTA) and dialyzed, with three changes, against 20 volumes of this buffer in 16 h. The preparations were aliquoted and stored at -70 °C. A second peak of unidentified inhibitor activity emerged from Sephacryl S-200 with apparent M(r) 50,000. This inhibitor activity was detected following gel permeation chromatography of extracts from transformed and mock-transformed cells that had been incubated in the absence or presence of IPTG and was therefore discarded.


RESULTS AND DISCUSSION

Amino Acid Sequencing

To establish the identity of I(2), we set out to determine the amino acid sequence of this protein. Initially, the amino acid sequences of two tryptic peptides (LNEQASEEILK, peptide I) and (QHEEPESFFTWFTDH, peptide II), generated and purified as described under ``Experimental Procedures,'' were determined. Comparison with amino acid sequences available at the PIR, SwissProt, and GenBank data bases indicated that peptides I and II were identical to residues 45-55 and 182-196 predicted for human SET, respectively(22) . Earlier, we reported (21) that the N-terminal amino acid sequence of I(2) (SDGADATSTK) showed 70% identity (differences are underlined) with residues 17-26 of SET(22) . Because the purified preparations of bovine kidney I(2) exhibit an apparent M(r) 20,000(21) , whereas SET exhibits an apparent M(r) 39,000 as estimated by SDS-PAGE (23, 24) , the results suggested that I(2) may have been derived from SET by proteolysis possibly during the purification procedure and that the three amino acid differences noted in the N terminus may be species and/or tissue related. In this connection, it is pertinent that the human SET gene encodes two transcripts of 2.0 and 2.7 kilobases that result from the use of alternative polyadenylation sites. However, both transcripts contain identical open reading frames(2, 22) .

Expression and Purification of SET

To test whether SET inhibits PP2A in a manner corresponding to I(2), a cDNA coding for human SET was generated and placed into a pET-21a vector under the control of the T7lac promotor as described under ``Experimental Procedures.'' Consistent with the possibility that SET inhibits PP2A, IPTG-induced expression of this cDNA in bacteria resulted in about a 10-fold increase in PP2A inhibitor activity as determined following poly-L-lysine-agarose chromatography (Fig. 1). Because of interference from an unidentified endogenous inhibitor(s), differences in PP2A inhibitor activity in bacterial extracts from control and IPTG-treated cells could not be distinguished. This (these) endogenous inhibitor(s) was (were) not a product(s) of the introduced cDNA because similar activity was detected in extracts of bacteria that had been mock-transformed or transformed with the vector alone.


Figure 1: Induction of PP2A inhibitor. Extracts from 500-ml cultures of control (A) and IPTG-treated (B) bacteria containing SET cDNA were prepared as described under ``Experimental Procedures.'' Each extract was then applied onto a separate column (2.5 times 8.5 cm) of poly-L-lysine-agarose equilibrated with buffer B. Each column was washed with 500 ml of buffer B followed by buffer B containing 0.3 M NaCl. Inhibitor activity was recovered with buffer B containing 0.8 M NaCl. Fractions (3 ml) were collected, and PP2A inhibitor activity (bullet) was determined according to Li et al.(21) . The absorbance at 595 nm (circle) was determined according to Bradford (33) using 0.05-ml aliquots of the indicated fractions.



To more directly test the possibility that SET inhibits PP2A, a procedure was developed to purify the IPTG-induced PP2A inhibitor to apparent homogeneity from the bacterial extracts as described under ``Experimental Procedures.'' This procedure was based on the one employed previously in the purification of I(2) from extracts of bovine kidney (21) and included chromatography of the extracts on poly-L-lysine-agarose, precipitation with ethanol, and gel permeation chromatography on Sephacryl S-200, followed by trichloroacetic acid and ethanol precipitation. The purified preparations of the recombinant protein consisted of a single Coomassie Blue staining polypeptide of apparent M(r) 39,000 as estimated by SDS-PAGE (Fig. 2). N-terminal amino acid sequencing of these preparations (to the 10th residue) confirmed that the purified protein was SET. Typically, about 3 mg of this protein was obtained from a 500-ml culture of transformed cells.


Figure 2: SDS-PAGE pattern of purified recombinant SET (1 µg). PAGE was performed in slab gels (12% acrylamide) with 0.1% SDS and Tris/glycine buffer, pH 8.3(32) . Purification of SET was as described under ``Experimental Procedures.''



Effect of SET on PP2A Activity

The effect of the purified recombinant SET preparations on PP2A activity was examined next. These preparations inhibited PP2A potently (Fig. 3). Half-maximal inhibition of the phosphatase occurred at about 2 nM (Fig. 3), similar to the potent inhibition obtained with purified preparations of bovine kidney I(2)(21) . Previously, we showed that, by contrast to PP2A, purified preparations of PP1(C), PP2B, and PP2C were unaffected by I(2)(21) . Similar experiments revealed that recombinant human SET also exhibited little or no effect on the activities of PP1(C), PP2B, and PP2C (Fig. 3). Together, these results indicate that SET is a potent and specific inhibitor of PP2A. Because the recombinant SET preparations inhibited PP2A(1) (Fig. 3) PP2A(2) (not shown), and PP2A(C) (not shown), SET is analogous to I(2) in that it appears to act by binding to the C subunit of the phosphatase.


Figure 3: Effect of SET on protein phosphatases. The activities (0.005 unit) of PP2A(1) (bullet), PP1(C) (circle), PP2B (), and PP2C (up triangle) were measured as described (21) with P-labeled MBP as substrate in the presence of the indicated concentration of purified recombinant SET. The calculated molecular mass of 32,100 was used to determine the concentration of SET employed in the assays.



Earlier studies suggested a role for SET as a transcriptional activator because it enhanced adenovirus core DNA replication in HeLa cell extracts(24) . The molecular basis of this effect was not determined, although our results raise the possibility that it may have been a consequence of PP2A inhibition. A role for SET in antigen-mediated responses was also suggested because this protein appeared to bind to a peptide (CFIIKGLRKSNAAERRGPL) patterned on an amino acid sequence present in the cytoplasmic C-terminal region of the DR2alpha chain of human histocompatibility class II receptor(23) . However, the functional significance of this interaction and whether it occurs with the intact receptor were not determined. Based on the results presented herein, we recommend that SET be renamed I(2) to indicate its function and to distinguish it from I(1), which is the product of a distinct gene. (^2)This revised nomenclature is used in the remaining discussion.

The results presented in this communication provide a firm basis for further characterization of the physiological role of I(2). Tissue and species distribution studies indicate that, by analogy with PP2A(3, 4, 5, 6) , I(2) is ubiquitous(36) , suggesting its potential importance for controlling the activity of the phosphatase in diverse cells and tissues. Interestingly, I(2) undergoes phosphorylation on unidentified serines in intact cells(36) . However, how this phosphorylation affects I(2) activity and whether or not this regulation responds to extracellular stimuli is unknown. Further studies are also needed on the mechanism of action of I(2). In this connection, the C terminus of I(2)(22) (and that of I(1)^2) is highly acidic (22) , and deletion of this acidic tail (residues 224-277) abolished the stimulation by I(2) of adenovirus core DNA replication in HeLa cell extracts(24) . However, whether deletion of the acidic tail abolishes the inhibition of PP2A by I(2) (and/or I(1)) remains to be determined. In this regard, it is pertinent that, based on the apparent M(r) (20,000) as estimated by SDS-PAGE and that only 16 amino acids are missing at the N terminus, the bovine kidney I(2) preparations appear to be proteolyzed largely at the acidic C terminus. Thus, it would appear that the acidic tail may not be important for inhibition. However, how removal of the 16 N-terminal amino acid residues affects the mobility of I(2) on SDS-PAGE is unknown. In this context, it is pertinent that, although the calculated molecular mass of I(2) is 32,100, this inhibitor protein exhibits an anomalous apparent M(r) (39,000) as determined by SDS-PAGE.

Previous studies have indicated a role for PP2A in tumorigenesis and the viral transformation of cells(3, 4, 5, 6) . Thus, PP2A is inhibited potently in vitro(37, 38) and in vivo(39) by the tumor promotor okadaic acid. In addition, the SV40 small tumor t antigen of several DNA tumor viruses replaces the B subunit and inhibits the phosphatase in transformed cells(40) . The results presented in this communication suggest that the I(2)-Nup214 gene fusion, which occurs in acute non-lymphocytic myeloid leukemia(22) , may also lead to altered regulation of PP2A and thus contribute to leukemogenesis. The chimeric protein predicted by this gene fusion contains residues 1-270 of the 277 amino acid residues of native I(2) and residues 813-2090 of the 2090 amino acid residues of Nup214. However, although Nup214 appears to be asymmetrically located on the cytoplasmic side of the nuclear envelope in normal cells(41, 42) , I(2)(23) and the I(2)-Nup214 fusion protein (41, 42) occur largely in the nucleus of normal and leukemic cells, respectively. Thus, it will be important to determine how, relative to normal cells, the activity of nuclear PP2A is affected in leukemic cells that express the I(2)-Nup214 fusion protein.


FOOTNOTES

*
This work was supported by American Cancer Society Grant BE-247 and National Science Foundation Grant MCB-9513672. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Massachusetts General Hospital, East Bldg. 149, 13th St., Charlestown, MA 02129.

To whom correspondence and reprint requests should be addressed: Dept. of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, P. O. Box 850, Hershey, PA 17033. Tel.: 717-531-4195; Fax: 717-531-7667; ZDAMUNI{at}CMP.HMC.PSU.EDU.

(^1)
The abbreviations used are: PP2A, protein phosphatase 2A; PP2A(C), the purified C subunit of PP2A; PP1(C), the purified catalytic subunit of protein phosphatase 1; PP2B, protein phosphatase 2B; PP2C, protein phosphatase 2C; MBP, myelin basic protein; Nup214, nucleoporin 214; PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl-1-thio-beta-D-galactopyranoside.

(^2)
M. Li, A. Makkinje, and Z. Damuni, manuscript in preparation.


ACKNOWLEDGEMENTS

Tryptic peptide generation, purification, and sequencing were performed at the Worcester Foundation Microsequencing Facility, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts. N-terminal amino acid sequencing was performed at the Milton S. Hershey Medical Center Macromolecular Core Facility.


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