From the Department of Biochemistry, MCP Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102
Received for publication, August 16, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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Signal transduction by the antigen
receptor complexes is critical for developmental progression of
B-lymphocytes, which are defined by assembly and sequential expression
of immunoglobulin genes, which in turn are regulated by the enhancer
elements. Although proximal antigen-receptor signal transduction
pathways are well defined, the precise nuclear factors targeted by
these signals remained unknown. Previous studies have demonstrated that
tissue-restricted transcription factors including PU.1 and PU. 1 interaction partner (PIP) function synergistically with c-Fos
plus c-Jun to stimulate the Signaling components of the antigen receptor complexes on the
surface of B cell progenitors
(pre-BCR)1 and B cells (BCR)
are necessary for the developmental progression of B-lymphocytes
(1-5). BCR engagement rapidly induces an array of signal cascades,
particularly the activity of three nonreceptor protein-tyrosine
kinase families including members of Src (Lyn, Fyn, and Blk), ZAP-70
(Syk), and Tec (Btk) (6-9). Functional deficiencies in any one of
these three family members of the protein-tyrosine kinase family result
in defective or aberrant function and impaired development of B cells
(10-12). Although various signal molecules have been shown to be
necessary for proper development and function of B cells, the effects
of these distinct signaling outputs on nuclear target molecules remain
to be elucidated. In fact, gene-targeting studies indicate that the
developmental progression of B cells is critically dependent on the
activity of various transcription factors, which bind to promoter and
enhancer elements of Ig genes (heavy and light chains),
suggesting a link between the signal transduction and transcription
factors during B-cell development (13-18). The enhancer elements are
not only implicated in regulation of the expression of Ig genes but are
also involved in somatic rearrangement and hypermutation of the same
genes (19-21). In fact, disruptions with the Previous studies have demonstrated that transcriptional activity of the
Our recent studies indicate that both PU.1 and PIP interact with
additional factors and function synergistically within the Here we demonstrate that MEKK1 stimulates the synergy between PU.1 plus
PIP and c-Fos plus c-Jun in 3T3 cells. In S194 plasmacytoma cells,
where the Construction of Plasmids--
Plasmid constructs containing the
TK promoter driving the expression of the enhancer core, Transient Transfections and Reporter Gene Analysis--
S1 94 plasmacytoma cells were grown in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% horse serum and
antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin,
henceforth known as Pen-Strep), whereas A20 B cells were maintained in
RPMI 1640 supplemented with 10% fetal calf serum, 5 µM
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation of PU.1 proteins was performed
essentially as described previously (25). Briefly, 3T3 cells were
transfected by the calcium phosphate method with 5 µg of plasmid
expressing either wild type or various PU.1 mutants. 24 h
post-transfection, cells were pulsed with 0.2 mCi/ml
35S-protein labeling mix
(EXPRE35S35S Protein Labeling Mix
L-35S-Met; PerkinElmer Life Sciences) for
2 h and then chased with cold methionine (0.5 mM).
Cells were washed twice with phosphate-buffered saline, harvested in a
lysis buffer (containing 20 mM Tris, pH 7.4, 50 mM NaCl, 0.5% SDS, 0.5% deoxycholate, 1 mM
dithiothreitol, 10 µg/ml leupeptin, 1 µg/ml pepstatin), and
sonicated, and clear cellular lysates were collected following
centrifugation. Cell lysates were normalized by trichloroacetic acid
precipitation of the labeled proteins. Approximately 10 × 106 cpm counts of each cell lysate were incubated with
anti-PU.1 antibodies (Santa Cruz Biotechnology Inc.) for 2 h at
room temperature, and the immune complexes were separated by protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech Inc.). The beads were
washed three times with a RIPA buffer (10 mM Tris, pH 7.4, 0.15 M NaCl, 1.0% IGEPAL CA-630, 1% deoxycholate,
0.1% SDS, and 0.5% aprotinin) and once with a high salt buffer (2 M NaCl, 10 mM Tris, pH 7.4, 1.0% IGEPAL
CA-630, and 0.5% deoxycholate). The immune-complexes were eluted by
boiling with SDS-polyacrylamide gel electrophoresis sample buffer,
resolved on 10% SDS-polyacrylamide gels, and subjected to autoradiography.
Retroviral Production--
293T retroviral packaging cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 1 mM sodium pyruvate, and Pen-Strep.
The 293T cells were transiently transfected at >50% confluence on
100-mm dishes with 5 µg of retroviral vector, pBabe GFP, or pBabe
GFP-Myr AKT along with 5 µg of Ecotropic packaging vector by the
calcium phosphate coprecipitation method as described above. 36 h
post-transfection, viral supernatants were collected and filtered, and
the viral titers were determined by infecting 3T3 cells.
Retroviral Infection--
70Z/3 pre-B cells were grown in RPMI
1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated
fetal calf serum, 5 µM Electrophoretic Mobility Shift Assays--
Binding reactions
were performed as previously described (41). Briefly, various
concentrations of nuclear extracts were pre-incubated with 2 µg of
poly(dI-dC) in a binding buffer (10 mM Tris, pH 7.6, 50 mM NaCl, 20% glycerol, 1 mM dithiothreitol, and 0.5 mM EDTA) for 5 min at room temperature.
Approximately 10,000 cpm of 32P-labeled DNA containing the
PU.1 plus PIP DNA-binding site (from the MEKK1 Potentiates the Functional Synergy between PU.1 Plus PIP and
c-Fos Plus c-Jun--
Previous studies have demonstrated that the
AKT Signals Stimulate Transcriptional Activity of the
AKT Stimulation of PU.1 Is Independent of Its Interaction with
PIP--
The above studies indicate that AKT signal greatly stimulates
the activity of PU.1 plus PIP site when compared with MEKK1 and induces
We next investigated whether Myr AKT activation of PU.1 is due to AKT
function. To test this, transient transfections were conducted in S194
plasma cells using the reporter plasmid containing the PU.1 binding
site (M 5.6) in the presence of various concentrations of Wt-AKT, Myr
AKT (the Myr signal assists in membrane localization, thereby
constitutively stimulating downstream molecules), or a kinase inactive
mutant Myr AKT (K AKT Activation of PU.1 Is Not Due to Altered PU.1 Expression, DNA
Binding, or Protein-Protein Interaction with PIP--
The above
studies strongly suggest that AKT activation of PU.1 is independent of
its interaction with PIP. If this is true, Myr AKT should have no
effect on PU.1 protein-protein interaction with PIP. To test this, we
examined the binding pattern of PU.1 and PIP following the expression
of Myr AKT by retroviral transduction in 70Z/3 pre-B cells, where the
enhancer is inactive to determine the effect of Myr AKT on PU.1.
GFP-tagged Myr AKT (GFP-Myr AKT) or GFP alone was transduced into 70Z/3
cells (pre-B) and allowed to grow for 36 h in regular growth
medium. Flow cytometric analysis indicated that nearly 90% of
the cells were infected by retrovirus expressing GFP-Myr AKT or GFP
alone. Cells were harvested, and mini-nuclear extracts were prepared
and subjected to gel electrophoretic mobility shift assays using an
oligonucleotide containing PU.1 and PIP binding sites as a probe.
Expression of GFP Myr AKT or GFP alone had no effect on the binding
ability of PU.1 and yielded a shift complex of identical size when
compared with the bound complex observed with the in vitro
translated protein (Fig. 6). Similarly,
no significant difference in the binding intensity of PU.1 and PU.1
plus PIP complexes was observed when compared with nuclear extracts
obtained from the 70Z/3 pre-B cells transduced with retroviruses
expressing GFP alone or GFP-Myr AKT. These results indicate that AKT
stimulation of PU.1 was not due to changes in expression levels, DNA
binding, or protein-protein interaction with PIP.
An Acid-rich Transactivation Domain Is Necessary for AKT
Stimulation of PU.1--
The above studies suggest that AKT-mediated
activation of PU.1 is independent of its interaction with PIP. If this
is true, deletion of the PEST domain may have little effect in the
stimulation of PU.1 activity, whereas removal of other amino acid
regions should result in a loss of AKT-mediated stimulation of PU.1. To test this, transfections were carried out in S194 cells with a reporter
plasmid containing a PU.1 binding site but carrying a mutation in the
PIP site (Fig. 4B; M5.6), along with plasmids expressing
various PU.1 mutants either alone or in the presence of wild type AKT.
AKT stimulation of the wild type PU.1 protein was considered 100%.
Expression of wild type PU.1 caused a slight increase in activity of
the reporter plasmid (Fig. 7). On the other hand, expression of the PU.1 mutant lacking the transactivation domain (
Further, to define the minimal amino acids of the transactivation
domain necessary for signal-mediated activation, we constructed PU.1
mutants lacking acid-rich ( AKT Stimulation of PU.1 Is Due to Phosphorylation-mediated
Modifications within the Transactivation Domain--
The acid-rich
transactivation domain of PU.1 contains two serine phosphorylation
sites (Ser41 and Ser45). If AKT stimulation of
PU.1 requires either of these sites, PU.1 mutants containing serine to
alanine mutations at positions 41, 45, or both should fail to respond
to AKT. To examine this, serine to alanine PU.1 mutants S41A, S45A, and
S41A/S45A were expressed with wild type AKT in S194 plasmacytoma cells
in the presence of reporter plasmid M5.6. In addition, we prepared an S37A mutant. These residues (Ser37, Ser41, and
Ser45) lie with a homology to the consensus sequence
(SXXXS) for phosphorylation by IKKs (53). Expression
of PU.1 mutant S45A had no effect on AKT-mediated induction. However,
expression of PU.1 mutant S41A greatly lowered the AKT induction,
whereas S37A showed a slight effect on induction of the reporter.
Consistent with these studies, PU.1 mutants S41A/S45A and
S37A/S41A/S45A blocked AKT-mediated activation (Fig. 7B).
The differences in AKT induction by the PU.1 mutants is not due to
differences in expression levels, because they were all expressed at
comparable levels when transfected in 3T3 cells (Fig. 7C).
In addition, the loss of stimulation of PU.1 mutant S41A or S41A/S45A
is not due to loss of PU.1 DNA binding, because these mutations are
able to bind DNA and recruit PIP to the Ligation of BCR Stimulates PU.1 Activity--
Gene disruption
studies have demonstrated that PU.1 is necessary for B cell
development. The block in B cell development occurs at very early
stages because the mutants (PU.1 The results presented here indicate that the transcriptional
activity of PU.1 can be stimulated by externally regulated signals including MEKK1 and AKT. However, AKT stimulation of PU.1 was markedly
higher when compared with activation observed in the presence of MEKK1.
Consistent with these results, AKT but not MEKK1 signals were able to
induce the transcriptional activity of the
E3'-enhancer in 3T3 cells. In this
study, we demonstrate that the functional synergy between these factors
is enhanced in response to mitogen-activated protein kinase
kinase kinase, in 3T3 cells, where the enhancer is inactive.
However in S194 plasmacytoma cells, mitogen-activated protein kinase
kinase kinase was able to stimulate the activity of PU.1 but unable to
induce the
E3'-enhancer activity. We have found that
Ras-phosphoinositide 3-kinase-dependent externally regulated
kinase, AKT, induces
E3'-enhancer activity in both pre-B and
plasmacytoma cells. AKT stimulation of the
E3'-enhancer is primarily
due to PU.1 induction and is independent of PU.1 interaction with PIP.
Activation of AKT had no effect on the expression levels of PU.1 or its
protein-protein interaction with PIP. Using a series of deletion
constructs, we have determined that the PU.1 acid-rich (amino acids
33-74) transactivation domain is necessary for AKT-mediated induction.
Substitution analyses within this region indicate that phosphorylation
of Ser41 is necessary to respond to AKT. Consistent
with these studies, ligation of antigen receptors in A20 B cells mimics
AKT activation of PU.1. Taken together, these results provide evidence
that PU.1 is induced by AKT signal in a phosphoinositide
3-kinase-dependent manner, leading to inducible or constitutive
activation of its target genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E3'-enhancer results
in loss of tissue and developmental stage specific rearrangement of
light chain genes (19).
E3'-enhancer is critically dependent on binding components of the
E3'-CRE (binds c-Fos, c-Jun, CREM, and ATF1), PU.1 plus PIP (binds
PU.1 and PIP) and E2A (binds E12/47) sites. Mutation of any one of
these binding sites greatly reduces enhancer activity (22-25). Among
the core binding proteins, the expression of PU.1 and PIP is restricted
to cells of hematopoietic lineages. Within the
E3'-enhancer, PU.1
recruits the binding of PIP, through protein-protein interaction to its
adjacent DNA-binding site (22, 23). The recruitment of PIP requires
phosphorylation of PU.1 at the amino acid, Ser148. Mutation
of this serine residue to alanine (S148A) prevents interaction of PU.1
with PIP as well as the binding of PIP to its adjacent DNA binding
sequences. This phosphorylation-mediated interaction of PU.1 appears to
induce a conformational change in PIP, thereby allowing it to recognize
DNA-binding sequences (26). Similar protein-protein interaction between
PU.1 and PIP has been detected in the Ig
light chain enhancer and
the CD20 promoter (27-29). Interestingly, PIP can bind independently
to the interferon-stimulated response element, ISRE (30, 31). PIP is expressed exclusively in the lymphoid lineages (27, 30, 32, 33).
Gene targeting studies indicate that PU.1 is essential for development
of both B cells and macrophages (13, 14), whereas PIP function is
necessary for maturation of B and T lymphocytes (34).
PU.1
/
mutants completely lack both lymphoid and myeloid
progenitors, whereas PIP
/
animals exhibit a block in
peripheral maturation of B cells and fail to produce antibodies in
response to antigenic stimulation. Similarly, T cells of the mutant
animals (PIP
/
) lack proliferative and cytotoxic
responses (34).
E3'-enhancer. Such factors include c-Fos plus c-Jun, which binds to
the
E3'-CRE site, and E2A proteins (E12/E47),
which binds to the E2A site (see Fig. 1). Through these interactions,
PU.1 participates in the assembly of an enhanceosome, a higher order nucleoprotein complex (25). Despite the ability to interact with
various transcription factors, PU.1 was found to be a weak transactivator. Studies of gene regulation have suggested that transcriptional activity of some factors requires
phosphorylation-mediated modifications (35, 36). For instance,
phosphorylation of serine residues at positions 63 and 73 of c-Jun is
important for its activity. These residues are rapidly phosphorylated
in response to oncoproteins (v-Sis and Raf) or exposure to UV radiation
(37), growth factors (38), or cytokines (tumor necrosis factor
) (39). Both Ser63 and Ser73 of c-Jun are
preferentially phosphorylated by JNK1, which is sequentially regulated
as result of activation of the Ras-PI3K-responsive protein kinase,
MEKK1. In fact, target disruption studies suggest that MEKK1 is
essential for JNK activation (40). Therefore, the possibility arose
that externally regulated signals could play an important role in
stimulation of Ig
E3'-enhancer activity. Therefore, we have focused
on the role of the Ras-PI3K-dependent signal molecules,
MEKK1 and AKT, on
E3'-enhancer activity and target sites within the
enhancer in the current studies.
E3'-enhancer is active, MEKK1 weakly stimulated activity
of the PU.1 plus PIP site but failed to induce enhancer activity.
Interestingly, the PI3K-dependent signal molecule, AKT, stimulated activity of the PU.1 plus PIP site and induced
E3'-enhancer activity in both pre-B and plasmacytoma cells.
Activation of the PU.1 plus PIP site in response to the AKT signal is
higher when compared with the stimulation observed in the presence of
MEKK1. Mutational analyses of the PU.1 plus PIP site indicated that
PU.1 responds to the AKT signals, and this response is independent of
its interaction with PIP, suggesting that AKT induction of the
E3'-enhancer is primarily due to PU.1 stimulation. By mutational analyses, we determined that AKT stimulation of PU.1 is mediated through the acid-rich (amino acids 33-74) region. Activation of PU.1
in response to AKT appears to be due to phosphorylation-mediated modifications within this region. Mutation of PU.1 serine 41 (which is
phosphorylated in vivo) to alanine impaired PU.1 induction by AKT signal. Consistent with these studies, we found that
cross-linking the BCR mimicked AKT activation of PU.1. These studies
indicate that PU.1 serves as a target molecule for Ras-PI3K-mediated
signals in B cells providing a role for PU.1 in B-cell proliferation
and humoral immunity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E3'-CRE,
PU.1/PIP, and E2A were previously described (41). Mutations of the PU.1
plus PIP binding sites were reported earlier (24). Wild type or
deletion mutants of c-Fos and c-Jun expression plasmids were kindly
supplied by Dr. Frank Rauscher (Wistar Institute, Philadelphia, PA).
Plasmids expressing MEKK
, MEKK
(K432M) were kindly provided by
Dr. Michael Karin (University of California, San Diego, CA). The
expression plasmids of AKT including HA-Wt AKT, HA-Myr AKT, and HA-Myr
AKT (K
) and retroviral vectors, pBabe GFP and pBabe GFP
Myr AKT were generously provided by Dr. Philip Tsichlis (Thomas
Jefferson University, Philadelphia, PA). The reporter plasmid, pT81-Luc
containing the minimal TK promoter driving expression of the luciferase
gene was kindly provided by Dr. Daniel Tanen (Harvard University,
Boston, MA). The pT81 Luc-M5.6 reporter plasmid containing the
multimerized PU.1 binding site was constructed by transferring M5.6
from the TK-CAT plasmid by BamHI and HindIII
digestion followed by cloning as a blunt end fragment into a blunt end
HindIII site of pT81-Luc. The PU.1 deletion mutants,
2-30,
33-74, and
75-100 were constructed by isolating them
from PURI plasmids (42) by EcoRI and ligating them into the
EcoRI site of an expression plasmid pCB6+
containing the CMV promoter (kindly supplied by Dr. Frank Rauscher, Wistar Institute, PA). The serine to alanine mutant S41A was isolated from Bluescript KS+ by EcoRI digest and cloned
into the EcoRI site of the pCB6+ expression
vector, whereas serine to alanine substitution mutants including S37A,
S45A, S41A/S45A, and S37A/S41A/S45A were prepared by the overlap
extension PCR method (43). Two substitute mutant primers were generated
for each desired amino acid, one on the top strand and one on the
bottom strand. The sequences of the mutant primers were as follows:
F37, 5'-GACTACTACGCCTTCGTGGGC-3'; R37, 5'-GCCCACGAAGGCGTAGTAGTC; F45,
5'-GATGGAGAAGCCCATAGCGAT; and R45, ATCGCTATGGGCTTCTCCATC. Two external
primers containing EcoRI sites were also used, corresponding
to the 5' and 3' ends of PU.1. The sequence of 5' and 3' end primers of
PU.1 were, 5'-GCGGAATTCAGCTGGATGTTACAGGCG-3' and
5'-GCGGAATTCTCAGTGGGGCGGGAGGCG-3', respectively.
Typically, the first PCR amplification was carried out with each mutant
primer and corresponding external primer to generate two DNA fragments, each with a newly substituted amino acid. The amplified fragments were
gel purified and subjected to the second PCR reaction in the presence
of 5' and 3' external primers of PU.1, and full-length cDNAs were
generated. The amplified products were digested with EcoRI
and ligated into the EcoRI site of the pCB6+
expression plasmid. Each PCR reaction was performed by using 2.5 units
of Taq Polymerase (Roche Molecular Biochemicals), 2.5 units
of Taq Extender (Stratagene), a deoxynucleoside triphosphate mix in a final concentration of 0.25 mM, 10 ng of template
DNA, and 250 ng of each primer. The wild type PU.1 cDNA was used as a template to generate the S37A and S45A mutants. The S41A/S45A mutant
was generated using S45A mutant primers and a plasmid containing S41A
mutant cDNA as a template. Subsequently, the PU.1 mutant containing
serine to alanine mutations S37A/S41A/S45A was generated using S37A
mutant primers and S41A/S45A PU.1 cDNA as a template during PCR
amplification. DNA sequences of all inserts were determined to confirm
the mutations and to verify that no new mutations were introduced by
PCR.
-mercaptoethanol and Penn Strep. 3T3 cells were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum
and Pen-Strep. Transfections in S194 cells were performed by the
DEAE-dextran (Amersham Pharmacia Biotech Inc.) procedure (44).
Transfections contained 4-5 µg of reporter plasmid, 1 µg of
-galactosidase expression plasmid pCH110 (45), and varying concentrations of MEKK1, AKT, or PU.1 expression plasmids. The maximum
amount of DNA was kept between 6 and 7 µg during all transfections. 3T3 cells were transfected by the calcium phosphate coprecipitation method of Graham and Van der Eb (46). The total amount of DNA per
transfection in 3T3 cells varied between 16 and 21 µg. Both S194 and
3T3 cells were harvested 48 h post-transfection, cells were lysed
by freezing and thawing and the
-galatosidase activity was
determined from each cellular extract. CAT assays were carried out
according to Gorman et al. (47) using normalized cell
extracts. Transfections in A20 B cells were performed using FuGene-6
reagent (Roche Molecular Biochemicals) with 3 µg of pT81 Luc reporter alone or containing multimerized PU.1 binding site (pT81 Luc-M5.6) along with 1 µg of
-gal expression plasmid. Following
transfection, cells were either unstimulated or stimulated with 5 µg/ml of F(ab')2 anti-mouse IgG (Jackson ImmunoResearch
Laboratories, PA). To block AKT activation, cells were incubated for 30 min in the presence of 100 nM wortmannin (PI3K inhibitor)
prior to addition of antibodies for stimulation. After 36 h, cells
were harvested, and luciferase activity was determined by reading in a
luminometer. The luciferase activities were corrected for transfection
efficiency by using the
-galactosidase activities.
-mercaptoethanol, and
Pen-Strep. Prior to retroviral infection, cells were washed and treated
with DEAE-dextran (1 mg/ml) for 30 min and exposed to retroviral
supernatants expressing GFP alone, or GFP-Myr AKT, for 4 h at
37 °C. Viral supernatants were removed, and cells were plated in
regular growth medium. 36 h post-infection, cells were washed with
phosphate-buffered saline, and mini-nuclear extracts were prepared
according to the method described by Schreiber et al.
(48).
E3'-enhancer) was then
added to the binding reaction, and incubation was continued for an
additional 25 min. The bound complexes were separated on 4%
nondenaturing polyacrylamide gels and exposed for autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E3'-enhancer requires multiple core binding proteins for its
activity (25). Such factors include c-Fos plus c-Jun, which binds to
the
E3'-CRE site, PU.1 plus PIP, which bind to PU.1, and PIP sites
and E2A proteins (E12/E47), which binds to the E2A
site (Fig. 1). Cotransfection of the
enhancer with either c-Fos plus c-Jun, PU.1 plus PIP, or E2A alone
resulted in no enhancer activity in 3T3 cells. However, mixture of
c-Fos, c-Jun, PU.1, and PIP caused a dramatic induction in enhance
activity in 3T3 cells, where it is normally inactive. Removal of any
one of these factors resulted in a loss of enhancer activity (25).
Transcriptional activation by c-Jun requires phosphorylation at serines
63 and 73. These sites are rapidly phosphorylated by a protein kinase,
JNK1 (49, 50), whose activity is regulated through the Ras-responsive
protein kinase, MEKK1 (51). Because c-Jun is important for the activity
of the
E3'-enhancer, we sought to determine the role of MEKK1 in
functional synergy and enhancer activity. If MEKK1 is important to
activity of the
E3'-enhancer, it should stimulate the enhancer when
expressed along with PU.1 plus PIP and c-Fos plus c-Jun. To test this,
transfections were carried out in 3T3 cells with a reporter plasmid
containing the enhancer core fragment along with PU.1, PIP, c-Fos, and
c-Jun either in the absence or presence of various concentrations of catalytically active MEKK1, MEKK
. Parallelly, transfections were carried out with the kinase inactive mutant, MEKK
(KM).
Expression of MEKK1 caused a significant increase in the functional
synergy between PU.1, PIP, c-Fos, and c-Jun and led to stimulation of activity of the enhancer in a concentration-dependent manner. On the
other hand, expression of MEKK
(KM) resulted in a loss of
enhancer activity (Fig. 2). Because MEKK1
stimulates activity of c-Jun, activation of the
E3'-enhancer may be
due to stimulation of the c-Jun alone or a combination of c-Jun and
PU.1 plus PIP.
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Fig. 1.
Schematic of the
E3'-enhancer with relative positions of the
E3'-CRE, PU.1 plus PIP, and E2A DNA-binding
sites.
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Fig. 2.
MEKK1 promotes the functional synergy between
PU.1 plus PIP and c-Fos plus c-Jun within the
E3'-enhancer. NIH 3T3 cells were transfected
by the calcium phosphate method with 5 µg of TKCAT reporter plasmid
containing the core fragment and 3 µg of expression plasmids of PU.1,
PIP, c-Fos, and c-Jun either in the absence or presence of increasing
amounts (0.1, 1.0, and 3.0 µg) of MEKK
, or kinase-inactive mutant
MEKK
(KM) as described under "Experimental
Procedures." Cells were harvested after 48 h, and CAT activities
were determined as described under "Experimental Procedures." The
level of the activity of the enhancer core in the presence of PU.1,
PIP, c-Fos, and c-Jun was considered as 1-fold. The transcriptional
activity of the core fragment in the presence of MEKK
or MEKK
(KM) was calculated relative to the activity observed when
the reporter was cotransfected with PU.1, PIP, c-Fos, and c-Jun alone.
Con., amount of MEKK1 expression vector.
E3'-enhancer--
Because, the
E3'-enhancer is B cell-specific
and the expression of binding factors including PU.1 and PIP is
restricted to hematopoietic lineages, we sought to determine the role
of MEKK1 on the activity of the
E3'-enhancer in S194 plasmacytoma
cells. MEKK1 is an intermediate signal molecule in the
mitogen-activated protein kinase pathway and becomes active in response
to Ras signals (51). Recent studies suggest that in B cells, AKT is a
major signal molecule that becomes active in response to BCR activation in a Ras-PI3K-dependent manner (52). Therefore, we examined the role of AKT in parallel with MEKK1 on the
E3'-enhancer activity. Transfections were carried out with reporter plasmids containing the
enhancer core fragment or multimerized (four copies) binding sites of
E3'-CRE, PU.1 plus PIP or E2A, in the absence or presence of
constitutively active forms of MEKK1 (MEKK
) or AKT (Myr AKT) in S194
plasmacytoma cells. As shown in Fig. 3,
expression of MEKK1 had no significant effect on activity of the core
fragment. However, activity of the PU.1 and PIP site was stimulated
(6-fold) in the presence of MEKK1. No significant MEKK1 induction of
E3'-CRE or E2A was observed. These results suggest that MEKK1 can
stimulate activity of the PU.1 plus PIP binding site but fails to
induce activity of the enhancer. Interestingly, expression of Myr AKT stimulated (5-7-fold) the activity of the enhancer core. This induction appears to be due to stimulation of the PU.1 plus PIP function (17-fold) because very little increase in activity of
E3'-CRE (3-fold) or E2A (4-fold) was observed in response to Myr
AKT. Activation of the reporter plasmid containing PU.1 and PIP binding
sites in the presence of Myr AKT is significantly higher (Student's
t test; p > 0.001) than activity observed
in the presence of MEKK1 (Fig. 3). These results indicate that the
E3'-enhancer can be stimulated by an AKT-mediated signal in
plasmacytoma cells. Similar results were obtained in 1-8 pre-B cells
(data not shown). AKT stimulation of the
E3'-enhancer is primarily due to activation of the PU.1/PIP site. The signal-mediated activation of the
E3'-enhancer suggests a regulatory mechanism that may control
activity of the enhancer during development of B cells.
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Fig. 3.
AKT stimulation of the
E3'-enhancer is mediated through the PU.1/PIP
site. A, a representative set of CAT assays showing the
effect of MEKK
and Myr AKT on the enhancer core and its binding
sites including the
E3'-CRE, PU.1 plus PIP, or E2A. Transient
transfections were carried out in S194 plasma cells with reporter
plasmids containing the enhancer core,
E3'-CRE, PU.1 plus PIP, or
E2A sites in either the absence or the presence of MEKK
or Myr AKT.
B, the activities observed with the enhancer core,
E3'-CRE, PU.1 plus PIP, or E2A in the absence of MEKK
or Myr AKT
is considered one. The relative effect of expression of MEKK
or Myr
AKT on the activities of
E3'-CRE, PU.1 plus PIP, or E2A were
calculated by comparing with the activity obtained by respective
reporter alone. Relative CAT activities were determined by averaging
three or more independent experiments. Error bars indicate
S.D.
E3'-enhancer activity in B lymphoid cells. Therefore, we focused on
studying the mechanism of AKT induction of PU.1 plus PIP site. Previous
studies have demonstrated that phosphorylation of PU.1 at position 148 is necessary for recruitment of PIP to the
E3'-enhancer.
Transcriptional activity of the enhancer is greatly reduced in the
presence of a PU.1 mutant containing a serine to alanine mutation at
position 148 apparently because of the inability of PU.1 S148A to
recruit PIP to its adjacent DNA-binding site (22, 23). Because AKT can
activate the PU.1 plus PIP binding site resulting in activation of the
E3'-enhancer, we wished to determine whether AKT activation is due
to induction of PU.1, PIP, or the augumentation of
protein-protein interaction between PU.1 and PIP. To test this,
reporter plasmids containing multimerized (four copies) 3-base pair
mutants (Fig. 4B; M5.1-M5.8) across the PU.1 and PIP binding sites were transfected in S194 plasma
cells either in the absence or in the presence of Myr AKT. Previous
studies demonstrated that the mutations, which disrupt the binding of
PU.1 also abolish the binding of PIP, whereas the mutations disrupting
the PIP binding site have no effect on binding PU.1 (22). Therefore, if
PU.1 is important for AKT-mediated stimulation, mutants that bind PU.1
alone should be stimulated to a level comparable with reporters
containing PU.1 plus PIP sites. As shown in Fig. 4B, mutants
M5.3 to M5.5, which essentially fail to form PU.1 or PU.1 plus PIP
complexes (22), were inactive and unable to respond to the AKT
function. However, mutant M5.6, carrying mutations in the PIP site
(binds only PU.1 not PIP), was greatly induced by Myr AKT. Similarly,
mutants M5.1, M5.2, M5.7, and M5.8, which form PU.1 and PU.1 plus PIP
complexes, were able to respond to Myr AKT at a level comparable with
the PU.1 binding only mutant (M5.6). These studies suggest that AKT
targets PU.1 and stimulates its activity.
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Fig. 4.
AKT signals stimulate the transcriptional
activity of PU.1 in S194 plasmacytoma cells. Transient
transfections were performed in S194 plasma cells with reporter
plasmids containing multimer binding sites of PU.1 plus PIP or serial
3-base pair linker scan mutants across PU.1 and PIP sequences, in
either the absence or the presence of Myr AKT. A,
representative CAT assay using extracts from cells transfected with
various linker scan mutants in the absence or presence of Myr AKT.
B, the nucleotide sequence of wild type or mutant constructs
(M5.1 to M5.8) and their relative abilities to bind PU.1 and PU.1 plus
PIP complexes is indicated by ( ) or (+) (22). The 3-base pair
substitution mutations in each mutant construct are
underlined. The fold of Myr AKT stimulation of each
construct was determined by comparison with the activity observed by
the respective reporter alone. The values represent the means of two
independent experiments.
), which contains a lysine to methionine
mutation (K179M). Expression of wild type Wt-AKT caused a nearly 6-fold
increase in activity of the reporter plasmid. Consistent with previous
studies, expression of Myr AKT caused a significant increase
(
17-fold) in activity of the reporter plasmid containing the PU.1
binding site (Fig. 5). On the other hand,
the kinase inactive mutant Myr AKT (K
) slightly lowered
PU.1 activity. These results indicate that AKT kinase activity is
needed for PU.1 activation and that membrane localization increases the
level of activity.
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Fig. 5.
PU.1 activity is stimulated by the AKT
signal. Transfections were performed in S194 plasma cells with the
TKCAT reporter containing a wild type (Wt.) PU.1 binding
site but carry mutation in PIP site (M5.6; Fig. 4B) along
with Wt AKT, Myr AKT, or Myr AKT (K ) at various
concentrations. The values represent the means of two independent
assays.
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Fig. 6.
DNA binding or protein-protein interaction of
PU.1 with PIP is not altered by the AKT signal. Nuclear extracts
were prepared from 70Z/3 cells that were transduced with a retrovirus
construct directing the expression of GFP or GFP-Myr AKT as described
under "Experimental Procedures." Binding assays were carried out
using an oligonucleotide containing the PU.1 and PIP sites of the
E3'-enhancer. PU.1 and PU.1 plus PIP complexes and free probe are
indicated.
33-100) resulted in a loss of activity. This protein functioned in a dominant-negative manner and blocked activity of the
endogenous protein (65% loss). A weak activity observed by
33-100
may be due to its ability to interact with other factors (25).
Interestingly, the PU.1 mutant lacking the PEST region (
PEST) did
not interfere with the endogenous protein but rather caused a slight
increase in the activity of the reporter plasmid in the presence of AKT
(Fig. 7B). Previous studies suggest that PEST regions are
involved in protein degradation. Therefore, it is possible that removal
of PEST domain may result in stabilization of PU.1 and thus serve as an
efficient target for externally regulated signals. The above results
suggest that the transactivation domain is necessary for signal
mediated activation, and this is independent of its interaction with
PIP.
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Fig. 7.
Phosphorylation-mediated modifications within
the acid-rich transactivation domain are required for AKT stimulation
of PU.1 in S194 plasmacytoma cells. A, schematic of
wild type (WT) and mutant PU.1 expression constructs used in
transient transfection assays. The sequences deleted or mutated are
indicated on the left. B, identification of amino
acid residues necessary for AKT induction of PU.1. Transient
transfections were carried out with 4 µg of a TKCAT reporter plasmid
containing the PU.1 binding site (M5.6; Fig. 4B) along with
1 µg of the indicated constructs in the absence or presence of 1 µg
of Wt AKT. 48 h following transfection, the cells were harvested,
and CAT activity was determined as described under "Experimental
Procedures." Data are representative of three to five independent
experiments. Error bars indicate S.D. C,
expression levels of various PU.1 mutants in NIH 3T3 cells. 3 µg of
wild type or each PU.1 mutant construct was individually transfected
into NIH 3T3 cells by calcium phosphate coprecipitation. Prior to
harvesting (24 h) the cells, proteins were metabolically labeled, and
cell lysates were prepared. Cell lysates were normalized by
trichloroacetic acid precipitation and subjected to immunoprecipitation
with PU.1 antibodies, and the immunocomplexes were resolved on 15%
SDS-polyacrylamide gel.
2-30 and
33-74) or glutamine-rich (
75-100) regions. Interestingly, expression of the deletion mutant
2-30 caused a slight decrease in AKT activation, whereas
33-74 mutant lowered (65% loss) AKT-mediated stimulation of PU.1. On the
other hand,
74-100 had no effect on AKT-mediated induction of the
reporter plasmid. These experiments suggest that amino acids 33-74 of
the transactivation domain are important for transcriptional activity
of PU.1 and are responsible for AKT-mediated stimulation.
E3'-enhancer (23). These
results suggest that AKT-mediated modification of PU.1 at amino acid
residue Ser41 is important for its induction, and mutation
of this single amino acid residue (S41A) fails to respond to the AKT.
/
) completely lack
lymphoid progenitor cells (13, 14). Because PU.1 is necessary for
expression of Ig genes, it suggests a critical role throughout B cell
development. Although PU.1 is important for B cell development, its
expression levels and phosphorylation-mediated modifications at various
stages are not yet established. Binding studies suggest that the
expression levels of PU.1 at pre-B and B cells are comparable (41).
This raises the possibility that the activity of PU.1 may be regulated
through post-translational modifications. The above studies establish
that AKT signals can induce the activity of PU.1. Recent studies
indicate that BCR cross-linking stimulates AKT in a
PI3K-dependent manner (52). If AKT regulates the activity
of PU.1, BCR stimulation should induce PU.1 activation. To test this
and to establish a direct physiological link between PI3K-AKT
activation and PU.1 induction, the transcriptional activity of PU.1 was
measured following BCR cross-linking in A20 B cells. A20 B cells were
transfected with a luciferase reporter alone (pT81 Luc) or containing
PU.1 binding site (pT81 Luc-M5.6) and left either unstimulated or
stimulated with anti-mouse F(ab')2 antibodies. 36 h
post-transfection, cells were harvested, and PU.1 activity was
determined. Interestingly, BCR cross-linking caused a nearly 10-fold
increase in PU.1 activity. No increase in activity of the reporter
alone was observed following BCR stimulation (Fig.
8). These studies suggest that BCR
signals target PU.1. We next examined whether PU.1 induction in
response to BCR cross-linking is due to activation of PI3 kinase
pathway. If PU.1 induction is due to stimulation of
PI3K-dependent stimulation of AKT, addition of wortmannin
(a PI3K inhibitor) should block PU.1 activity. To test this, A20 cells
were transfected with reporter plasmid containing the PU.1 binding
site, and cells were treated with wortmannin (100 nM) prior
to stimulation with anti-F(ab')2. Luciferase assays
indicated that treatment of wortmannin inhibited the activation of PU.1
that is induced by BCR stimulation (Fig. 8). Because PI3K activates AKT
and constitutively active AKT stimulates PU.1 activity, these data
suggest that AKT induces physiological signals that originate through
BCR stimulation leading to activation of PU. 1 (Fig. 9). Such signals
may play an important role both in B cell development as well as in the
regulation of the responses of mature B cells to antigenic
stimulation.
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Fig. 8.
BCR cross-linking stimulates PU.1
activity. A20 B cells were transfected with a leuciferase reporter
alone or containing PU.1 binding site (M5.6). Following transfection,
cells were either left unstimulated or stimulated by cross-linking the
BCR with anti-F(ab')2. After 36 h, cells were
harvested, and luciferase activities were measured. In experiments
involving treatment of cells with wortmannin, cells were treated with
100 nM wortmannin prior to BCR ligation. Shown is the fold
of induction in the presence or absence of BCR stimulation. The average
and S.D. shown are derived from three to five independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E3'-enhancer. Mutational
analyses indicated that the increase in PU.1 activity in response to
AKT was due to modifications within the transactivation domain (amino
acids 33-74) and was independent of PU.1 protein-protein interaction
with PIP. In addition, we demonstrated that BCR cross-linking
stimulates PU.1 activity in a AKT-dependent manner.
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Fig. 9.
Schematic of the proposed signal-mediated
activation of the E3'-enhancer.
Activation of Ras drives AKT signal pathway leading to activation of
PU.1. As a result, the functional synergy between PU.1 and other
enhancer core binding proteins leads to greater transcriptional
activation.
PU.1 functions as a transcriptional regulator by binding to a
purine-rich consensus sequence, GGAA, known as the PU box, in various
promoter/enhancer elements (54). Previous studies demonstrated that
PU.1 recruits the binding of PIP through
phosphorylation-dependent interaction with PU.1 at
Ser148. This interaction is important for the
transcriptional activity of the E3'-enhancer (22, 23). However,
stimulation of PU.1 observed in response to the AKT signal was not due
to an increase in PU.1 protein-protein interaction with PIP. This is
clearly evidenced by the fact that the relative fold of activation
observed with the reporter plasmid containing a PU.1 binding site alone was comparable with the activity of the reporter containing binding sites for PU.1 and PIP. In addition, PU.1 mutants lacking sequences needed for recruitment of PIP were still stimulated by AKT. Instead, PU.1 activation domain sequences were required for maximal AKT activation. Together, these data suggest that AKT activation of PU.1 is
mediated through stimulation of the PU.1 activation domain. Deletional
analyses of the transactivation domain indicated that the acid-rich
(amino acids 33-74) region is important for AKT-mediated activation.
Ser to Ala mutational analyses indicated that S41A but not S45A is
important for PU.1 induction. However, the significance of
phosphorylation of Ser41 is yet to be determined in B
cells. In macrophages, phosphorylation of PU.1 at position
Ser41 has been shown to be important for proliferation
(55). Possibly AKT induction of PU.1 may result in
phosphorylation-mediated modification of Ser41, yielding a
high affinity interaction with the basal transcriptional machinery.
Alternatively, the AKT signal could stimulate the transcriptional activity of PU.1 indirectly by recruiting a cofactor through the phosphorylationdependent protein-protein interaction. Finally, AKT stimulation of PU.1 may be due to phosphorylation-mediated modification of other transcriptional factors interacting with PU.1.
This possibility is unlikely because no change in the binding pattern
of PU.1 was observed in the presence of AKT stimulation.
Functional studies indicate that PU.1 regulates the activity of various
genes in both B cells and macrophages. In recent years, a number of
PU.1 target genes have been identified in both B cells and myeloid
lineages. In B cells, PU.1 binding is important for transcriptional
activity of the immunoglobulin heavy and light chain ( and
)
enhancers (22, 23, 56) and the J chain (57), mb-1 (58), CD20 (29), and
CD72 (59) promoter elements. Similarly, PU.1 is necessary for promoter
functions of a number of myeloid specific genes including CD11b (60,
61), CD18 (62), c-Fes (63, 64), granulocyte colony-stimulating receptor
(65), macrophage colony-stimulating receptor, c-fms (66-68),
macrophage scavenger receptor (69, 70), FC
RIb (71), FC
RIIIA (72), interleukin-1
(73), and interleukin-18 (74). Targeted disruption of
PU.1 in mice severely impairs the development of lymphoid (B cells) and
myeloid (macrophages and neutrophils) lineages. The block in the
development of these lineages occurs at very early stages, because the
mutant animals completely lack lymphoid and myeloid progenitor cells,
rendering it difficult to determine the precise role of PU.1 during
stage- and lineage-specific gene expression. Recently, Harinder
Singh's laboratory demonstrated that reconstitution of PU.1 expression
by retroviral transduction in PU.1 deficient hematopoietic progenitor
cells allowed them to differentiate into macrophages and B cells
in vitro (75). Interestingly, the progenitor cells
expressing high levels of PU.1 were found to preferentially
differentiate into macrophages, whereas the cells with low levels of
PU.1 were committed to differentiate into pre-B cells. These studies
suggest that the variation in expression and/or function of PU.1
regulates the lineage commitment of hematopoietic progenitors between
lymphoid and myeloid development. Expression of the PU.1 mutant lacking
the acid-rich transactivation (
74-100) domain rescued the
development of B cells but substantially reduced the ability of
PU.1
/
hematopoietic progenitors to differentiate into
macrophages. Interestingly, PU.1 mutants lacking acid-rich regions
(
2-100 and
2-74) failed to rescue the development of both B
cells and macrophages, indicating that an acid-rich transactivation
domain is important for development of both B cells and macrophages
(75). The fact that the acid-rich domain is necessary for
transactivation and for stimulation of PU.1 in response to AKT warrants
a possible role of externally regulated signals at the level of
cellular development.
An important finding of this study is that PU.1 is stimulated in
response to AKT and MEKK1 signals. MEKK1 is an intermediate signal
molecule of the mitogen-activated protein kinase pathway and is
involved in cross-talk with various signal pathways, including JNK and
p38. In addition, MEKK1 has been implicated in nuclear localization of
NF-B through stimulation of protein serine kinases (IKKs). On the
other hand, AKT is a downstream effector of PI3K, which becomes active
in a Ras-dependent manner (52). AKT has been shown to play
an important role in the regulation of cell survival signals in
response to deprivation of growth factors, cytokines, and oncogenic Ras
(76). This is mediated through phosphorylation-dependent
inhibition of proteins associated with apoptosis, including Bad,
caspase-9, and the transcription factor Forkhead (77-79). Recent
studies indicate that AKT also promotes survival by activating NF-
B
(77-80). The AKT activation of NF-
B is due to stimulation of IKKs
and is independent of MEKK1 and NIK, providing an alternate pathway
allowing growth factors such as platelet-derived growth factor to
activate NF-
B (81-83). In fact, tumor necrosis factor and
interleukin-1 have been shown to stimulate the PI3K-AKT pathway (84,
85). Together, these studies indicate that both MEKK1 and AKT signals
activate NF-
B through common signal molecules (IKKs). Consistent
with these studies, we demonstrate that the transcriptional activity of
PU.1 is stimulated in an AKT or MEKK1-dependent manner,
suggesting that PU.1 induction may be mediated through stimulation of
IKKs. However, PU.1 stimulation detected in the presence of Mry AKT is
higher when compared with activity observed in the presence of MEKK1.
This may be due to a prolonged activation signal by AKT or delay in the
suppression of AKT signals. Alternatively, activation of PU.1 in
response to AKT may involve multiple mechanisms including stimulation
and interaction of PU.1 with other cofactors. Overexpressed MEKK1 or
AKT may associate with other cellular proteins leading to the
activation of PU.1. The fact that kinase inactive mutants of MEKK
(KM) or Myr AKT (K
) failed to stimulate PU.1
suggests that activation of PU.1 in response to AKT and MEKK1 may be
due to direct or their downstream signal molecules.
Ligation of BCR stimulates AKT signals in a PI3K-dependent
manner (52, 86). In the present study, we provided evidence that PU.1
is a target of the PI3K-AKT signaling pathway and becomes active in
response to BCR activation. The loss of PU.1 induction in the presence
of wortmannin indicates that kinase signals are important for PU.1
induction in vivo. Because activation of BCR signal pathways
and PU.1 are both essential for proper development of B cells, the
transcriptional activation of PU.1 by the PI3K-AKT pathway may be an
important mechanism by which antigen receptor complexes control the
developmental progression of B cells. In fact, activated Ras signals
are sufficient to promote the differentiation of Rag-1/
mutant pro-B cells to B cells. The Rag-1
/
mutant B
cells lack Ig H chain genes but display extensive light chain
rearrangement (87). Because AKT becomes active in a
Ras-PI3K-dependent manner and stimulates the activity of
the
E3'-enhancer, induction of
light chain genes in response to
Ras-mediated signals may be due to activation of various transcription
factors including PU.1.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Philip Tsichlis for generously supplying various AKT expression plasmids and Dr. Michael Karin for kindly providing expression plasmids of MEKK1. We thank Drs. Harinder Singh, Philip Tsichlis, Jane Azizkhan-Clifford, and Michael Atchison for helpful suggestions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI 46308 (to J. M. R. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
School of Medicine, MCP Hahnemann University, 245 N. 15th St.,
Philadelphia, PA 19102. E-mail:
jagan.pongubala@drexel.edu.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007482200
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ABBREVIATIONS |
---|
The abbreviations used are:
BCR, B cell
receptor;
PIP, PU. 1 interaction partner;
PI3K, phosphoinositide
3-kinase;
PCR, polymerase chain reaction;
CAT, chloramphenicol
acetyltransferase;
GFP, green fluorescent protein;
MEKK1, mitogen-activated protein kinase kinase kinase;
IKK, IB
kinase signalsome.
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