Transcription Factor STAT5A Is a Substrate of
Bruton's Tyrosine Kinase in B Cells*
Sandeep
Mahajan
§,
Alexei
Vassilev§,
Nancy
Sun§,
Zahide
Ozer¶
,
Chen
Mao**, and
Fatih M.
Uckun
From the Molecular Signal Transduction Laboratory,
Parker Hughes Cancer Center and the Departments of
§ Biochemistry, ¶ Molecular Biology, Immunology,
and ** Structural Biology, Parker Hughes Institute,
St. Paul, Minnesota 55113
Received for publication, May 29, 2001
 |
ABSTRACT |
STAT5A is a molecular
regulator of proliferation, differentiation, and apoptosis in
lymphohematopoietic cells. Here we show that STAT5A can serve as a
functional substrate of Bruton's tyrosine kinase (BTK). Purified
recombinant BTK was capable of directly binding purified recombinant
STAT5A with high affinity (Kd = 44 nM),
as determined by surface plasmon resonance using a BIAcore biosensor
system. BTK was also capable of tyrosine-phosphorylating ectopically
expressed recombinant STAT5A on Tyr694 both in
vitro and in vivo in a Janus kinase 3-independent
fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F
mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src
homology 2 (SH2) and SH3 domains did not alter the BTK-mediated
tyrosine phosphorylation. Recombinant BTK proteins with mutant
pleckstrin homology, SH2, or SH3 domains were capable of
phosphorylating STAT5A, whereas recombinant BTK proteins with
SH1/kinase domain mutations were not. In pull-down experiments, only
full-length BTK and its SH1/kinase domain (but not the pleckstrin
homology, SH2, or SH3 domains) were capable of binding STAT5A.
Ectopically expressed BTK kinase domain was capable of
tyrosine-phosphorylating STAT5A both in vitro and in
vivo. BTK-mediated tyrosine phosphorylation of ectopically
expressed wild type (but not Tyr694 mutant) STAT5A enhanced
its DNA binding activity. In BTK-competent chicken B cells,
anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was
prevented by pretreatment with the BTK inhibitor LFM-A13 but not by
pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor
ligation resulted in enhanced tyrosine phosphorylation of STAT5 in
BTK-deficient chicken B cells reconstituted with wild type human BTK
but not in BTK-deficient chicken B cells reconstituted with
kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in
enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells
from wild type mice but not in BTK-deficient B cells from XID mice. In
contrast to B cells from XID mice, B cells from JAK3 knockout mice
showed a normal STAT5A phosphorylation response to anti-IgM
stimulation. These findings provide unprecedented experimental evidence
that BTK plays a nonredundant and pivotal role in B cell antigen
receptor-mediated STAT5A activation in B cells.
| |
INTRODUCTION |
Bruton's tyrosine kinase
(BTK),1 a member of the TEC
family of cytoplasmic protein-tyrosine kinases (PTKs), is intimately
involved in signal transduction pathways regulating survival,
activation, proliferation, and differentiation of B-lineage lymphoid
cells (1-5). BTK participates in signal transduction pathways
initiated by the binding of a variety of extracellular ligands to their cell surface receptors (2). Following ligation of B cell antigen receptors, BTK activation by the concerted actions of the PTKs LYN and
SYK is required for induction of phospholipase C-
2-mediated calcium
mobilization (2). Mutations in the human btk gene are the
cause of X-linked agammaglobulinemia, a male immune deficiency disorder
characterized by a lack of mature, immunoglobulin-producing, peripheral
B cells (6, 7). In mice, mutations in the btk gene have been
identified as the cause of murine X-linked immune deficiency (8).
BTK has an N-terminal region consisting of a pleckstrin homology (PH)
domain followed by a proline-rich TEC homology domain. The PH domain is
the site of activation by phosphatidylinositol phosphates and G-protein
subunits and inhibition by protein kinase C (9). The remaining
portion of BTK contains Src homology (SH) domain SH3, followed by SH2
and a C-terminal SH1/kinase domain (KD). The SH2 domain mediates
binding to tyrosine-phosphorylated peptide motifs on other molecules,
and the SH3 domain mediates binding to proline-rich motifs. Mutations
in the SH1 domain, SH2 domain, and PH domain of human BTK have been
found to cause maturational blocks at early stages of B cell ontogeny
leading to XLA (10). BTK-deficient mice generated by introducing PH
domain or SH1 domain mutations in embryonic stem cells exhibit
defective B cell development and function (11). Thus, different domains
of BTK are important for its physiologic functions.
Proximal events involving Src family PTK and SYK that lead to BTK
activation following stimulation of the B cell antigen receptor are
well known, but only very few signaling molecules have been identified
as downstream substrates of BTK (2, 12). Recent observations suggest
the involvement of BTK in signal transduction pathways affecting gene
transcription (13). Recent studies have further revealed a
nucleocytoplasmic shuttling system for BTK that has implications
regarding potential targets inside the nucleus, which may be critical
in gene regulation during B cell development and differentiation (14).
An unresolved question in B cell immunology is the nature of the
molecular coupling mechanisms that link the proximal signals that are
triggered by the engagement of the B cell antigen receptor to
downstream signal transduction pathways affecting gene transcription.
BCR stimulation leads to the activation of transcription factor NF-
B
in a BTK-dependent fashion, which in turn regulates genes
controlling B cell growth (15). Recently, the BTK substrate
phospholipase C-2 was shown to couple BTK to the NF-B signaling
pathway (16). BTK has also been shown to regulate the nuclear
localization and transcriptional activity of the multifunctional
transcription factor BAP-135/TFII-I (17). These results illustrate that
the identification of substrates for BTK in B cells is a critical step
to a better understanding of the molecular mechanism(s) of lymphocyte
activation through the antigen receptor.
Signal transducers and activators of transcription (STAT)
proteins are a family of DNA-binding proteins that were initially identified during a search for interferon-
- or
interferon--stimulated gene transcription targets (18-20).
Different ligands and cell activators employ specific STAT family
members (21-23). The basic model for STAT activation suggests that in
unstimulated cells, latent forms of STATs are predominantly localized
within the cytoplasm. Ligand binding induces STAT proteins to associate
with intracellular phosphotyrosine residues of transmembrane receptors
(24, 25). Once STATs are bound to receptors, they are phosphorylated by Janus family or Src family PTK (26). STAT proteins then dimerize through specific reciprocal SH2-phosphotyrosine interactions (27) and
may form complexes with other DNA-binding proteins (28). STAT complexes
translocate to the nucleus and interact with DNA response elements to
enhance transcription of target genes (29). STAT5 is a molecular
regulator of proliferation, differentiation, and apoptosis in
hematopoietic cells (30). B cells abundantly express STAT5, and
engagement of the B cell antigen-receptor results in activation of
STAT5 (31). B cells from mice bearing subcutaneous mammary
adenocarcinoma tumors are severely impaired in their ability to
generate antigen-specific responses (32). Interestingly, purified B
cells isolated from these immunocompromised tumor-bearing mice
exhibited a marked decrease in the expression level of STAT5 without a
concomitant change in the expression levels of STAT1, -3, and -6 (32).
The observed correlation of the loss of B cell function with the
selective decrease in STAT5 expression suggested that regulation of the
STAT5 signaling pathway may provide a molecular mechanism for
modulating the antigen-specific B cell responses. STAT5A-deficient mice
showed decreased proliferation of splenocytes to interleukin (IL)-2
stimulation, which was reported to result from defective induction
of IL-2 receptor chain (33). Recently, STAT5A and STAT5B doubly
disrupted mice have been generated (34), and Sexl et al.
reported that STAT5A/B contribute to interleukin-7-induced B cell
precursor expansion (35). The expansion of B cell precursors is
disrupted at the level of late pro-B and pre-B cells in
STAT5A/B-deficient mice (35). These mice also have reduced numbers of
circulating B cells in their blood (35).
Notably, Bmx/Etk, another member of the TEC PTK family, was shown to
induce activation of the STAT signaling pathway in transfected COS
cells and insect ovary cells (36). More recently, Bmx/Etk was
identified as a critical mediator of Src-induced cell transformation and STAT3 activation (37). Expression of Bmx/Etk in a human hepatoma
cell line Hep3B resulted in a significant increase in its transforming
ability, and this effect was abrogated by dominant negative inhibition
of STAT3 (37), indicating that Bmx/Etk links Src to STAT3 activation
and that Src-Etk-STAT3 is an important pathway in cellular
transformation. While these results set a precedent for activation of
STAT proteins by a member of the TEC family PTK, the significance of
these findings for the B cell physiology has not been investigated. The
purpose of the present study was to examine the role of BTK in STAT5
activation in B cells that follows B cell antigen receptor ligation.
Our experimental data presented herein provide unprecedented evidence
that STAT5A is a substrate of BTK and that engagement of the B cell
antigen receptor activates STAT5A in a BTK-dependent (but
not JAK3-dependent) fashion. Thus, our study provides
experimental evidence that BTK couples the STAT signaling pathway to
the B cell antigen receptor. Our findings prompt the hypothesis that
STAT5A links BTK-mediated signaling events that follow the engagement
of the B cell antigen receptor to downstream gene activation. A
compromised coupling between BTK and STAT5A might lead to aberrant
expression of the STAT5A-responsive genes and might contribute to the
XLA and XID phenotypes.
| |
MATERIALS AND METHODS |
Mice, Cell Lines, Reagents, and Biochemical Assays--
Control,
XID, and JAK3 knockout mice were purchased from Jackson Laboratories.
Mouse splenocytes were isolated, starved in RPMI at a density of
10 × 106 cells/ml for 2 h, and then stimulated
either with murine anti-µ antibodies (10 µg/ml; Sigma) or
recombinant IL-2 (20 ng/ml; R & D Systems) for the indicated times.
KL-2 is an Epstein-Barr virus-transformed human lymphoblastoid cell
line (4). The Sf21 (IPLB-Sf21-AE) insect cell line (Invitrogen) derived from Spodoptera frugiperda ovarian
cells was used to isolate and propagate recombinant viral stocks and produce recombinant proteins of interest. The cells were maintained at
26-28 °C in Grace's insect cell medium containing 10% fetal bovine serum and 1% antibiotic/antimycotic (Life Technologies, Inc.).
Stock cells were maintained in suspension at 0.2-1.6 × 106/ml in 600 ml of total culture volume in 1-liter Bellco
spinner flasks at 60-90 rpm. Cell viability was maintained at >95%
as determined by trypan blue dye exclusion. The establishment of BTK-deficient DT-40 lymphoma B cell clones has been previously described (38). Lack of BTK expression in BTK-deficient DT-40 cells was
confirmed by both immune complex kinase assays and Western blot
analysis (38). Mutations in the human btk cDNA were
introduced by PCR using Pfu polymerase (Stratagene) and
confirmed by sequencing. Wild type and catalytic domain mutant
btk cDNAs were subcloned into pApuro expression vector
and electroporated into BTK-deficient cells. The PTK activity of BTK
immune complexes, as measured by in vitro
autophosphorylation, was abrogated by the catalytic domain mutation.
Equal amounts of BTK proteins were detected by Western blot analysis in
all of the BTK-deficient DT-40 clones transfected with wild type or
mutated human btk genes, but no BTK protein was detectable
in the untransfected BTK-deficient DT-40 cells (38). Cells were starved
in serum-free medium at a density of 10 × 106
cells/ml for 2 h and then stimulated with an anti-chicken IgM antibody (M4 antibody, Southern Biotechnology, Inc.; 10 µg/ml). STAT5A and TEC antibodies were obtained from Transduction Laboratories, Inc.; JAK3 antibodies were from Upstate Biotechnology, Inc.; and anti-BTK antibodies were produced as described (39). The
structure-based design, synthesis, and characterization of the JAK3
inhibitory dimethoxyquinazoline compound HI-P131 (40) (Calbiochem
catalog no. 420101) and BTK inhibitory leflunomide metabolite analogue LFM-A13 (5) (Calbiochem catalog no. 435300) were previously described
in detail (5, 40).
Immunoprecipitations, immune complex protein kinase assays, and
immunoblotting using the ECL detection system (Amersham Pharmacia Biotech) were conducted as described previously (3-5, 38-40). Phosphoamino acid analysis (41), phosphotryptic peptide mapping (41),
and cyanogen bromide (CNBr) peptide analysis (42) have been performed
as described previously. For two-dimensional phosphotryptic peptide
mapping, 32P-labeled protein bands were excised and
subjected to enzymatic digestion with 100 µg/ml trypsin (Sigma)
overnight in 50 mM ammonium bicarbonate. Supernatants were
dried by centrifugal evaporation, and dried samples were resuspended in
4 µl of a buffer containing 7.8% glacial acetic acid and 2.5%
formic acid. Labeled peptides were separated on thin layer
phosphocellulose plates (Eastman Kodak Co.) by electrophoresis at pH
1.9 for 30 min at 1000 V, followed by ascending chromatography in a
buffer containing 37.5% n-butanol, 7.5% glacial acetic
acid, and 25% pyridine. Subsequently, air-dried plates were exposed to
Kodak XAR-5 film. For electrophoretic mobility shift assays, cell
extracts were prepared according to the previously published method
(43). Briefly, cells were lysed in WCE lysis buffer (50 mM
Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol,
1 mM EDTA, 0.5 mM
Na3VO4, and protease inhibitors). An annealed
oligonucleotide corresponding to the GAS-like STAT5 binding site in the
-casein gene promoter (5'-AGATTTCTA GGAATTCAAATC-3') purchased from
New England Biolabs was end-labeled with T4 polynucleotide kinase using
[-32P]ATP (Amersham Pharmacia Biotech). A total of 10 µg of cell extract and 0.5 ng of labeled oligonucleotide were used
per reaction. The cell extract and 200 ng of poly(dI-dC) (Amersham
Pharmacia Biotech) were first incubated on ice for 15 min followed by
another 15-min incubation on ice after adding the labeled
oligonucleotide. The reaction products were run on a 5% polyacrylamide
gel in 0.5× Tris borate-EDTA buffer and visualized by autoradiography.
Recombinant Baculovirus Expression System--
The murine wild
type STAT5A gene in pBluescript SKII+ vector was a kind gift from
J. N. Ihle (St. Jude Children's Hospital, Memphis, TN).
The STAT5A gene was excised from this plasmid by EcoRI digestion and cloned into pFastBac1 (PFB) donor vector
(Life Technologies). The resulting pFastbac-stat5a
recombinant plasmid (PFB-stat5a) was then used to generate
the recombinant baculovirus by site-specific transposition in
Escherichia coli DH10Bac competent cells (Life
Technologies), which harbor a baculovirus shuttle vector (bacmid),
bMON14272 with a mini-attTn7 target site for site-specific
transposition. The bacterial colonies containing recombinant bacmids
were identified by disruption of the lacZ gene. High
molecular weight miniprep DNA was prepared from selected E. coli clones containing recombinant bacmid and transfected into Sf21 cells using Cellfectin reagent (Life Technologies, Inc.) in
a 60-mm dish (1.5 × 106 cells/dish). On day 5 after
transfection, the supernatants from each dish were collected and saved
for subsequent infections and plaque purification. Similarly, the genes
encoding Y694F mutant STAT5A, Y694F-Y STAT5A, wild type BTK, TEC, and
JAK3 were excised from pBluescript plasmid, cloned into PFB vector and
processed as described above for expression in Sf21 cells. The
BTK kinase domain (BTK-KD) gene was amplified from wild type BTK by PCR
using 5'-primer (AGCCATGGGAattgatccaaaggacctcac) with a NcoI
site and 3'-primer (ATAAGCTTtcaggattcttcatccatcaca) with a
HindIII site. The PCR used Taq polymerase (Life
Technologies, Inc.) and conditions of 95 °C denaturing for 3 min,
followed by 30 cycles of 94 °C denaturing for 1 min, 60 °C
annealing for 1 min, 72 °C extension for 2 min, and then running for
an additional 10 min at 72 °C. The PCR product was cloned into the
PCR2.1 vector (BTKKD/PCR2.1) using the Invitrogen TA cloning technique.
The identity of the insert was confirmed by DNA sequencing.
Subsequently, the BTKKD/PCR2.1 was completely digested with
NcoI and HindIII. The 800-base pair fragment was
purified on a 0.8% agarose gel (Qiagene kit) and ligated to the
pFastBac HTb donor plasmid (Life Technologies), followed by digestion
with NcoI and HindIII to generate pFastBac HTb:
BTKKD. Recombinant viral clones were identified visually and purified
from plaque assay plates containing 1.5% SeaPlaque GTG agarose (FMC
Bioproducts) after 5-6 days at 28 °C. Recombinant viral clones were
then amplified in T-25 flask cultures (4.0 × 106/flask on day 0) for 3 days; infected cells were
identified morphologically and then screened for production of the
respective recombinant proteins by SDS-PAGE on 10% polyacrylamide gels
stained with Coomassie Brilliant Blue R. The various recombinant
baculoviruses were inoculated into 25 × 106
Sf21 cells, and the cells were harvested 48 h later as
detailed earlier (44). When coexpression experiments were performed, the amounts of different viruses were adjusted following preliminary experiments to yield equivalent expression of the necessary proteins.
For large scale production of wild type or mutant forms of STAT5A and
BTK, 1-liter cultures of Sf21 cells in 3-liter Bellco spinner flasks (1.0-1.2 × 106 cells/ml) were
infected with the recombinant plasmids PFB-stat5a or
PFB-btk during logarithmic growth with 25 ml of viral stock solution (~2.5 × 108 plaque-forming
units/ml). This infection protocol results in a multiplicity of
infection of ~6. The viral stock solutions were passaged no more than
three times following plaque purification prior to use in these large
scale infections in order to minimize the presence of defective
interfering particles. Cultures were incubated at 28 °C and stirred
at 80-100 rpm using a magnetic stirrer (Bellco Glass Inc., Vineland,
NJ) for 48 h. Infected cells were harvested by gentle
centrifugation in a Beckman GS-6 centrifuge at 500 × g
for 7 min at room temperature. Cells from 1-liter cultures were divided
into two aliquots (~4 g of wet cell paste) and immediately flash-frozen at 
80 °C and stored at 80 °C until purification of the recombinant proteins.
Gel Filtration Analysis--
Sf21 insect cells
coexpressing STAT5 and BTK were washed twice with assembly buffer (10 mM PIPES, 1 mM EDTA, pH 7.2) and lysed in lysis
buffer (0.01 M Tris-Cl, pH 8.2, 1 mM EDTA, 0.15 M NaCl, 1% Nonidet P-40, 2 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, and 1 mM Na3VO4) for
2 h on ice. Cytosolic fractions were recovered after
centrifugation at 18,000 × g for 15 min. Supernatants
were analyzed with fast protein liquid chromatography on a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min. 0.5-ml fractions were collected and subjected to Western blot
analysis to determine the elution patterns and tyrosine phosphorylation
status of STAT5 and BTK proteins, as described above.
STAT5A Homology Model--
A molecular model of STAT5A from
residue 210 to 687 was initially constructed based on its homology with
both STAT1 and STAT3 using the Swiss-Model program (45).
Subsequently, the model was extended to include all coordinates from
residue 163 to 710 and then refined using the Homology module of the
InsightII program (MSI, San Diego) and a Silicon Graphics INDIGO2
computer (Silicon Graphics, Mountain View, CA). Specifically, the
homology modeling of STAT5A was carried out in two steps as follows. 1)
The most reasonable sequence alignment between STAT5A and a coordinate template based on the STAT1 and STAT3 crystal structures was determined. 2) New coordinates were assigned to the STAT5A residues according to the template coordinates (based on the sequence
alignment), followed by the determination of the loop coordinates and
an energy minimization of the entire structure. Finally, the
constructed model of STAT5A was subjected to visual inspection in CHAIN
(46) and energy minimization using the X-plor program (version 3.1, A. T. Brunger, Yale University Press). The coordinates of the BTK
kinase domain have been experimentally determined and will be published
elsewhere. The model of the BTK kinase domain complexed with the tail
segment of STAT5A was based on the ternary structure of the insulin
receptor kinase domain and a peptide substrate.
Site-directed Mutagenesis--
Point mutations were introduced
into the stat5a cDNA by PCR (47). The codon for arginine
618 within the SH2 domain was mutated to a codon for lysine. The codons
for the conserved tryptophans at positions 571 and 573 within the SH3
domain were mutated to leucine. The codon for tyrosine 694 that is the
equivalent of tyrosine 701 in murine STAT1, a previously known
phosphorylation site by JAK kinases (48), was mutated to a codon for
phenylalanine. The codons for the tyrosine residues 665, 668, 682, and
683 that flank tyrosine 694 and appear to be potential phosphorylation sites were also mutated to a codon for phenylalanine. All mutations were confirmed by sequence analysis. Point mutations introduced into
the btk gene (39) have been reported earlier.
Expression and Purification of MBP-BTK and GST-BTK Fusion
Proteins--
cDNAs encoding full-length BTK and its kinase or PH
domains with PCR-generated 5' and 3' BamHI sites were cloned
into the E. coli expression vector pMAL-C2 with the
isopropyl-1-thio--D-galactopyranoside-inducible Ptac
promoter to create an in frame fusion between these coding sequences
and the 3'-end of the E. coli malE gene, which
codes for maltose-binding protein (MBP). cDNAs encoding the SH2,
SH3, or SH2 + SH3 domains with PCR-generated 5' and 3' BamHI
sites were cloned into the E. coli expression vector pGEX-2T
with the isopropyl-1-thio--D-galactopyranoside-inducible
Ptac promoter to create an in frame fusion between these coding
sequences and the 3'-end of the E. coli glutathione
S-transferase (GST) gene. The generated recombinant plasmids
were transformed into the E. coli strain DH5. Single
transformants were expanded in 5 ml of LB medium (1% tryptone, 1%
NaCl, 0.5% yeast extract) containing ampicillin (100 µg/ml) by
overnight culture at 37 °C. Expression of the fusion proteins was
induced with 10 mM
isopropyl-1-thio--D-galactopyranoside. The cells were
harvested by centrifugation at 4500 × g in a Sorvall RC5B centrifuge for 10 min at 4 °C, lysed in sucrose-lysozyme buffer
(20 mM Tris, pH 8.0, 150 mM NaCl, 10% sucrose,
1 mM EDTA, 20 mM lysozyme), and further
disrupted by sonication. After removal of the cell pellets by
centrifugation at 35,000 × g for 1 h at 4 °C,
GST-BTK fusion proteins were purified by glutathione-Sepharose chromatography, whereas MBP-BTK fusion proteins were purified from the
culture supernatants by amylose affinity chromatography (4).
Pull-down Assays with MBP-BTK and GST-BTK Fusion
Proteins--
GST-BTK fusion proteins were noncovalently bound to
glutathione-agarose beads (Sigma) and MBP-BTK fusion proteins were
noncovalently bound to amylose beads under conditions of saturating
protein, as previously described (4). In brief, 50 µg of each protein was incubated with 50 µl of the beads for 2 h at 4 °C. The
beads were washed three times with 1% Nonidet P-40 buffer. Nonidet
P-40 lysates of KL2 human Epstein-Barr virus-transformed lymphoblastoid cells were prepared as described (4), and 500 µg of the lysate was
incubated with 50 µl of fusion protein-coupled beads for 2 h on
ice. The fusion protein adsorbates were washed with ice-cold 1%
Nonidet P-40 buffer and resuspended in reducing SDS sample buffer.
Samples were boiled for 5 min and then fractionated on SDS-PAGE.
Proteins were transferred to Immobilon-P (Millipore Corp.) membranes,
and membranes were immunoblotted with anti-STAT5A antibody
(Transduction Laboratories), according to previously described
procedures (3-5, 38-40).
Purification of STAT5A and BTK Proteins--
Frozen Sf21
insect cells previously infected with PFB-btk (~24 g of
cell paste; 3 liters of culture) were resuspended in 100 ml of
extraction buffer (20 mM sodium phosphate, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, and 1× CompleteTM
protease inhibitors (Roche Molecular Biochemicals)), incubated on ice
for 15 min, and then sonicated three times for 30 s each time
using a Branson sonifier 250 at 50% duty cycle, 30% output while on
ice. All subsequent steps were performed at 4 °C unless otherwise
noted. The extract was then centrifuged at 100,000 × g
for 1 h in a Beckman Optima LE-80K ultracentrifuge using a 70Ti rotor. Following centrifugation, the supernatant was filtered through a
0.22-µm membrane filter (S100) and applied to a 5 × 13.5-cm
Heparin-Toyopearl (Sigma) affinity column (column volume 250 ml) which
had been equilibrated with buffer A (20 mM sodium phosphate, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM DTT). Following application of the filtered soluble cell
extract, the column was washed with 500 ml of buffer A plus 200 mM NaCl. Protein was then eluted from the column with a
1000-ml linear gradient from 0.2 to 1.0 M NaCl in buffer A,
at a flow rate of 2.5 ml/min, and 15-ml fractions were collected.
Eluted fractions were analyzed using SDS-PAGE and immunoblotting.
Fractions containing anti-BTK immunoreactive material were pooled
(~80 ml) and dialyzed twice against 2 liters of buffer A to remove
NaCl and then applied to a Sepharose Q HP26/10 ion exchange column
(column volume 50 ml; Amersham Pharmacia Biotech) that had been
equilibrated with buffer A. Protein was eluted from the Sepharose Q
column using a 250-ml linear gradient from 0 to 0.5 M NaCl
in buffer A, at a flow rate of 2 ml/min, and 4-ml fractions were
collected. Eluted fractions were again analyzed using SDS-PAGE and
immunoblotting. Fractions containing anti-BTK immunoreactive material
were pooled and dialyzed twice against 2 liters of buffer A to remove
NaCl and then applied to a Mono S HR 5/5 ion exchange column (column
volume 1 ml; Amersham Pharmacia Biotech). Protein was eluted from the
Mono S column with a 40-ml linear gradient from 0 to 0.4 M
NaCl in buffer A at a flow rate of 0.5 ml/min, and 1-ml fractions were
collected. BTK was eluted at ~0.25 M NaCl. Fractions
containing BTK were then concentrated individually to a final
concentration of ~2 mg/ml of >95% protein with a total recovery of
1 mg from 4 liters of Sf21 cell culture fluid.
Similarly, frozen Sf21 insect cells previously infected with
PFB-stat5a, PFB-stat5a-[Y694F], or
PFB-stat5a-[Y694F-Y] (~24 g of cell paste; 3-liter
culture) were resuspended in 100 ml of extraction buffer (10 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM
EGTA, 2 mM DTT, 0.1% Triton X-100, and 1×
CompleteTM protease inhibitors (Roche Molecular
Biochemicals), incubated on ice for 15 min, and then sonicated three
times for 30 s each. The extract was then centrifuged at
100,000 × g for 1 h, and the supernatant was
filtered through a 0.22-µm membrane filter (S100) and applied to a
2.5 × 15-cm Q Sepharose High Performance (Amersham Pharmacia
Biotech) column. Following application of the filtered soluble cell
extract, the column was washed with 200 ml of Hepes buffer (10 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT) plus 0.1 M NaCl. Protein was
then eluted from the column with a 400-ml linear gradient from 0.18 to
0.3 M NaCl in Hepes buffer, at a flow rate of 1 ml/min, and
8-ml fractions were collected. Eluted fractions were analyzed using
SDS-PAGE and immunoblotting. Fractions containing anti-STAT5A
immunoreactive material were pooled and dialyzed twice against 2 liters
of Hepes buffer and then applied to a 2 × 10-cm Heparin-Toyopearl
(Sigma) affinity column that had been equilibrated with Hepes buffer.
Elution of protein was performed with a linear gradient of 0.05-0.32
M NaCl in buffer A at a flow rate of 1 ml/min, and 4-ml
fractions were collected. Eluted fractions were analyzed using SDS-PAGE
and immunoblotting. Fractions containing STAT5A (or mutant STAT5A) were
combined, concentrated using Centriprep 30 (Millipore Corp.) to ~2
ml, and then applied to a HiLoad 26/60 Superdex 200 size exclusion
column (Amersham Pharmacia Biotech) preequilibrated with 10 mM Hepes, pH 7.4, 1 mM DTT. STAT5A (or mutant
STAT5A) was eluted with ~350 ml of 10 mM Hepes, pH 7.4, 1 mM DTT and concentrated using Centriprep 30 to a protein
concentration of 10 mg/ml.
For purification of the BTK-KD, frozen Sf21 cells expressing
this protein were thawed in a 37 °C water bath, resuspended in 5 volumes of the lysis buffer (50 mM Tris/HCl, pH 8.5, 100 mM KCl, 2 mM DTT, and 1 mM
phenylmethylsulfonyl fluoride), sonicated for 1 min, and centrifuged at
30,000 × g for 45 min. The supernatant was loaded on a
nickel column, which was equilibrated with a solution containing 10 mM Tris/HCl buffer at pH 8.0, 500 mM KCl, and
0.5 mM DTT at a flow rate of 0.75-0.5 ml/min. The column
was washed with 20 bed volumes of buffer B (20 mM Tris/HCl,
pH 8.5, 500 mM KCl, 15-20 mM imidazole, and 2 mM DTT) and then washed with 10 bed volumes of buffer C (20 mM Tris/HCl, pH 8.5, 1 M KCl, and 2 mM DTT). Subsequently, it was washed again with 2 bed
volumes of buffer B. The protein was eluted with buffer D (20 mM Tris/HCl, 100 mM KCl, 150 mM
imidazole, and 2 mM DTT). The fractions that contained BTK-KD were pooled together and dialyzed against a
solution that contained 20 mM Tris/HCl, 100 mM
NaCl, 2 mM DTT, and 1 mM EDTA. Then the protein
was concentrated to 5 mg/ml, digested with rTEV protease of 6 µg/mg
at 4 °C overnight, and then concentrated to 3 ml, which was then
loaded on a Superdex 200 column (26/60), which was equilibrated with a
solution of 20 mM Tris/HCl, pH 8.5, 50 mM NaCl,
and 2 mM DTT at a flow rate of 0.3 ml/min.
Monitoring of Binding Interactions Using Surface Plasmon
Resonance Technology--
A BIAcore 2000 surface plasmon
resonance-based biosensor system (Amersham Pharmacia Biotech Biosensor
AB) was used to measure the kinetic parameters for the interactions
between soluble recombinant STAT5A protein (analyte) and the
immobilized recombinant BTK protein (ligand). BTK or bovine serum
albumin (control protein) was covalently linked to the dextran on the
surface of research grade CM5 sensor chips via primary amino groups
using the amine coupling kit from Amersham Pharmacia Biotech according
to the manufacturer's instructions (Amersham Pharmacia Biotech),
yielding a resonance signal of 2700 resonance units (RU) (1 RU
corresponds to an immobilized protein amount of 1 pg/mm2
surface). Unreacted moieties on the surface were blocked with ethanolamine. The STAT5A protein samples were diluted in PBS buffer (1 mM KH2PO4, 10 mM
Na2HPO4, 0.137 M NaCl, 2.7 mM KCl, 0.005% Tween P-20, pH 7.4) to a final
concentration of 50 nM before the injection. Purified
STAT5A protein samples in HBS-EP buffer (0.01 Hepes, pH 7.4, 0.15 M sodium chloride, 3 mM EDTA, 0.005%
polysorbate 20 (v/v)) was injected in a total volume of 75 µl
containing 20-80 µg of purified protein. Samples were injected at
25 °C at a flow rate of 15 µl/min onto the sensor chip surface on
which the BTK protein had been immobilized or onto a control surface on
which bovine serum albumin had been immobilized. Binding surface was regenerated by washing with 2 M NaCl.
The primary data were analyzed using the BIAevaluation software
(version 3.0) supplied with the instrument (Biacore, Inc.). To prepare
the data for analysis, base lines were adjusted to zero for all curves,
and injection start times were aligned. Background sensorgrams were
then subtracted from the experimental sensorgrams to yield curves
representing specific binding. All of the kinetic data were fit most
adequately by assuming a simple bimolecular reaction model for
interaction between soluble analyte and immobilized ligand, equivalent
to the Langmuir isotherm for adsorption to a surface. For determination
of kon, only the middle portion of the
association curve was used for fitting. For determination of
koff, only the initial portion of the curve
encompassing the fast dissociation phase was used for fitting. The
goodness of fit was assessed by inspecting the statistical value
2 and the residuals (observed calculated). The
2 values were low (<2), and the residuals randomly
distributed about zero.
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RESULTS AND DISCUSSION |
Physical Interactions between STAT5A and BTK--
In
co-immunoprecipitation experiments using Triton X-100 whole cell
lysates from KL-2 human B cells, we discovered that BTK immune
complexes contain STAT5A (Fig.
1A; see A.2,
first lane) and STAT5A immune complexes contain
BTK (Fig. 1B; see B.1, first lane). These results indicated that BTK is constitutively
associated with STAT5A in B cells. Interestingly, this physical
association appeared to be markedly reduced after engagement of the B
cell antigen receptor. No STAT5A was detected in the BTK immune
complexes from whole cell lysates prepared after 20 min of stimulation
with an anti-IgM antibody (Fig. 1A.2, lane
2), although there was as much BTK in these immune complexes
as in those prepared before anti-IgM stimulation (Fig.
1A.1), and the Western blot analysis of the whole cell
lysates with the same anti-STAT5A antibodies did not indicate any
change in the amount of cellular STAT5A (Fig. 1A.3).
Similarly, virtually no BTK was detected in the STAT5A immune complexes
from whole cell lysates prepared after anti-IgM stimulation (Fig.
1B.1, lane 2), although there was as
much STAT5A in these immune complexes as in those prepared before
anti-IgM stimulation (Fig. 1B.2), and the Western blot
analysis of the whole cell lysates with the same anti-BTK antibodies
did not indicate any change in the amount of cellular BTK (Fig.
1B.3). These observations are reminiscent of the reported
association between BTK and the multifunctional transcription factor
BAP-135/TFII-I in unstimulated (but not anti-IgM-stimulated) B cells
(17).
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Fig. 1.
A and B, constitutive
association of BTK with STAT5A. A, detection of STAT5A in
BTK immune complexes. BTK was immunoprecipitated from TX-100 lysates of
KL-2 human B cells before (IgM) or 20 min after
(+IgM) stimulation with an anti-IgM antibody and
subjected to immunoblotting with anti-BTK (A.1) and
anti-STAT5A (A.2) antibodies. B, detection of BTK
in STAT5A immune complexes. STAT5A was immunoprecipitated from TX-100
lysates of KL-2 human B cells before (IgM) or 20 min after
(+IgM) stimulation with an anti-IgM antibody and subjected to
immunoblotting with anti-BTK (B.1) and anti-STAT5A
(B.2) antibodies. Whole cell lysate proteins were also
subjected to immunoblotting with anti-STAT5A (A.3) and
anti-BTK (B.3) antibodies. C, binding of BTK
fusion proteins to STAT5A protein in human B cells. BTK-(1-659)
(full-length protein), BTK-(408-659) (kinase domain), and BTK-(2-137)
(PH domain) were fused to MBP. BTK-(219-268) (SH3 domain) and
BTK-(281-377) (SH2 domain) were fused to GST. B, MBP-BTK
and GST-BTK fusion proteins were used in pull-down binding assays to
examine their ability to interact with STAT5A protein in lysates of KL2
human Epstein-Barr virus-transformed B lymphoblastoid cells, as
described under "Materials and Methods." Fusion protein adsorbates
and control samples (CON (1), cell lysate + amylose beads
with no fusion protein added; CON (2), cell lysate + glutathione-agarose beads, no fusion protein added) as well as a KL2
whole cell lysate sample were resolved by SDS-PAGE, immunoblotted with
anti-STAT5A antibody, and developed with ECL. D and
E, physical association between ectopically expressed BTK
and STAT5A in Sf21 cells. D, gel filtration analysis.
Sf21 cells coexpressing BTK and STAT5A were lysed and cytosolic
fractions were recovered then analyzed using fast protein liquid
chromatography at a flow rate of 0.5 ml/min. Fractions (0.5 ml) were
collected and subjected to Western blotting with antibodies specific
for BTK and STAT5A. Proteins were visualized using ECL and analyzed by
densitometry. The numbers 20-34 correspond to
fractions, and arrows below the blot
designate the predicted size of the complexes based upon size
standards. E, coimmunoprecipitation analysis. STAT5A
(STAT5A IP) and BTK immune complexes (BTK IP) as
well as whole cell lysates (WCL) were subjected to Western
blot (WB) analyses using anti-STAT5A and anti-BTK
antibodies. The arrowheads indicate the positions of the BTK
and STAT5A protein bands.
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We performed "pull-down" experiments with full-length
MBP-BTK-(1-659) and truncated MBP-BTK (MBP-BTK-(408-659), BTK
kinase domain; MBP-BTK-(2-137), BTK PH domain) or GST-BTK fusion
proteins (GST-BTK-(219-268), BTK SH3 domain; GST-BTK-(281-377), BTK
SH2 domain) corresponding to different domains of BTK in an attempt to
elucidate the structural requirements for BTK binding to STAT5A. Only
the full-length BTK and BTK KD were able to bind and pull down
STAT5A from lysates of KL2 human Epstein-Barr virus-transformed B
lymphoblastoid cells (Fig. 1C). Thus, the kinase domain
appears to mediate the binding of BTK to STAT5A.
We next set out to examine the in vivo interactions between
ectopically expressed BTK and STAT5A proteins in a heterologous expression system. To confirm that a physical association exists between BTK and STAT5A when they are coexpressed in Sf21 cells, we examined whether BTK and STAT5A are present as native complexes Sf21 cells cotransfected with PFB-stat5a + PFB-btk. Lysates of Sf21 cells were size-fractionated
by fast protein liquid chromatography on a Superdex 200 size exclusion
column (Amersham Pharmacia Biotech), and the elution patterns of BTK
and STAT5A proteins were examined by immunoblotting with antibodies
recognizing the individual proteins. The relative amounts of each
protein per fraction were determined by densitometric scanning. More
than 90% of the BTK protein eluted in fractions 27-29, corresponding
to apparent native molecular masses of 75-150 kDa (Fig. 1,
D.1 and D.2). The remainder of the BTK protein
eluted in fractions 21-26, which correspond to apparent native
molecular masses of >270 kDa. Notably, these fractions also contained
STAT5A protein (Fig. 1D). These results suggested the
existence of 270-350-kDa native complexes containing BTK and STAT5A.
We next used Western blot analyses to evaluate the interaction between
BTK and STAT5A in these Sf21 cells coexpressing both BTK and
STAT5A. BTK immune complexes from these whole cell lysates contained
STAT5A (Fig. 1E.1, lane 2), and STAT5A
immune complexes from these whole cell lysates contained BTK (Fig.
1E.2, lane 1). Taken together, these
initial findings indicated that, when ectopically coexpressed in
Sf21 cells, BTK and STAT5A proteins can physically associate
with each other.
We next sought to examine the ability of purified recombinant BTK to
bind purified recombinant STAT5A by surface plasmon resonance, which
permits direct measurements of the association and dissociation kinetics of binding interactions. Fig. 2
shows representative BIAcore sensorgrams of
concentration-dependent solution phase >95% pure STAT5A
binding to immobilized >95% pure BTK on BIAcore sensor chips. The
binding traces were analyzed assuming a single bimolecular binding
equilibrium between BTK and STAT5A proteins. The maximum resonance
signal (Rmax) increased in a
concentration-dependent fashion from 49.6 RU at 20 µg/ml
STAT5A to 169 RU at 40 µg/ml STAT5A and 238 RU at 80 µg/ml STAT5A.
The kinetics of the binding was independent of the STAT5A
concentration. The average on-rate (kon) was
3.0 ± 0.9 × 104 M
1
s1, the average off-rate (koff)
was 11.1 ± 0.3 × 104 s1, and
the average affinity constant (KD = koff/kon) was 44.4 ± 13.0 nM. These results provide unprecedented
experimental evidence that BTK is capable of directly binding STAT5A
with a relatively high affinity.
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Fig. 2.
Solution phase binding interactions between
BTK and STAT5A. A representative BIAcore sensorgram shows the
binding of purified recombinant STAT5A to the purified recombinant BTK
protein immobilized on a CM5 sensor chip. STAT5A protein was injected
at time 0 and exposed to the surface for 300 s (association
phase), followed by a 5-min flow of Hepes-EP buffer during which
dissociation could be observed.
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Tyrosine Phosphorylation of STAT5A by BTK--
We have performed a
series of experiments to further confirm and extend these initial
observations. Sf21 cells transfected with PFB-stat5a
expressed the recombinant murine STAT5A protein, as detected by
anti-STAT5A immunoblotting of STAT5A immune complexes from whole cell
lysates prepared 48 h after transfection (Fig. 3A). By comparison, control
Sf21 cells transfected with PFB did not show any evidence of
STAT5A expression, demonstrating that the anti-STAT5A antibody used for
immunoblotting does not recognize an endogenous insect STAT5A protein.
The anti-phosphotyrosine Western blot analysis of the STAT5A immune
complexes from Sf21 cells transfected with PFB-stat5a
did not show any evidence of tyrosine phosphorylation. Thus, insect
kinases in Sf21 cells do not significantly phosphorylate
recombinant STAT5A in vivo. We next sought to determine if
coexpression of STAT5A with BTK leads to tyrosine phosphorylation of
STAT5A in vivo. Controls included Sf21 cells
co-transfected with PFB-stat5a and recombinant plasmids for
TEC or JAK3. As shown in Fig. 3B.1 (first
lane) and Fig. 3C.1 (second
lane), anti-phosphotyrosine Western blot analysis of STAT5A immune complexes from Sf21 cells cotransfected with
PFB-stat5a + PFB-btk showed tyrosine
phosphorylation of STAT5A protein. The level of tyrosine
phosphorylation of STAT5A in Sf21 cells coexpressing BTK was
similar to that in Sf21 cells coexpressing JAK3. In contrast, no
STAT5A tyrosine phosphorylation was observed in Sf21 cells coexpressing TEC kinase (Fig. 3B.1, second
lane, and Fig. 3C.1., third
lane). STAT5A was expressed at similar levels in each of the
transfectants (Fig. 3B.2 and 3C.2), and all three
tyrosine kinases were enzymatically active in Sf21 cells, as
shown by their ability to phosphorylate an exogenous substrate
(GST-Ig for Tec family members and -casein for JAK3) (Fig.
3B.3). The ability of both BTK and JAK3 to
tyrosine-phosphorylate STAT5A was dependent on the enzymatic activity
of their respective kinase domains. As shown in Fig. 3D,
coexpression of kinase-dead mutants of BTK (Y551F, BTKmKD)
or JAK3 (K851E, JAK3mKD) did not result in tyrosine
phosphorylation of STAT5A.
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Fig. 3.
In vivo phosphorylation of
ectopically expressed STAT5A by BTK in a heterologous expression
system. A, expression of STAT5A in Sf21 cells.
Sf21 cells were transfected with PFB or PFB-stat5a.
After 48 h, cells were lysed, lysates were immunoprecipitated with
anti-STAT5A antibodies, and immune complexes were subjected to
Western blot analysis with anti-STAT5A or anti-phosphotyrosine
(APT) antibodies. B and C,
coexpression of STAT5A with Tec family tyrosine kinases or JAK3 in
Sf21 cells. STAT5A was co-expressed with BTK, TEC, or JAK3.
STAT5A was immunoprecipitated, and immune complexes were subjected to
Western blot analysis with anti-phosphotyrosine (B.1 and
C.1) or anti-STAT5A (B.2 and C.2)
antibodies. The enzymatic activities of the expressed tyrosine kinases
were measured by phosphorylation of exogenous substrates (GST-Ig for
Tec family members and -casein for JAK3) in the presence of
[-32P]ATP (B.3). D, requirement
for an active kinase domain for BTK-induced or JAK3-induced tyrosine
phosphorylation of STAT5A. STAT5A was co-expressed with JAK3,
kinase-inactive JAK3 mutant, BTK, or kinase-inactive BTK mutant. STAT5A
was immunoprecipitated, and immune complexes were subjected to Western
blot analysis with anti-phosphotyrosine (D.1) or anti-STAT5A
(D.2) antibodies.
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We next used in vitro kinase assays to determine if BTK can
phosphorylate STAT5A.
BTK immune complexes from whole cell lysates of Sf21 cells
transfected with PFB-btk were mixed with purified STAT5A (10 µg) and 10 µCi of [-32P])ATP in the presence of 5 mM each of Mncl2 and MgSO4. TEC
from PFB-tec-transfected Sf21 cells and JAK3 from
PFB-jak3-transfected Sf21 cells were also included
for comparison. The kinase reactions were stopped after 10 min, STAT5A
was immunoprecipitated, and its phosphorylation status was assessed by
autoradiography. As shown in Fig.
4A, BTK was capable of
phosphorylating STAT5A. The phosphorylation level of BTK-treated STAT5A
was similar to that of JAK3-treated STAT5A. In contrast to BTK, TEC did
not phosphorylate STAT5A. The amount of STAT5A was virtually identical
in each kinase reaction (Fig. 4B) and all three tyrosine
kinases, including TEC, were enzymatically active, as measured by
autophosphorylation (Fig. 4C). As shown in Fig.
4D, phosphoamino acid analysis of the BTK-phosphorylated
STAT5A protein confirmed that BTK phosphorylates STAT5A exclusively on
tyrosine residues. CNBr cleavage of BTK-phosphorylated STAT5A protein
yielded a single phosphopeptide of ~17 kDa (Fig. 4, E and
F). This fragment contains the tyrosine residues at
positions 665, 668, 682, 683, and 694 as well as the SH2 domain and
a portion of the SH3 domain.
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Fig. 4.
A-C, in vitro
phosphorylation of STAT5A by BTK. Sf21 cells were transfected
with PFB-btk, PFB-tec, or PFB-jak3.
A, cell lysates from various transfectants were
immunoprecipitated with specific antibodies, and the immune-complexes
were mixed with purified STAT5A and [-32P]ATP. The
kinase reactions were stopped by adding radioimmune precipitation
buffer (20 mM MOPS, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1%
SDS), centrifuged at 12,000 rpm, and the supernatants were
immunoprecipitated with an anti-STAT5A antibody. The phosphorylation
status of the immunoprecipitated STAT5A was determined by
autoradiography. B, the amount of STAT5A in each kinase
reaction was determined by STAT5A immunoprecipitation and anti-STAT5A
immunoblotting. C, the enzymatic activity of the
immunoprecipitated tyrosine kinases was measured by autophosphorylation
in immune complex kinase assays. D-F, phosphoamino acid
analysis (PAA) and cyanogen bromide (CNBr)
cleavage analysis of BTK-phosphorylated STAT5A. BTK was
immunoprecipitated from lysates of PFB-btk-transfected
Sf21 cells and an immune complex kinase assay was
performed with purified STAT5A and [-32P]ATP. The
reaction was stopped with radioimmune precipitation buffer, and STAT5A
was immunoprecipitated and run on a polyacrylamide gel. The
32P-labeled STAT5A band was isolated and subjected to PAA
(shown in D) or CNBr cleavage analysis (shown in
E). The positions of ninhydrin-stained phosphoamino acid
standards (phosphoserine (PS), phosphotyrosine
(PY), and phosphothreonine (PT)) are indicated
with circles. The CNBr cleavage map of STAT5A is depicted in
F, with arrowheads pointing to the positions of
methionines.
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Identification of Tyrosine 694 of STAT5A as the Target
Phosphorylation Site for BTK Kinase Domain--
We constructed a
molecular model of the STAT5A structure (residues 163-703) based on
its homology with STAT1 and STAT3 crystal structures (Fig.
5). A flexible or -strand-like
conformation is a desired structural feature for effective
phosphorylation of protein substrates by protein kinases. Similar to
the structural conformation revealed by the STAT1 and STAT3 crystal
structures, our model indicates that the tail segment of STAT5A from
residue 693 to residue 703 (694 included) adopts a -strand-like
conformation, which is therefore suitable for binding interactions with
the BTK kinase domain. Tyr694 of STAT5A is in close contact
with the BTK catalytic residues including Asp521,
Arg525, and Asn526. In addition,
Gly693, Val695, and Lys695 of
STAT5A are near the BTK residues Pro560 (backbone),
Phe559, and Lys558, respectively. Based on our
model, these residues interact with each other and constitute the
binding affinity between STAT5A and the BTK kinase domain. Our model
also indicates that the SH2 and SH3 domains of BTK may sterically clash
with the N-terminal domain of STAT5A (residues 1-150), which is
predicted to reside near the coiled-coil domain and may be accessible
by the less bulky BTK kinase domain.
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Fig. 5.
Molecular model of STAT5A. Molecular
model of the STAT5A structure is shown as a surface representation
(A) and ribbon representation (B). The
structure of STAT5A (residues 163-703) was constructed based on its
homology with STAT1 and STAT3 crystal structures. Different domains
are shown in different colors: the coiled-coil domain in
red, the DNA binding domain in gold, the linker
domain in green, and the SH2 domain in blue. The
DNA double strand is used to mark the DNA binding location and is shown
as a stick model (A, multicolor) and a
space-filling model (B, yellow for phosphate and
blue and white for two different DNA strands,
respectively). All five tyrosine residues on the SH2 domain and tail
segment that were subjected to phenylalanine mutations are shown in
different colors and labeled accordingly. Among them, only
Tyr694 (red) is fully exposed to the solvent
environment. The other four tyrosine residues are at least half-buried,
consistent with our experimental data suggesting that
Tyr694 is most accessible to BTK phosphorylation. Other
tyrosine residues including Tyr225 and Tyr568
(not shown) are situated on a helix of the coiled-coil domain.
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According to this model, of the five tyrosine residues in the SH2
domain and tail segment of STAT5A, only Tyr694 is fully
exposed to the solvent environment and therefore is the most accessible
tyrosine residue for phosphorylation by BTK. All tyrosine residues
except for Tyr694 are either sterically inaccessible or
adopt a helical conformation unsuitable for phosphorylation. For
example, Tyr682 is surrounded by His588,
Pro587, and Lys681; Tyr683 is
surrounded by Ser680 and Lys675 and possibly
sterically occluded by a loop formed by residues 685-700;
Tyr665 is surrounded by Arg659,
Lys675, and Ile667; and Tyr668 is
surrounded by Pro674, Pro611, and
Ile667. Both Tyr225 and Tyr568
adopt a helical conformation that is not suitable for phosphorylation. Tyr225 is the only tyrosine (other than Tyr694)
that may be eligible for phosphorylation and more exposed than the
aforementioned tyrosine residues. However, Tyr225 is
located on a -strand as a part of a -sheet structure on the DNA
binding domain. Although sufficiently exposed for phosphorylation, Tyr225 is situated on a relatively rigid -sheet
structure. More importantly, Tyr225 is less accessible than
Tyr694 because it is close to the N-terminal domain of
STAT5A according to the full model of the STAT1 protein (61). These
modeling studies taken together with the CNBr cleavage data prompted
the hypothesis that Tyr694 of STAT5A is the target
phosphorylation site for BTK.
In order to determine the site of BTK-mediated phosphorylation in
STAT5A, tyrosine residues present in the 18-kDa CNBr fragment at
positions 665, 668, 682, 683, and 694 were mutated to phenylalanine, as
described under "Materials and Methods." In addition, mutations were also made in the SH2 and SH3 domains to determine the role of
these domains for in vivo recognition and phosphorylation of STAT5A by BTK. The various STAT5A mutants were coexpressed in Sf21 cells with wild type BTK. Fig.
6A shows that BTK tyrosine phosphorylates the Y665F, Y668F, and Y682F,Y683F mutants of STAT5A but
not the Y694F mutant of STAT5A. Notably, STAT5A mutations in the SH2
(R307K) and SH3 (Y251L,Y252L) domains did not alter the BTK
phosphorylation of STAT5A. STAT5A mutants were expressed at similar
levels (Fig. 6B), and the enzymatic activity levels of BTK
in the presence of the various STAT5A proteins were comparable (Fig.
6C). Similarly, BTK immunoprecipitated from whole cell
lysates of PFB-btk-transfected Sf21 cells
phosphorylated wild type STAT5A (Fig. 6D, first
lane), but it failed to phosphorylate the Y694F mutant of
STAT5A (Fig. 6D, second lane),
although the amounts of STAT5A proteins (Fig. 6E) and level
of BTK kinase activity (Fig. 6F) were virtually identical in
these in vitro kinase reactions. Notably, mutation of the
Phe694 residue back to a tyrosine residue restored the
ability of BTK to phosphorylate STAT5A (Fig. 6D,
third lane). Taken together, these results
uniquely implicate Tyr694 of STAT5A as the BTK
phosphorylation site.
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Fig. 6.
A-C, tyrosine phosphorylation of mutant
STAT5A proteins by ectopically expressed wild type BTK in a
heterologous expression system. Mutant STAT5A proteins were
co-expressed in Sf21 cells with wild type BTK. STAT5A proteins
were immunoprecipitated, and the immune complexes were subjected to
Western blot analysis with anti-phosphotyrosine (A) or
anti-STAT5A (B) antibodies. The enzymatic activity of the
expressed BTK protein was measured by examining the autophosphorylation
of BTK in the anti-BTK immunoprecipitates from the Sf21 lysates
in the presence of [-32P]ATP (C). The
positions of BTK, STAT5A, and prestained molecular mass markers (in
kilodaltons) are indicated with arrowheads. D-F,
requirement for an intact Tyr694 residue for BTK-mediated
tyrosine phosphorylation of STAT5A. D, Sf21 cells
expressing wild type BTK were lysed and immunoprecipitated, and the BTK
immune complexes were mixed with purified wild type STAT5A
(BTKWT + STAT5A), Y694F mutant of STAT5A (BTKWT + STAT5AY694F), or Y694F-Y mutant of
STAT5A(BTKWT + STAT5AY694F-Y) in the presence
of [-32P]ATP. The in vitro kinase reactions
were stopped by adding radioimmune precipitation buffer, and the
phosphorylated STAT5A protein was visualized by autoradiography.
E, after stopping the kinase reactions, the kinase reaction
mixtures were centrifuged at 12,000 rpm, and the supernatants were
immunoprecipitated with an anti-STAT5A antibody. The immune complexes
were subjected to Western blot analysis with the anti-STAT5A antibody.
F, the enzymatic activity of BTK in the kinase reactions was
measured by examining its autophosphorylation as well as its ability to
phosphorylate GST-Ig in immune complex kinase assays.
G-J, tyrosine phosphorylation of STAT5A by ectopically
expressed BTK is independent of JAK3. Wild type BTK, JAK3 and their
kinase-inactive mutants were co-expressed with STAT5A in Sf21
cells. Cells were lysed after 48 h of incubation, lysates were
immunoprecipitated with anti-STAT5A (G and H),
anti-JAK3 (I), or anti-BTK (J) antibodies and
subjected to immunoblotting with anti-phosphotyrosine antibodies
(G), anti-STAT5A antibody (H), anti-JAK3 antibody
(I), or anti-BTK antibody (J).
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We next examined the ability of BTK to phosphorylate STAT5A in the
presence and absence of wild type or kinase-inactive JAK3. As shown in
Fig. 6G, both BTK (lane 3) and JAK3
(lane 1) (but not their inactive kinase domain
mutants; see lanes 2 and 4) were capable of phosphorylating STAT5A. By comparison, no tyrosine phosphorylation of STAT5A was evident in Sf21 cells
co-expressing kinase inactive mutants of BTK as well as JAK3
(lane 6). Notably, wild type BTK was capable of
phosphorylating STAT5A even when co-expressed with the kinase-inactive
mutant of JAK3 (lane 8), which does not
phosphorylate STAT5A (lane 2). Thus, BTK-mediated tyrosine phosphorylation of STAT5A cannot be attributed to JAK3. Similarly, neither the wild type nor the kinase-inactive mutant of BTK
affected the ability of the co-expressed wild type JAK3 kinase to
phosphorylate STAT5A (lanes 5 and 7).
The immunoprecipitable STAT5A protein expression levels for the various
Sf21 cell samples were virtually identical (Fig. 6H).
The presence of co-expressed kinase proteins JAK3 (Fig. 6I)
and BTK (Fig. 6J) was confirmed by Western blot analysis.
Taken together, these results do not lend any support to the suggestion
that BTK induces tyrosine phosphorylation of STAT5A indirectly by first
transphosphorylating and activating JAK3. However, it is conceivable
that these two tyrosine kinases cooperate in regulation of the
STAT5A-linked signaling pathways. One might also speculate that
co-expression of these two tyrosine kinases in Sf21 cells would
have resulted in additive tyrosine phosphorylation of STAT5A if the
expression level of each kinase alone was insufficient to cause optimal
phosphorylation of STAT5A.
Tyrosine phosphorylation of STAT5A by JAK3 results in enhanced DNA
binding activity. In order to determine if the BTK-mediated tyrosine
phosphorylation also augments the DNA binding activity of ectopically
expressed STAT5A in a heterologous expression system, we performed
electrophoretic mobility shift assays using a 32P-labeled
oligonucleotide representing a GAS-like element from the -casein
gene promoter region. As shown in Fig.
7A, extracts from
untransfected Sf21 cells (lane 1) or
Sf21 cells transfected with PFB-stat5a alone
(lane 2) did not contain any proteins capable of
binding the probe causing a mobility shift. By comparison, a
significant mobility shift of the 32P-labeled
oligonucleotide probe was observed in extracts from Sf21 cells
co-expressing wild type BTK and STAT5A (lane 5).
These findings indicate that BTK-mediated tyrosine phosphorylation of ectopically expressed STAT5A enhances its DNA binding activity. The
level of STAT5A activation in these extracts was virtually identical to
that observed in extracts of Sf21 cells co-expressing wild type
JAK3 and STAT5A (compare lanes 5 and
10). The observed mobility shifts of the oligonucleotide
probe were not caused by endogenous insect STAT proteins, because no
mobility shifts were observed when we used extracts from Sf21
cells expressing wild type BTK alone (lane 3) or
wild type JAK3 alone (lane 9). BTK-mediated activation of STAT5A was dependent on the enzymatic activity of BTK,
since no mobility shifts were observed in extracts of Sf21 cells
co-expressing STAT5A and a kinase-inactive mutant of BTK (lane 6). In accordance with the above detailed
data identifying Tyr694 as the BTK phosphorylation site of
STAT5A, co-expression of BTK was unable to activate the Y694F mutant of
STAT5A (lane 8). These results demonstrate that
STAT5A is a functional target for BTK.
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Fig. 7.
A, activation of STAT5A by ectopically
expressed BTK. Wild type or Y694F mutant STAT5A was coexpressed with
wild type or kinase-inactive mutant BTK, or wild type JAK3. Control
Sf21 cells were either not transfected or transfected with
expression plasmids for STAT5A, mutant STAT5A, BTK, mutant BTK, or JAK3
alone. Whole cell extracts were prepared and tested for the presence of
active STAT5A by electrophoretic mobility shift assays using an
annealed oligonucleotide corresponding to the GAS-like STAT5 binding
site in the -casein gene promoter (5'-AGATTTCTAGGAATTCAAATC-3'). The
position of shifted complex of activated STAT5A is indicated with an
arrowhead. B-E, requirement for an intact BTK
kinase domain for the in vivo tyrosine phosphorylation of
STAT5A by ectopically expressed BTK. STAT5A was coexpressed in
Sf21 cells with the indicated site-specific mutants of BTK.
STAT5A was immunoprecipitated and STAT5A immune complexes were
subjected to either anti-phosphotyrosine (B) or anti-STAT5A
(C) immunoblotting. BTK was immunoprecipitated, and BTK
immune complexes were subjected to in vitro kinase assays
with [-32P]ATP (E) or anti-BTK
immunoblotting (D). F and G,
recombinant BTK kinase domain phosphorylates STAT5A. F,
purified BTK-KD was examined for its ability to phosphorylate purified
STAT5A in in vitro kinase assays (F.1). Control
reactions contained no STAT5A (lane 1) or no BTK
(lane 2). Samples were examined for BTK-KD
content (F.2) and STAT5A content (F.3) by Western
blot analysis. G, BTK-KD and STAT5A were coexpressed in
Sf21 cells, and STAT5A immunoprecipitated from Sf21 whole
cell lysates was examined for tyrosine phosphorylation by
anti-phosphotyrosine (APT) Western blot analysis.
Lane 1, STAT5A immunoprecipitates (IP)
from Sf21 cells transfected with PFB-stat5a;
lane 2, STAT5A immunoprecipitate from Sf21
cells transfected with PFB-stat5a plus
PFB-btk-kd; lane 3, BTK
immunoprecipitate from Sf21 cells transfected with
PFB-btk-kd; lane 4, whole cell lysate
Sf21 cells transfected with PFB-stat5a plus
PFB-btk-kd.
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We next used the baculovirus expression system to compare the ability
of mutant BTK proteins with point mutations of conserved residues in
the PH (R28C), SH1/kinase domain (Y551F, autophosphorylation site
mutant; K430E, ATP binding site mutant), SH2 (R307K), and SH3
(Y251L,Y252L) domains to phosphorylate STAT5A in vivo. As shown in Fig. 7B, recombinant BTK proteins with mutant PH,
SH2, or SH3 domains were capable of tyrosine phosphorylating the
co-expressed wild type STAT5A protein, whereas recombinant BTK proteins
with SH1/kinase domain mutations were not. The expression levels of STAT5A (Fig. 7C) and mutant BTK proteins (Fig.
7D) were similar for all Sf21 cells co-expressing
STAT5A and a mutant BTK protein. No BTK kinase activity was detected in
BTK immune complexes from Sf21 cells expressing the kinase
domain mutants of BTK (Fig. 7E). Taken together with the
results of the pull-down experiments, these findings indicate that the
PH, SH2, and SH3 domains of BTK do not significantly contribute to its
physical and functional interactions with STAT5A. The ability of BTK to
phosphorylate STAT5A requires both an intact ATP-binding site and an
intact autophosphorylation site within the kinase domain.
We next examined the ability of purified recombinant BTK-KD to
phosphorylate purified recombinant STAT5A using in vitro
kinase assays. As shown in Fig. 7F.1, STAT5A did not show
any significant level of phosphorylation in the absence of BTK-KD, and
BTK-KD was capable of tyrosine-phosphorylating STAT5A in the absence of
other proteins. The amounts of STAT5A in these kinase reactions were
virtually identical (Fig. 7F.3). We next sought to determine if coexpression of STAT5A with BTK-KD in Sf21 cells leads to
tyrosine phosphorylation of STAT5A in vivo. As shown in Fig.
7G (fourth lane), anti-phosphotyrosine
Westen blot analysis of whole cell lysates of Sf21 cells
coexpressing STAT5A and BTK-KD showed two major tyrosine-phosphorylated
protein bands corresponding to STAT5A and BTK-KD. STAT5A
immunoprecipitated from whole cell lysates of Sf21 cells
expressing STAT5A alone showed no tyrosine phosphorylation (Fig.
7G, lane 1). When compared with this
STAT5A protein, STAT5A immunoprecipitated from whole cell lysates of
Sf21 cells coexpressing STAT5A and BTK-KD showed markedly
enhanced tyrosine phosphorylation (Fig. 7G, lane
2). Taken together, these findings provided direct evidence
that the KD of BTK is capable of tyrosine-phosphorylating STAT5A both
in vitro and in vivo. Thus, the KD of BTK is
sufficient for the functional interactions between BTK and STAT5A.
Activation of a BTK-dependent and JAK3-independent
Mechanism of STAT5A Tyrosine Phosphorylation in B Cells after
Engagement of the B Cell Antigen Receptor--
B cells abundantly
express STAT5, and engagement of the B cell antigen-receptor results in
activation of STAT5 (31). We first sought to confirm these results by
examining the phosphorylation status of anti-STAT5A immunoreactive
protein (presumed chicken STAT5) in DT40 chicken B cells after
stimulation with the anti-chicken IgM antibody M4. As shown in Fig.
8A, M4 stimulation resulted in
enhanced tyrosine phosphorylation of STAT5 (lane
2). Notably, pretreatment of DT40 cells with the BTK
inhibitor LFM-A13, which does not inhibit JAK3 (5) abrogated the
M4-induced STAT5 phosphorylation (lane 3). By
comparison, the JAK3 inhibitor HI-P131, which does not inhibit BTK
(40), did not prevent the M4-induced STAT5 phosphorylation in DT40
cells (lane 4). Anti-STAT5A immunoblotting
confirmed that the amounts of immunoprecipitated STAT5A in these
samples were comparable (Fig. 8B). These findings provided
circumstantial evidence that BTK (but not JAK3) plays an important role
in STAT5 tyrosine phosphorylation after B cell antigen receptor
ligation.
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Fig. 8.
A and B, pretreatment of
chicken B cells with the BTK inhibitor LFM-A13 prevents
anti-IgM-induced tyrosine phosphorylation of STAT5A. Cells were
stimulated with anti-IgM (M4) antibody (10 µg/10 × 106 cells/ml) in the presence of 100 µM
LFM-A13 (a BTK inhibitor that does not inhibit JAK3) or 100 µM HI-P131 (a JAK3 inhibitor that does not inhibit BTK)
for 30 min. Cells were lysed and immunoprecipitated with STAT5A, and
STAT5A immune complexes were subjected to Western blot analyses with
anti-phosphotyrosine (A) or anti-STAT5A (B)
antibodies. The positions of BTK and STAT5A are marked with
arrowheads. C-F, requirement for an active BTK
for the anti-IgM-induced in vivo tyrosine phosphorylation of
STAT5A. BTK-deficient DT40 chicken B cells reconstituted with wild type
human BTK (C-F) and BTK-deficient DT40 chicken B cells
reconstituted with inactive kinase domain-mutant human BTK
(C'-F') were stimulated with M-4 anti-chicken
IgM antibody (20 µg of M-4/ml/10 × 106 cells) and
lysed at the indicated times. Cell lysates were immunoprecipitated with
anti-BTK (C, D, C', and D')
or anti-STAT antibodies (E, F, E', and
F') and immunoblotted with anti-phosphotyrosine
(C, E, C', and E'),
anti-BTK (D and D'), or anti-STAT5A antibodies
(F and F') as indicated. G and
H, B cell antigen receptor-mediated versus IL-2
receptor-mediated tyrosine phosphorylation of STAT5A in B cells from
wild type, XID, and JAK3 knockout mice. Splenocytes from normal and XID
mice (G) and normal and JAK3 knockout mice (H)
were stimulated with an anti-mouse IgM antibody (10 µg/10 × 106 cells/ml) or recombinant IL-2 (20 ng/10 × 106 cells/ml) for 10 min. Cells were lysed and
immunoprecipitated with an anti-STAT5A antibody, and the STAT5A immune
complexes were subjected to immunoblotting with anti-phosphotyrosine
(G and H, upper panels) or
anti-STAT5A (G and H, lower
panels) antibodies.
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We next studied the role of BTK in B cell antigen receptor-mediated
STAT5 phosphorylation by comparing the STAT5 tyrosine phosphorylation
levels after M4 antibody stimulation in BTK-deficient DT40 cells
reconstituted with wild type human BTK versus BTK-deficient DT40 cells reconstituted with kinase-inactive mutant BTK. Notably, tyrosine phosphorylation of BTK (Fig. 8C) preceded the
tyrosine phosphorylation of STAT5A (Fig. 8E) in
BTK-deficient DT40 cells reconstituted with wild type human BTK. By
comparison, B cell antigen receptor ligation failed to trigger tyrosine
phosphorylation of BTK (Fig. 8C') or STAT5 (Fig.
8E') in BTK
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ABSTRACT
INTRODUCTION
