(Received for publication, February 29, 1996; and in revised form, March 19, 1996)
From the
Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor
proteins designated I and
I
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
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.
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-RAP)(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) ()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
and PP2A
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
, 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
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
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 and I
(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
, PP2B, and
PP2C(21) , the other major mammalian protein serine threonine
phosphatases(3, 4, 5, 6) .
Furthermore, I
and I
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
and
I
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 on a firm basis. In this communication, we
show that the purified bovine kidney preparations of
I
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
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.
P-Labeled MBP was prepared by incubation with the
protamine kinase (27) exactly as described(21) . Assay
of PP2A
, PP2A
, PP2A
,
PP1
, 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
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) .
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 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 (
) was determined according to Li et
al.(21) . The absorbance at 595 nm (
) 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 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
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.''
Figure 3:
Effect of SET on protein phosphatases. The
activities (0.005 unit) of PP2A (
), PP1
(
), PP2B (
), and PP2C (
) 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 DR2 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
to indicate its
function and to distinguish it from I
, which
is the product of a distinct gene. (
)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. Tissue and species distribution studies
indicate that, by analogy with
PP2A(3, 4, 5, 6) ,
I
is ubiquitous(36) , suggesting its
potential importance for controlling the activity of the phosphatase in
diverse cells and tissues. Interestingly, I
undergoes phosphorylation on unidentified serines in intact
cells(36) . However, how this phosphorylation affects
I
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
. In this connection, the C terminus of
I
(22) (and that of
I
) is highly acidic (22) , and deletion of this acidic tail (residues
224-277) abolished the stimulation by I
of adenovirus core DNA replication in HeLa cell
extracts(24) . However, whether deletion of the acidic tail
abolishes the inhibition of PP2A by I
(and/or I
) remains to be determined.
In this regard, it is pertinent that, based on the apparent M
(
20,000) as estimated by SDS-PAGE and that
only 16 amino acids are missing at the N terminus, the bovine kidney
I
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
on SDS-PAGE is unknown. In this context,
it is pertinent that, although the calculated molecular mass of
I
is 32,100, this inhibitor protein exhibits
an anomalous apparent M
(
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-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
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
(23) and the I
-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
-Nup214
fusion protein.