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Carboxyl-Truncated STAT5b Is Generated by a Nucleus-Associated Serine
Protease in Early Hematopoietic Progenitors
By Johann Meyer, Manfred Jücker, Wolfram Ostertag, and Carol Stocking
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 a forms of STAT5A/5B (p96/p94) to generate carboxyltruncated b 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.
r 1998 by The American Society of Hematology.
C
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 b 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 STAT5b reported in early hematopoietic
cells.6 This study was initiated to determine if an alternative
mechanism is involved in the generation of the truncated
STAT5b isoforms. We have identified a nucleus-associated
protease present in early hematopoietic cells that cleaves
activated STAT5a to generate STAT5b. The importance of this
serine endopeptidase in regulating hematopoiesis is discussed.
YTOKINES 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 b forms that, unlike the
long a 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 STAT5b, 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 STAT3b.7
A number of studies have clearly shown that the truncated b
form is the predominant phosphorylated STAT5 form observed
after IL-3, GM-CSF, or Epo stimulation in early hematopoietic
Blood, Vol 91, No 6 (March 15), 1998: pp 1901-1908
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 (23) in
10% fetal calf serum and IL-3. IL-3 was obtained from conditioned
From the Department of Cell and Virus Genetics, Heinrich-PetteInstitut für experimentelle Virologie und Immunologie an der Universität Hamburg, Hamburg, Germany.
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-PetteInstitute, 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.
r 1998 by The American Society of Hematology.
0006-4971/98/9106-0039$3.00/0
1901
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1902
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 3 107 cells were washed twice with phosphatebuffered 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 270°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 Na-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 3 103 cpm/fmol. The STAT5 binding site of the bovine b-casein
promoter was used as a probe (58-AGATTTCTAGGAATTCAAATC38).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 0.253 TBE (45 mmol/L Tris-borate, 1
MEYER ET AL
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 STAT5a. 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 affınity 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 b-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 33 SDS loading buffer
was added to samples that were subsequently separated by SDS-PAGE
(7.5%), blotted onto nitrocellulose membrane, and visualized with
antibody.
RESULTS
Truncated STAT5b is the preferential activated isoform
observed in multipotent hematopoietic progenitors. Activated
b 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 STAT5b. Nuclear extracts were separated by SDSPAGE 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 STAT5a (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 b forms detected in FDC-P1 and FDC-Pmix cells and
the a forms in FDC-P1M cells were able to bind DNA, proteins
binding to oligonucleotides containing the STAT5 recognition
sequences from the b-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 STAT5a and STAT5b isoforms bound the STAT5
recognition sequence. This was further confirmed by EMSA
(Fig 1C). Two distinct complexes were observed in the two
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PROTEOLYTIC CLEAVAGE OF STAT5
1903
Full-length STAT5a is the predominant form in the cytoplasm
of early progenitors. It is conceivable that both a and b 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 STAT5b in the nucleus of IL-3–stimulated
FDC-P1, full-length STAT5a was the major form present in the
cytoplasm as determined by Western blot analysis (Fig 2).
Indeed, only trace amounts of STAT5b were detectable. Significantly, when cytoplasm extracts were analyzed by EMSA, only
low levels of both STAT5a and STAT5b 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 STAT5b detected in the cytoplasm is phosphorylated
and probably enroute to the nucleus. Although it is conceivable
that the b 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.
Proteolytic activity in nuclear extracts of FDC-P1 cells
cleaves the full-length STAT5a found in FDC-P1(M) nuclear
extracts to generate STAT5b. 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 exclu-
Fig 1. Expression of full-length and truncated STAT5 proteins in
hematopoietic cell lines. (A) Nuclear extracts were prepared from
unstimulated (2) or IL-3–stimulated (1) A4, 15S, FDC-P1 and FDCP1(M) cells. Full-length (STAT5a) or truncated (STAT5b) 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 (2)
and stimulated with IL-3 (1) for 30 minutes. Nuclear extracts were
prepared and incubated with multimerized b-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 b-casein GAS element was added
to nuclear extracts from IL-3–stimulated FDC-P1 and FDC-P1(M) cells
and subsequently incubated either without (2) or with antibodies
directed against the C-terminus (C) or N-terminus (N) of STAT5.
Complexes were analyzed by an EMSA.
types of cells reflecting homodimerization of the short a forms
or the b 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 a-forms were
detected (Fig 1C). This confirms that the short b forms
expressed in FDC-P1 and FDC-Pmix cells represent a carboxyltruncated form of full-length STAT5a. In addition, these results
verify that expression of the short b is a hallmark of early
hematopoietic progenitors.
Fig 2. Full-length STAT5a 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 (2) and
stimulated with IL-3 (1) 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 (a) and truncated (b) STAT5 proteins are
indicated on the right.
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1904
sively 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 STAT5a. In accordance with the hypothesis that a protease
associated with the nucleus cleaves STAT5a, 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 STAT5a from FDC-P1(M) was converted to
STAT5b (Fig 3, compare lanes 4 and 5). The levels of
conversion of STAT5a to STAT5b 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 STAT5b 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
STAT5a (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
STAT5a 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 STAT5a to generate a carboxyltruncated STAT5b.
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
Fig 3. Cleavage of full-length STAT5a in FDC-P1(M) cells by a
nuclear-associated protease from FDC-P1 cells. Unstimulated (2) or
IL-3–stimulated (1) 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 STAT5a observed in
lane 10 are due to the addition of high levels of STAT5a 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).
MEYER ET AL
Fig 4. Both activated and nonactivated forms of STAT5a are
cleaved by the protease in FDC-P1 nuclear extracts. The different
forms of STAT5a 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).
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
STAT5a isolated from FDC-P1(M) cells to STAT5b (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 STAT5a
was the only prerequisite for cleavage (cellular colocalization)
or if activation by IL-3 (eg, phosphorylation or dimerization)
was also required. STAT5a 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 STAT5a 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,
STAT5a 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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PROTEOLYTIC CLEAVAGE OF STAT5
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 Ca12 or Zn12 (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 FDCP1(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 STAT5b bands (77/80 kD; data not
shown). In conclusion, the nucleus-associated protease found in
early hematopoietic cells cleaves a specific internal peptide
bond.
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
Fig 5. Detection of a proteolytic C-terminal STAT5 fragment in
FDC-P1 cells. Nuclear extracts of unstimulated (2) or IL-3–stimulated
(1) FDC-P1 and FDC-P1(M) cells were analyzed by Western blotting
using a C-terminal anti-STAT5 antibody (C-17). The full-length STAT5a
protein is indicated on the right. The proteolytic C-terminal fragment
of STAT5 is indicated on the left.
1905
Fig 6. The protease activity in FDC-P1 nuclear extracts can be
inhibited by PMSF. Both unstimulated (2) or IL-3–stimulated (1)
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).
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.
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 STAT5b
was detectable, approximately equal levels of STAT5a and
STAT5b 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 STAT5b
could not be inhibited by PMSF. This is in contrast to our results
obtained in mixing experiments in which incubation with PMSF
entirely inhibited STAT5a 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 STAT5a present in the nucleus of
FDC-P1 cells is converted to STAT5b in vivo.
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1906
MEYER ET AL
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 STAT5b 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
STAT5a to the nucleus and not for inducing conformational
changes required for MSA recognition.
Earlier reports have shown that early hematopoietic progenitors preferentially express STAT5b.6,20 This was clearly shown
in human primary monocyte cultures in which differentiation
induction leads to a switch from the coexpression of both
STAT5a and STAT5b 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
STAT5b 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 STAT5b in synergy with other
transcription factors upregulates genes important in the mitogenic response, whereas STAT5a 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
STAT5b 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 STAT5b showed a
delayed and reduced activation of these two genes. Importantly,
IL-3 stimulation of FDC-P1(M) cells (and consequent activation of STAT5a) induces the Osm expression to levels more
than fivefold higher than in stimulated FDC-P1 cells, in which
activated STAT5b 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 STAT5a and STAT5b. The identification of other target
genes of STAT5 is necessary to clarify the role of STAT5b 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
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PROTEOLYTIC CLEAVAGE OF STAT5
of a pivotal transcription factor of cytokine regulation. Although
we have shown that activated STAT5a 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.
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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1998 91: 1901-1908
Carboxyl-Truncated STAT5β Is Generated by a Nucleus-Associated Serine
Protease in Early Hematopoietic Progenitors
Johann Meyer, Manfred Jücker, Wolfram Ostertag and Carol Stocking
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