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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1901-1908
Carboxyl-Truncated STAT5 Is Generated by a Nucleus-Associated
Serine Protease in Early Hematopoietic Progenitors
By
Johann Meyer,
Manfred Jücker,
Wolfram Ostertag, and
Carol Stocking
From the Department of Cell and Virus Genetics,
Heinrich-Pette-Institut für experimentelle Virologie und
Immunologie an der Universität Hamburg, Hamburg, Germany.
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ABSTRACT |
Hematopoiesis is tightly controlled by a family of cytokines that
signal through a related set of receptors. The pleiotropic and
overlapping response of a cell to different cytokines is reflected in
the number and complex pattern of activated signal transducers. Of
special interest is STAT5, which is stimulated by a large and diverse
set of cytokines. In addition to the two highly homologous proteins,
STAT5A and STAT5B, encoded by duplicated genes, expression and
activation of a dominant-negative, carboxyl-truncated form has also
been described in early hematopoietic progenitors. We show here that a
protease expressed in early hematopoietic cells cleaves the  forms
of STAT5A/5B (p96/p94) to generate carboxyl-truncated  forms
(p80/p77). Inhibition studies assigned this protease to the serine
class of endopeptidases. Cell fractionation experiments showed that the
protease is associated with the nucleus in a constitutively activated
form and does not require an activated STAT5 substrate. The ability of
a protease to modulate the specificity of an activated transcription
factor is unprecedented and underlines the importance of proteases in
regulation of cell functions.
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INTRODUCTION |
CYTOKINES ACT AS extracellular signals in
myelopoiesis and lymphopoiesis to activate cell function and to
maintain homeostasis in the adult tissue.1,2 These
polypeptide ligands bind cell-surface receptors that trigger a cascade
of signaling pathways, including the JAK/STAT pathway.3
Activation of the receptor-associated Janus protein tyrosine kinases
(JAKs) results in the phosphorylation of a unique family of
transcription factors termed the signal transducers and activators of
transcription (STATs).4,5 Phosphorylation triggers
dimerization, transport to the nucleus, and DNA binding. There are
seven STATs known (including both proteins encoded from the duplicated
STAT5A and STAT5B genes) that can be activated by more than 30 different ligands in various cell systems, resulting in diverse
biological effects. It is clear that the specificity of the response is
dictated by both direct and indirect interactions with other
transcription factors present in the different cell types. In the case
of STAT1, STAT3, and STAT5, specificity can also be mediated by short
forms that, unlike the long forms, can uniquely interact with
specific transcription factors or inhibit transcription in a dominant
negative fashion.6-10
STAT5 is expressed in a wide range of tissue and is involved in a
variety of responses observed in both hematopoietic and nonhematopoietic cells. In addition to its important role in prolactin signaling in mammary glands,11 STAT5 is activated by
cytokines that regulate the proliferation and differentiation of
myeloid (interleukin-3 [IL-3], IL-5, granulocyte-macrophage
colony-stimulating factor [GM-CSF], and
thrombopoietin),6,12,13 erythroid (erythropoietin [Epo]),14,15 and lymphoid lineages (IL-2 and
IL-15).16,17 Two highly related genes encode the STAT5A and
STAT5B proteins, which are greater than 95%
identical.6,12,18 These proteins differ at the carboxyl
terminus, a region that is highly variable among other STAT proteins
and thought to be involved in transcriptional activation.4
No evidence has yet been presented that define functional differences
between these proteins,12 although differentiation-specific differences in activation patterns have been observed.19 In contrast, the carboxyl-truncated forms, termed STAT5, have been shown to act in a dominant negative fashion to inhibit
transactivation9 and may interact with a unique set of
transcription factors, in analogy with STAT3.7
A number of studies have clearly shown that the truncated form is
the predominant phosphorylated STAT5 form observed after IL-3, GM-CSF,
or Epo stimulation in early hematopoietic cells.6,12,14,20 Significantly, these results were observed in both mice and humans, and
with both primary and established cells. A previous report showed that
the form can be generated from an alternatively spliced message
(the last intron remaining unspliced),9 consistent with
transcripts detected in rat liver and mammary glands.21,22 However, the levels of this alternative message were quite low and
inconsistent with the almost exclusive activation of STAT5 reported
in early hematopoietic cells.6 This study was initiated to
determine if an alternative mechanism is involved in the generation of
the truncated STAT5 isoforms. We have identified a
nucleus-associated protease present in early hematopoietic cells that
cleaves activated STAT5 to generate STAT5. The importance of this
serine endopeptidase in regulating hematopoiesis is discussed.
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MATERIALS AND METHODS |
Cell culture.
The multipotent FDC-Pmix independent isolates A4 and
15S23,24 and the factor-dependent FDC-P1(M) (clone 4) and
FDC-P125 cell lines were used for these studies. The latter
two cell lines were obtained from T.M. Dexter (Paterson Laboratories,
Manchester, UK) and D. Metcalf (Walter and Eliza Hall Institute,
Melbourne, Australia), respectively, and differ in their
responsiveness to GM-CSF, their infectivity with ecotropic
retroviruses, and cell surface markers (C. Laker and C.S., unpublished
results). FDC-Pmix cells were maintained in Iscove's
modified Dulbecco's medium (IMDM; GIBCO, Paisley, UK) supplemented
with IL-3 and 20% horse serum, whereas FDC-P1 and FDC-P1(M) cells were
held in modified Eagle's medium (2×) in 10% fetal calf serum
and IL-3. IL-3 was obtained from conditioned medium from cells
transfected with a bovine papilloma virus vector carrying the IL-3 gene
and was used at concentrations necessary for maximum stimulation.
Preparation of cell extracts.
Cell extracts were prepared from cells under three different
conditions: (1) 20 hours (or 12 hours for FDC-Pmix) after removal of
IL-3; (2) after stimulation of starved cells (20 or 12 hrs) with IL-3
for 30 minutes; or (3) under normal proliferating conditions in the
presence of IL-3 (approximately 24 hours after the last addition of
fresh medium and factor). To prepare nuclear and cytosolic extracts, 5 × 107 cells were washed twice with phosphate-buffered
saline containing 1 mmol/L orthovanadate and pelleted at 1,000g
for 5 minutes. The cell pellet was resuspended in a hypotonic buffer
containing 20 mmol/L HEPES, pH 7.6, 10 mmol/L KCl, 1 mmol/L
MgCl2, 0.5 mmol/L dithiothreitol (DTT), 0.1% Triton X-100,
20% glycercol, 1 mmol/L Pefabloc (Boehringer Mannheim, Mannheim,
Germany), 5 µg/mL leupeptin (Sigma, Deisenhofen,
Germany), 5 µg/mL pepstatinA (Biomol, Hamburg, Germany), 200 KIU/mL aprotinin (ICN, Eschwege,
Germany), and 1 mmol/L sodium orthovanadate. Cells were
lysed by 20 strokes in a glass Dounce homogenizer. The homogenate was
centrifuged at 2,000g for 5 minutes. The supernatant (cytosolic
extract) was centrifuged again for 15 minutes at 14,000 rpm to clear
lysate and flash frozen in liquid nitrogen. The pelleted nuclei were extracted in a hypertonic nuclear extract buffer (NEB; 20 mmol/L HEPES,
pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L DTT, 0.1% Triton
X-100, 20% glycerol) containing proteinase inhibitors (as listed
above) for 15 minutes at 4°C. The extracts were then centrifuged at
14,000 rpm for 5 minutes. Aliquots were frozen in liquid nitrogen and
stored at  70°C. Buffer volumes were adjusted so that the
protein concentrations of nuclear and cytosolic extracts reflected the
cellular ratio. The purity of the cell fractionation was confirmed in
Western blot analysis using antisera against laminB (kindly provided by
W. Bohn, Heinrich-Pette-Institut, Hamburg, Germany), which
is specific for the nucleus and with either anti-c-src (sc-18; Santa
Cruz Biotechnology, Santa Cruz, CA) or anti-SHC (#6-203;
Upstate Biotechnology Inc [UBI; Lake Placid, NY]). Only low levels of cross-contamination were observed (<1%).
In experiments in which cell extracts from different cells were
combined, extracts were incubated on ice for 15 minutes after mixing
(1:1 or 1:10) and then frozen in liquid nitrogen. To enhance protease
activity, in some cases extracts were incubated for an additional 2 minutes at 37°C before freezing. Freezing was found to destroy
proteolytic activity.
The following proteinase inhibitors were screened for inhibition
activity: phenylmethylsulfonyl fluoride (PMSF;  1 mmol/L; Sigma),
Pefabloc (4 mmol/L; Boehringer Mannheim), E64 (10 µg/mL; Boehringer
Mannheim); 3,4 Dichloroisocoumarin (DCI; 200 µmol/L; Sigma);
N-tosyl-L-phenyalanine chloromethylketone (TPCK; 0.1 mmol/L; Boehringer Mannheim), and
N -p-tosyl-L-lysine
chloromethalketone (TLCK; 1 mmol/L; Boehringer Mannheim). In all cases,
the given concentration was added to all buffers used during all stages
of cell extract isolation, in addition to the above-listed proteinase
inhibitors, except Pefabloc. Concentrations chosen were based on the
highest recommended dose given by the manufacturer. EDTA (2 mmol/L) was
added to one set of buffers.
Electrophoretic mobility shift assays (EMSA).
Oligonucleotides were end-labeled with polynucleotide kinase to a
specific activity of 5 × 103 cpm/fmol. The
STAT5 binding site of the bovine -casein promoter was used as a
probe (5 -AGATTTCTAGGAATTCAAATC-3).11 Nuclear extracts (1.2 µg) were incubated at room temperature for 30 minutes in a volume of 20 µL with 16 fmol end-labeled oligonucleotides and 2 µg of poly(dI.dC) in 10 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L EDTA, and 5%
glycerol. Reaction mixtures were then electrophoresed at 10 V/min on a
6% polyacrylamide gel in 0.25× TBE (45 mmol/L Tris-borate, 1 mmol/L EDTA). For supershift assays, antibody (1 µg) was added after 15 minutes and incubated for another 15 minutes at room temperature.
Immunoprecipitations, Western blot analysis, and antibodies.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot analysis as well as immunoprecipitations were
performed as described previously.26 Rabbit polyclonal antisera against STAT5b (C-17; Santa Cruz Biotechnology) was used to
immunoprecipitate STAT5. To detect STAT5 in Western blots, antisera
(C-17 and N-20) and monoclonal antibody (p89), purchased from Santa
Cruz Biotechnolgy and Transduction Laboratories (Lexington, KY), respectively, were used. Antisera against
phosphorylated tyrosine residues (4G10) purchased from UBI was used to
confirm phosphorylation of STAT5. Filters were developed using the
enhanced chemiluminescence (ECL) system according to the
manufacturer's protocol (Amersham, Braunschweig,
Germany).
Oligonucleotide affinity purification of activated STAT5.
Nuclear extracts (500 µg protein) were incubated with Sepharose beads
coupled to multimerized oligonucleotides containing the STAT5 binding
sites from the -casein gene promoter. Binding reactions were
performed in the presence of 30 µg poly(dI.dC) and 30 µg
poly(dA.dT) for 1.5 hours at 4°C in NEB supplemented with 60 mmol/L
NaCl. After two washes with NEB containing 60 mmol/L NaCl and one wash
containing 100 mmol/L NaCl, proteins bound to the Sepharose beads were
eluted with 400 mmol/L NaCl in NEB. One-third volume 3× SDS
loading buffer was added to samples that were subsequently separated by
SDS-PAGE (7.5%), blotted onto nitrocellulose membrane, and visualized
with antibody.
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RESULTS |
Truncated STAT5 is the preferential activated isoform observed
in multipotent hematopoietic progenitors.
Activated forms of STAT5 have previously been detected in both
primary and established myeloid progenitors and precursors, the
expression of which has been correlated with a more immature phenotype.
To further investigate this isoform, we screened cells of several
IL-3-dependent cell lines for expression of STAT5. Nuclear extracts
were separated by SDS-PAGE electrophoresis and probed with STAT5
antisera after transfer to nylon membranes. Consistent with the
hypothesis that the short form is preferentially expressed in early
progenitors, only the truncated forms (p80/p77) of STAT5A and STAT5B
were detected in the multipotent FDC-Pmix cells
(Fig 1A). Similar results were obtained
with FDC-P1 cells, which do not express any lineage-specific markers.
In contrast, predominantly full-length STAT5 (p96/p94) isoforms were
detected in the IL-3-dependent FDC-P1M cells (Fig 1A), which lack
markers of early progenitors. No significant difference in the levels of STAT isoforms was observed in the nucleus of IL-3 stimulated as
compared with IL-3-deprived cells; however, a slower migrating band
could be observed in stimulated cell extracts, indicative of
phosphorylation. To confirm that both the activated forms detected
in FDC-P1 and FDC-Pmix cells and the forms in FDC-P1M cells were
able to bind DNA, proteins binding to oligonucleotides containing the
STAT5 recognition sequences from the -casein gene promoter were
purified by affinity chromatography (Fig 1B). Consistent with earlier
results that phosphorylation is required for DNA binding, STAT5 was
only purified in stimulated cell extracts. Significantly, both STAT5
and STAT5 isoforms bound the STAT5 recognition sequence. This was
further confirmed by EMSA (Fig 1C). Two distinct complexes were
observed in the two types of cells reflecting homodimerization of the
short forms or the forms. Importantly, the DNA-STAT5 complexes
from both cell types could be supershifted with antibodies directed
against the amino-terminus of STAT5, but antibody directed against the
carboxyl-terminus of STAT5 only interacted with the complex detected in
FDC-P1M, in which only -forms were detected (Fig 1C). This confirms
that the short forms expressed in FDC-P1 and FDC-Pmix cells
represent a carboxyl-truncated form of full-length STAT5. In
addition, these results verify that expression of the short is a
hallmark of early hematopoietic progenitors.
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| Fig 1.
Expression of full-length and truncated STAT5 proteins in
hematopoietic cell lines. (A) Nuclear extracts were prepared from unstimulated () or IL-3-stimulated (+) A4, 15S, FDC-P1 and
FDC-P1(M) cells. Full-length (STAT5) or truncated (STAT5) STAT5
proteins were detected by Western blot analysis using monoclonal
anti-STAT5 antibodies (89p). (B) FDC-P1 and FDC-P1(M) cells were grown
in IL-3-containing medium (Med) or starved for 20 hours () and
stimulated with IL-3 (+) for 30 minutes. Nuclear extracts were
prepared and incubated with multimerized -casein oligonucleotides
coupled to Sepharose beads. Bound proteins were eluted and analyzed by Western blotting with anti-STAT5 antibodies (89p). (C) A labeled DNA
probe containing the -casein GAS element was added to nuclear extracts from IL-3-stimulated FDC-P1 and FDC-P1(M) cells and
subsequently incubated either without () or with antibodies directed
against the C-terminus (C) or N-terminus (N) of STAT5. Complexes were analyzed by an EMSA.
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Full-length STAT5 is the predominant form in the cytoplasm of
early progenitors.
It is conceivable that both and forms of STAT5 are present in
early progenitors, but only the short form is phosphorylated and
transduced to the nucleus. Cytoplasm extracts were thus examined. Despite the exclusive presence of activated STAT5 in the nucleus of
IL-3-stimulated FDC-P1, full-length STAT5 was the major form present in the cytoplasm as determined by Western blot analysis (Fig 2). Indeed, only trace amounts of
STAT5 were detectable. Significantly, when cytoplasm extracts were
analyzed by EMSA, only low levels of both STAT5 and STAT5 were
found to bind DNA, the predominant form being the truncated form (data
not shown). These results indicate that the majority (if not all) of
truncated STAT5 detected in the cytoplasm is phosphorylated and
probably enroute to the nucleus. Although it is conceivable that the
form in the FDC-P1 cytoplasm is the preferred substrate for JAK activation, a more likely explanation is that the long-form is altered
after phosphorylation or transport to the nucleus.
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| Fig 2.
Full-length STAT5 proteins are expressed in both
FDC-P1 and FDC-P1(M) cells. (A) FDC-P1 and FDC-P1(M) cells were grown
in IL-3-containing medium (Med) or starved for 20 hours () and
stimulated with IL-3 (+) for 30 minutes. Cytosolic extracts were
analyzed by Western blotting using anti-STAT5 antibodies (89p). (B) The nitrocellulose filter shown in (A) was stripped and reprobed with anti-STAT5 antibodies directed against the C-terminus of STAT5. Locations of full-length () and truncated () STAT5 proteins are
indicated on the right.
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Proteolytic activity in nuclear extracts of FDC-P1 cells cleaves the
full-length STAT5 found in FDC-P1(M) nuclear extracts to generate
STAT5.
One likely explanation for the presence of different STAT5 isoforms in
the nucleus versus cytoplasm is the presence of a protease that either
specifically recognizes phosphorylated forms of STAT5 and/or is
exclusively associated with the nucleus. Nuclear and cytosolic extracts
from FDC-P1 cells were thus incubated with nuclear extracts from
FDC-P1(M) that contain full-length phosphorylated STAT5. In
accordance with the hypothesis that a protease associated with the
nucleus cleaves STAT5, Western blot analysis of nuclear extracts of
FDC-P1(M) cells incubated with equal amounts of nuclear extracts
prepared from FDC-P1 cells showed that all STAT5 from FDC-P1(M) was
converted to STAT5 (Fig 3, compare lanes
4 and 5). The levels of conversion of STAT5 to STAT5 were
proportional with the amount of nuclear extracts added to the sample
(data not shown), consistent with an enzymatic activity. Furthermore, this analysis showed that the shortened form, generated by incubation with nuclear extracts from FDC-P1 cells, was the same length as STAT5 in FDC-P1 cells (Fig 3, compare lanes 2 and 5). In contrast, nuclear extracts incubated with cytosolic extracts of FDC-P1 showed only a slight reduction in levels of STAT5 (Fig 3, compare lanes 4 and 7). To rule out the possibility that the cytoplasm contained a
specific protease inhibitor, cytoplasm extracts from both FDC-P1(M) and
FDC-P1 cells were added to nuclear extracts of FDC-P1 cells before
incubation with FDC-P1(M) nuclear extracts containing the STAT5
template. No inhibition of the protease activity in FDC-P1 nuclear
extracts was observed (Fig 3, compare lane 5 with 9 and 10). Thus, the
slight levels of protease activity observed with cytosol extracts is
most likely due to trace levels of nuclear contamination. In
conclusion, a protease activity associated with the nucleus in FDC-Pmix
and FDC-P1 cells is able to cleave full-length STAT5 to generate a
carboxyl-truncated STAT5.
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| Fig 3.
Cleavage of full-length STAT5 in FDC-P1(M) cells by a
nuclear-associated protease from FDC-P1 cells. Unstimulated () or IL-3-stimulated (+) FDC-P1 and FDC-P1(M) cells were lysed and nuclear (N) and cytoplasmic (C) extracts were prepared. Extracts from
FDC-P1 and FDC-P1(M) cells were analyzed either separately (lanes 1 through 4) or mixed and incubated for 15 minutes on ice (lanes 5 through 10). STAT5 proteins were detected by Western blotting using
anti-STAT5 antibodies (89p). The trace levels of STAT5 observed in
lane 10 are due to the addition of high levels of STAT5 in FDC-P1(M)
cytosolic extracts (see Fig 2). This could be eliminated by raising the
incubation temperature for 2 minutes at 37°C (data not shown).
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Protease activity is independent of IL-3 stimulation and does not
require an activated substrate.
The predominant localization of the protease in the nuclear extract
could imply that, in analogy with STAT family members, IL-3 stimulation
leads to its activation and translocation to the nucleus. We thus
determined if FDC-P1 nuclear extracts prepared from starved cells also
contained proteolytic activity. Significantly, nuclear extracts from
either stimulated or IL-3-deprived cells converted STAT5 isolated
from FDC-P1(M) cells to STAT5 (Fig 3, compare lanes 5 and 6). Thus,
the protease is in an active form in the nucleus of both stimulated and
unstimulated cells.
We next asked the question if nuclear transport of STAT5 was the
only prerequisite for cleavage (cellular colocalization) or if
activation by IL-3 (eg, phosphorylation or dimerization) was also
required. STAT5 was isolated by immunoprecipitation from either the
nuclei of stimulated FDC-P1(M) cells (active form) or from the
cytoplasm of starved FDC-P1(M) cells (inactive form) and incubated with
nuclear extracts of FDC-P1 cells. Western blot analysis after SDS-PAGE
showed that both forms could be used as a substrate by the protease in
FDC-P1 nuclear extracts (Fig 4, lanes 1 through 5). Under the conditions used, not all of the inactive form was
cleaved. This may indicate a preference by the protease for the active
form or be due to the higher levels of STAT5 in the cytosolic
extracts (compare lanes 2 and 4 in Fig 4). To confirm the active state
of the nuclear STAT but not the cytosolic STAT, PTy antisera was used
to detect phosphorylated tyrosine residues. As expected, STAT5
isolated from the nucleus but not cytosol was recognized by the
antisera (Fig 4, compare lanes 6 and 8). These results confirm that
colocalization of the protease with the STAT5 substrate is sufficient
for cleavage; modification of STAT5 by IL-3 stimulation is not
required.
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| Fig 4.
Both activated and nonactivated forms of STAT5 are
cleaved by the protease in FDC-P1 nuclear extracts. The different forms of STAT5 were isolated from either stimulated FDC-P1(M) nuclear extracts (N) or unstimulated cytosol extracts (C) by
immunoprecipitation using antisera that recognizes the carboxyl
terminus of STAT5b (C-17). The Sepharose pellet was dissolved in
nuclear buffer and aliquots were either mixed with 1/10 volume of
nuclear extract from unstimulated FDC-P1 cells or nuclear extract
buffer. Extracts were incubated on ice for 15 minutes and then
transferred to 37°C for 2 minutes before freezing. STAT5 proteins
were detected by Western blotting using anti-STAT5 antibodies (89p) or
anti-P-Ty (4G10).
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The FDC-P1 protease is an endopeptidase and its activity is inhibited
by a serine protease inhibitor.
Proteolytic activity can be due to either exopeptidases or
endopeptidases. The latter can further be classified according to
essential catalytic residues at their active sites, as well as their
dependency on cofactors, such as Ca+2 or Zn+2
(reviewed in Bond and Butler27). Cleavage by an
endopeptidase should lead to the generation of two or more peptides,
depending on the number of cleavage sites. To determine if the observed proteolytic activity was due to an endonuclease or exonuclease, we
separated FDC-P1 nuclear extracts by SDS-PAGE under conditions that
would allow the detection of lower molecular weight peptides. Consistent with internal cleavage, a band of approximately 14 kD was
observed in nuclear extracts of FDC-P1 cells, but not FDC-P1(M) cells
when probed with antisera recognizing an epitope near the carboxyl
terminus (Fig 5). In contrast, probing the
same extract with antisera recognizing the area around the SH2 domain
detected only the STAT5 bands (77/80 kD; data not shown). In
conclusion, the nucleus-associated protease found in early
hematopoietic cells cleaves a specific internal peptide bond.
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| Fig 5.
Detection of a proteolytic C-terminal STAT5 fragment in
FDC-P1 cells. Nuclear extracts of unstimulated () or
IL-3-stimulated (+) FDC-P1 and FDC-P1(M) cells were analyzed by
Western blotting using a C-terminal anti-STAT5 antibody (C-17). The
full-length STAT5 protein is indicated on the right. The proteolytic
C-terminal fragment of STAT5 is indicated on the left.
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We thus sought to further classify the protease detectable in FDC-P1
and FDC-Pmix nuclear extracts. Although a battery of protease
inhibitors were routinely added to all buffers during the preparation
and analysis of cell extracts (see the Materials and Methods for
details), none of these effectively inhibited the observed proteolytic
activity. We thus added one of several other protease inhibitors to our
basic buffer systems, and in one case EDTA, and analyzed the FDC-P1
nuclear extracts either alone or in mix experiments as described above.
Taken together, this series of experiments can be summarized as
follows. Neither the site-directed inhibitor of active-site cysteine
residues (E64), the specific inhibitor of aspartic proteinases
(pepstatin), nor the addition of EDTA altered the protease activity of
FDC-P1 cells, indicating that the protease is, most likely, not a
member of either the cysteine, aspartic, or metallo-proteinases. In
support of its classification as a serine protease, the protease
activity could be inhibited with saturating (>1 mmol/L)
concentrations of PMSF, which is a relatively specific and broad
inhibitor of serine proteases (Fig 6,
compare lanes 2 and 3). Interestingly, other proteases that inhibit
serine proteases, including DCI, TPCK, TLCK, leupeptin, and Pefabloc,
did not inhibit the protease, although the highest recommended
concentrations were used. However, this is perhaps not unexpected,
because these compounds inhibit distinct classes of serine proteases.
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| Fig 6.
The protease activity in FDC-P1 nuclear extracts can be
inhibited by PMSF. Both unstimulated () or IL-3-stimulated (+)
FDC-P1 and FDC-P1(M) cells were lysed in the presence or absence of the protease inhibitor PMSF (P) and nuclear (N) extracts were prepared. Extracts from FDC-P1 and FDC-P1(M) cells were analyzed either separately (lanes 9 through 14) or mixed and incubated for 15 minutes
on ice (lanes 1 through 8). STAT5 proteins were detected by Western
blotting using anti-STAT5 antibodies (89p).
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The presence of protease activity in the cell extracts raised the
question if cleavage occurred in vitro, ie, during the preparation of
the extracts, but not in vivo. To address this, nuclear extracts of
FDC-P1 cells were analyzed that had been exposed to saturating concentrations of PMSF at all stages of extraction. In contrast to
nuclear extracts prepared with 0.2 mmol/L PMSF or 4 mmol/L Pefabloc, in
which only STAT5 was detectable, approximately equal levels of
STAT5 and STAT5 were detectable in Western blot analysis (Fig 6,
compare lanes 7 and 8). Although these results clearly show that in
vitro cleavage does occur, a significant portion of STAT5 could not
be inhibited by PMSF. This is in contrast to our results obtained in
mixing experiments in which incubation with PMSF entirely inhibited
STAT5 cleavage (Fig 6, compare lanes 2 and 3, and see figure
legend), confirming that the concentration of PMSF used is sufficient
for complete inhibition. In conclusion, these results show that
STAT5 present in the nucleus of FDC-P1 cells is converted to
STAT5 in vivo.
| |
DISCUSSION |
The cell-specific response to a particular cytokine stimulation
reflects various parameters of the cell, largely, but not exclusively,
determined by an endogenous program of differentiation.28 One of the important consequences of cytokine-receptor stimulation is
the transcriptional activation of previously quiescent genes. The
identification of the STAT family of transcription factors, which are
directly activated by cytokine stimulation, has provided an important
tool to identify mechanisms by which differential gene expression is
achieved through signaling of a common receptor. Various mechanisms
have been shown, including the differential activation of STAT family
members due to specificity determined by receptor subunits (reviewed in
Darnell29), quantitative differences in STAT
transcriptional activation modulated by the duration or strength of
receptor signaling or degree of serine phosphorylation,30 and functional interactions with other transcription
factors.31,32 Modification of the STAT proteins themselves,
due either to transcriptional or posttranscriptional mechanisms, is
clearly another mechanism by which the target gene specificity can be
altered.
The work presented here has functionally identified a protease that
modifies the action of STAT5 in early hematopoietic cells by cleavage
of the carboxyl terminus. We propose the name MSA (Modulator of Stat
Activity) for this protease. During the course of this work, this
protease activity was also reported by Azam et al.33 Our
findings extend their work by determining the localization of the
protease, defining its substrate, and determining the role of cytokine
stimulation for its activity. Although a previous report has proposed
that truncated STAT5 isoforms are generated at the level of transcript
splicing,9 we were unable to detect the proposed
alternative transcripts in the cells we examined (J.M. and C.S.,
unpublished results). However, we cannot exclude that
this mechanism is used to generate STAT5 in other cell types.
MSA is found in nuclear extracts and most probably has a serine
catalytic domain, thus belonging to one of the larger family of
proteases. Although a large number of protease inhibitors were screened, we were able to only identify one, PMSF, that inhibited its
activity. Although serine proteases are generally found in secretory or
specialized granules, MSA is unique in that it is associated with the
nucleus. To date, only a limited number of proteases have been
localized to the nucleus.34-36 Molecular cloning of the
gene encoding this protease may thus lead to the identification of a
unique family of proteases that regulate the activity of transcription
factors.
MSA is in an activated state in both IL-3 stimulated and nonstimulated
cells; thus, cytokine receptor stimulation is not necessary for its
activation. However, stimulation involving one of the JAK family
members is necessary to activate STAT5 and induce its translocation
into the nucleus, where it is cleaved by MSA. Interestingly, neither
phosphorylation nor dimerization is necesary for STAT to be used as a
substrate of MSA. JAK stimulation is thus only necessary for the
translocation of STAT5 to the nucleus and not for inducing
conformational changes required for MSA recognition.
Earlier reports have shown that early hematopoietic progenitors
preferentially express STAT5.6,20 This was clearly shown in human primary monocyte cultures in which differentiation induction leads to a switch from the coexpression of both STAT5 and STAT5 to the exclusive expression of the full-length form. The work presented
here extends this observation. Of particular interest is the high
expression levels of STAT5 in the two independent isolates of the
multipotent FDC-Pmix cells. These cells are able to differentiate into
both erythroid and myeloid lineages (G, M, Eo, and Meg) and exhibit an
open-chromatin structure for lymphoid-specific genes24,28,37 and thus clearly represent an early
hematopoietic progenitor or stem cell. The high expression levels of
MSA in these cells may reflect a mechanism by which the cell is able to
translate cytokine stimulation into a mitogenic signal coupled with
self-renewal versus differentiation. A role of proteases and protease
inhibitors in early hematopoietic differentiation has been suggested by
other studies in which the downregulation of genes encoding granzyme B
and serpin2A during differentiation of FDC-Pmix cells was
reported.38,39 Interestingly, upregulation of MSA has
occurred in 10 of 15 factor-independent mutants of FDC-P1(M) cells
(J.M., W.O., and C.S., unpublished results). The ability
of MSA to modulate the activity of a pivotal transcription factor in
cytokine stimulation suggests a possible mechanism by which proteases
can regulate differentiation and self-renewal. Although at this point
purely speculative, it could be envisaged that truncated STAT5 in
synergy with other transcription factors upregulates genes important in
the mitogenic response, whereas STAT5 is more effective in the
regulation of genes involved in differentiation. This would explain the
conflicting reports of STATs role in these two important functions of
cytokine stimulation.40-42
STAT5 could differentially regulate gene expression by exerting a
dominant negative effect on the transcription of a subset of target
genes9,30 or by interacting with a unique subset of
transcription factors.7,31 Previous studies have shown that
carboxyl-truncated STAT5 proteins are unable to transactivate Cis and Osm,9,41 two target genes shown to
be upregulated by STAT5 in early hematopoiesis.43,44 Azam
et al33 have also verified that cells expressing
exclusively STAT5 showed a delayed and reduced activation of these
two genes. Importantly, IL-3 stimulation of FDC-P1(M) cells (and
consequent activation of STAT5) induces the Osm expression
to levels more than fivefold higher than in stimulated FDC-P1 cells, in
which activated STAT5 is predominantly present (J.M. and C.S.,
unpublished results). Taken together, these results are
consistent with the idea that MSA is able to modulate STAT activity.
However, we were unable to detect significant difference between levels
of Cis transcripts between the two cells types after IL-3
stimulation. This discrepancy most likely reflects interactions with
different transcription factors that may also be activated by IL-3
stimulation or that are able to interact with both STAT5 and
STAT5. The identification of other target genes of STAT5 is
necessary to clarify the role of STAT5 in early hematopoietic cells.
Proteases have long been known to play important roles in regulating
cell proliferation, differentiation, and apoptosis. There are several
examples in which proteases play an important role in the regulation of
transcription factors, either by directing degradation (reviewed in
Ciechanover45) or by activating latent transcription
factors in the cytoplasm.46-48 We have described here a
novel protease that modulates the activity of a pivotal transcription
factor of cytokine regulation. Although we have shown that activated
STAT5 is a substrate of MSA, we cannot rule out that other
transcription factors in the nucleus are also regulated by this
protease. Purification and molecular cloning of this protease should
provide valuable insight into its regulation and specificity.
| |
FOOTNOTES |
Submitted August 11, 1997;
accepted October 30, 1997.
This work is a part of the doctoral thesis of J.M. at the Faculty of
Biology, University of Hamburg and was supported by grants from the
Thyssen Foundation and the Deutsche Forschungsgemeinschaft (SFB545).
The Heinrich-Pette-Institut is financially supported by Freie und
Hansestadt Hamburg and the Bundesministerium für Gesundheit.
Address reprint requests to Carol Stocking, PhD,
Heinrich-Pette-Institute, Martinistr. 52, D-20251 Hamburg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are indebted to Bernd Groner and Fabrice Gouilleux for
introducing us to the world of STATs. We also thank Drs B. Groner, J. Heukeshoven, and J. Ihle for stimulating discussions.
| |
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Abstract
Introduction
Abstract