|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 14, 12144-12150, April 5, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Biology, Boston College,
Chestnut Hill, Massachusetts 02467
Received for publication, January 4, 2002, and in revised form, January 29, 2002
A requirement for cyclin D2 in
G1-to-S phase progression has been definitively
established in mature B cells stimulated via the B cell antigen
receptor (BCR). However, the identity of constituents of the BCR
signaling cascade that leads to cyclin D2 accumulation remains
incomplete. We report that inhibition of mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked
BCR-induced activation of extracellular signal-regulated kinase (ERK).
Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate
BCR-induced cyclin D2 expression at the mRNA and protein levels.
Disruption of endogenous heat shock protein 90 (hsp90) function with
geldanamycin abrogated BCR-induced cyclin D2 expression and
proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of
MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of
BCR signaling. These results suggest that the MEK1/2-ERK pathway is
essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase- or protein kinase C-dependent pathways.
Binding of antigen to the B cell antigen receptor
(BCR)1 can lead to
proliferation, differentiation, or lymphocyte death by clonal deletion
(1). These distinct cell fates are determined, at least in part, by the
developmental stage of the lymphocyte and through the coordinate
regulation of signaling pathways by the BCR in response to binding of
monovalent versus polyvalent antigen. BCR signaling is
triggered by the activation of the Src family protein-tyrosine
kinases (PTKs), Bruton's tyrosine kinase (Btk), and Syk (1, 2). These
PTKs, in turn, coordinate the activation of multiple signal
transduction cascades, including phospholipase C (PLC)- We and others have shown previously that cyclin D2, and to a lesser
extent cyclin D3, are induced in response to BCR cross-linking (13,
14). Definitive evidence in support of cyclin D2 in BCR-induced proliferation came from studies with cyclin D2-deficient mice, which
exhibit normal levels of splenic B cells in comparison with wild-type
littermates; however, ex vivo splenic B cells fail to proliferate in response to BCR cross-linking (15). These observations suggest that the principal initial determinant of cell cycle
progression following BCR engagement in mature B cells lies in the
sustained accumulation of cyclin D2. Despite these findings, little is
known about the nature of signaling pathways that couple BCR to cyclin D2 accumulation in splenic B cells. In many nonlymphoid cell types, activation of the Ras-coupled mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular
signal-regulated kinase (ERK) pathway promotes cell cycle entry and
proliferation (reviewed in Refs. 4 and 5). This is achieved, in part, through Ras signaling de novo transcription of the cyclin D1
gene (16, 17). Ras, together with the phosphatidylinositol 3-kinase/Akt pathway, can increase D-type cyclin protein availability by enhancing mRNA translation; this pathway also negatively regulates
ubiquitin-dependent proteasomal degradation of cyclin D1
(18, 19).
Evidence has recently been obtained supporting a role for MEK1/2 and
ERK in BCR-induced proliferation of quiescent splenic B cells (20).
This signaling pathway is also activated during BCR-induced apoptosis
in WEHI-231 B cells and autoreconstituted splenic B cells, which
exhibit an immature B cell phenotype; however, specific inhibitors of
MEK1/2 do not block BCR-induced G1 phase arrest or
apoptosis in these lymphocyte models (20). These findings suggest that
activation of the MEK1/2-ERK pathway is a requisite for cyclin D2
expression and hence G1-to-S phase progression following BCR cross-linking on mature B cells. This view has been challenged by
studies in mice with deletional mutations of the B cell
signalosome. Splenic B cells from B cell linker (BLNK)- and
Vav-deficient mice fail to proliferate in response to BCR cross-linking
due to impaired cyclin D2 expression (21, 22). BCR signaling in these
mutant mice exhibits impaired PLC- Antibodies and Reagents--
Protein A/G-agarose, anti-cyclin
D2, anti-mouse CDK4, anti-rabbit IgG, and anti-mouse IgG-horseradish
peroxidase antibodies (Abs) were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Human pRb mAb (clone G3-245) was
obtained from Pharmingen (San Diego, CA). Anti-c-Raf-1 and anti-hsp90
Abs were from Transduction Laboratories (Lexington, KY) and StressGen
Biotechnologies Corp. (Victoria, Canada), respectively. Anti-actin mAb
(A-2066) was obtained from Sigma. The phospho-pRb
(Ser807/Ser811), phospho-p44/42 MAPK
(Thr202/Tyr204), phospho-MEK1/2
(Ser217/Ser221), phospho-p38 MAPK
(Thr180/Tyr182), phospho-SAPK/JNK
(Thr183/Tyr185), phospho-CREB
(Ser133), phospho-Akt (Ser473), and anti-p44/42
MAPK Abs were purchased from Cell Signaling Technology (Beverly, MA).
Enhanced chemiluminescent reagents were from Kirkegaard and Perry
(Gaithersburg, MD). F(ab')2 fragments of goat anti-mouse
IgM were obtained from Jackson Immunoresearch Laboratories (West Grove,
PA). MG-132, PD98059, and U0126 were obtained from
Calbiochem-Novabiochem. Geldanamycin was purchased from Alexis
Biochemicals (San Diego, CA). All other chemicals were obtained from Sigma.
Preparation of Splenic B Lymphocytes--
Balb/c mice at 8-12
weeks were obtained from Taconic Laboratories (Germantown, NY) and
housed at Boston College. Mice were cared for and handled at all times
in accordance with National Institutes of Health and Boston College
institutional guidelines. Mature B lymphocytes were isolated from
spleens by depletion of T cells with anti-Thy-1.2 plus rabbit
complement (Accurate Chemical and Scientific Corp., Westbury, NY) (13);
macrophages (and other adherent cells) were removed by plastic
adherence. Red blood cells and nonviable cells were removed by
sedimentation on Lympholyte M (Accurate Chemical and Scientific Corp.).
B cells were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD)
supplemented with 10 mM HEPES, pH 7.5, 2 mM
L-glutamine, 5 × 10 Immunoblotting and Immunoprecipitation--
B lymphocytes were
solubilized at 2 × 107/100 µl in 0.1% Triton
X-100-containing buffer as described (13). Cellular proteins were
separated through polyacrylamide-SDS gels and transferred to
Immobilon-P membrane (Millipore Corp., Bedford, MA). Immunodetection of
proteins was carried out as previously described (13).
Immunoprecipitation was performed in 0.5% Nonidet P-40-solubilized
cell lysates according to the method described by Zhang et
al. (23). Lysates were incubated with 1.5 µg of the indicated
Abs (5 h), followed by the addition of 50 µl of a 1:1 slurry of
protein A/G-agarose (90 min). The protein A/G-agarose was collected by
centrifugation and washed eight times with 1 ml of ice-cold Nonidet
P-40 buffer. The immunoprecipitates were dissociated in SDS-PAGE sample
buffer, separated through a 10% polyacrylamide-SDS gel, and
immunoblotted. Where indicated, the resulting autoradiograms were
analyzed by densitometry using a Molecular Dynamics Personal
Densitometer equipped with ImageQuant software (Molecular Dynamics,
Inc., Sunnyvale, CA).
RNA Isolation and RT-PCR--
Total cellular RNA was isolated
from 2 × 107 splenic B cells using Ultraspec Reagent
(Biotecx Laboratories, Inc., Houston, TX) denaturing solution following
the manufacturer's protocol. Purified RNA was treated with 1 unit of
DNase I using DNA-free (Ambion, Inc., Austin, TX). First-strand
cDNA synthesis was carried out with 2 µg of total RNA using the
RETROscriptTM first strand synthesis kit (Ambion, Inc.)
following the manufacturer's protocol. The concentration of cDNA
in each sample was adjusted to produce similar amounts of product
following quantitative PCR with primers specific for
Inhibition of MEK1/2 Blocks BCR-induced ERK
Activity in Splenic B Cells--
PD98059 has been shown to act
in vivo as a highly selective inhibitor of MEK1 activation
and the ERK1/2 cascade, without affecting the related MEK family
kinases, MKK3, MKK6, SEK/MKK4, or their immediate downstream effector
kinases, JNK and p38 MAPK (25). Likewise, U0126 is a highly selective
inhibitor of MEK1 and MEK2 and downstream ERK1/2, without affecting p38
MAPK, JNK, or numerous other protein kinases (e.g. protein
kinase C, CDK4, or Raf-1) (26). The three major classes of mammalian
MAPKs are rapidly and transiently activated following BCR cross-linking
in B cells (1, 20, 27). To determine the specificity of PD98059 and U0126 as inhibitors of BCR-induced ERK1/2 activity in ex
vivo splenic B lymphocyte cultures, whole cell extracts were
prepared from quiescent B cells, and cells were mitogenically
stimulated with 10 µg/ml F(ab')2 fragments of mouse
anti-IgM (anti-Ig) pretreated for 60 min in the presence or absence of
PD98059 or U0126. The concentrations of MEK1/2 inhibitors used herein
were selected based on a recent report demonstrating that 10 µM PD98059 or 10 µM U0126 inhibited
proliferation of splenic B cells stimulated with anti-Ig
(20).2 Immunoblotting was
carried out with highly specific anti-p44/p42 ERK, anti-SAPK/JNK, and
anti-p38 MAPK Abs that recognize the corresponding dually
phosphorylated threonine and tyrosine motifs, indicative of
catalytically active MAPKs. Treatment with either 10 µM
PD98059 or 10 µM U0126 blocked BCR-induced ERK1/2
phosphorylation in splenic B cells when measured at 20 min (Fig.
1A). U0126 appeared to be more
effective than PD98059 as an inhibitor of BCR-induced ERK1/2 phosphorylation at the concentration tested. The membrane was reprobed
with anti-p44/p42 ERK Ab to confirm that the differences in
phosphorylation were not due to changes in total cellular ERK. By
contrast, BCR-induced phosphorylation of SAPK/JNK or p38 MAPK was not
measurably reduced by inhibition of MEK1/2 (Fig. 1A). The
extracts were also probed with anti- BCR Induction of Cyclin D2 mRNA Is Dependent on
MEK1/2-ERK Activity--
Several laboratories, including our
own, have shown that cross-linking the BCR induces cyclin D2 protein
expression (13, 14). By contrast, little is known about the regulation
of cyclin D2 mRNA expression in mature B cells. A report by Reid
and Snow (28) indicates that mitogenic stimulation of splenic B cells with phorbol diester plus ionomycin leads to expression of cyclin D2
mRNA within 4 h, with maximal levels occurring by 18 h.
Based on these results, RNA was isolated from splenic B cells
stimulated with 10 µg/ml anti-Ig for 4 and 18 h, and cyclin D2
gene expression was measured by RT-PCR. Control B cells express a
relatively low level of cyclin D2 mRNA, which increased ~2- and
7-fold in response to BCR cross-linking at 4 and 18 h,
respectively (Fig. 1B). To test whether activation of the
MEK1/2-ERK pathway was required for BCR-induced cyclin D2 mRNA
accumulation, B cells were cultured in medium alone or stimulated with
10 µg/ml anti-Ig, after a 60-min preincubation in the presence of 10 µM U0126. Anti-Ig-induced cyclin D2 mRNA accumulation
was completely suppressed by 10 µM U0126 at the 4- and
18-h time points (Fig. 1C). Treatment with 10 µM PD98059 also led to a reduced accumulation of cyclin
D2 mRNA in response to anti-Ig stimulation at 4 and 18 h (Fig.
1D); however, PD98059 did not completely suppress cyclin D2
mRNA induction, which may reflect its inability to completely
suppress BCR-induced ERK activation (Fig. 1A). These data
suggest that MEK1/2-ERK activity is required for the accumulation of
cyclin D2 mRNA following BCR cross-linking in splenic B cells.
BCR Induction of Cyclin D2 Protein but Not CDK4 Requires Active
MEK1/2-ERK--
By having observed that the
accumulation of cyclin D2 mRNA in response to BCR cross-linking is
dependent upon an active MEK1/2-ERK pathway, we next determined whether
these effects were mirrored at the level of cyclin D2 protein.
Quiescent splenic B cells were pretreated with U0126 or PD98059 as
above and then stimulated with 10 µg/ml anti-Ig for 18 h. The
presence of 10 µM U0126 led to a nearly complete
inhibition of anti-Ig-induced cyclin D2 protein accumulation (Fig.
2A). PD98059 also reduced
cyclin D2 protein accumulation in response to anti-Ig stimulation,
although its inhibitory effect was not as great in comparison with
U0126 (Fig. 2B). The reduction in cyclin D2 protein in the
presence of the MEK1/2 inhibitors did not arise from changes in the
total amount of cellular protein being compared across conditions, as
evidenced by equal levels of
CDK4 represents the primary G1-CDK target of cyclin D2 in
mature splenic B cells, and the initial phosphorylation of endogenous pRb is mediated by cyclin D2-CDK4 holoenzyme complexes (5, 13).
Therefore, we sought to determine if the levels of CDK4 protein and pRb
phosphorylation were affected in B cells treated with MEK1/2
inhibitors. Western blot analysis of whole cell lysates from quiescent
B cells stimulated with 10 µg/ml anti-Ig revealed that the
accumulation of CDK4 protein was not affected by treatment with 10 µM U0126 at 18 h (Fig. 2C). Although CDK4
protein levels are not affected by U0126 or PD98059 (data not shown),
the lack of sustained cyclin D2 accumulation is probably sufficient to prevent activation of endogenous CDK4. This is consistent with the
finding that treatment with 10 µM U0126 is sufficient to
inhibit anti-Ig-induced hyperphosphorylation of endogenous pRb (Fig.
2D). We also monitored activity of in vivo D-type
cyclin-associated CDK activity in splenic B cells using a highly
specific Ab that recognizes phosphorylation of pRb on
Ser807/Ser811 by CDK4/6 (29, 30). Splenic B
cells treated with 10 µM U0126 exhibited a nearly
complete inhibition of BCR-induced pRb phosphorylation on serine
residues 807/811, suggesting that the MEK1/2-ERK pathway is required
for endogenous CDK4/6 activity (Fig. 2E).
Geldanamycin Impairs Signaling through MEK1/2-ERK
in Response to BCR Cross-linking--
To gain further support for the
involvement of the MEK1/2-ERK pathway in BCR signaling to cyclin D2
accumulation, we sought to determine if the benzoquinone ansamycin
anti-tumor drug geldanamycin (GA) inhibits activation of the MEK1/2-ERK
pathway via depletion of cellular Raf-1. hsp90 is a selective target of
GA, and upon binding it leads to destabilization of hsp90 client
proteins (31, 32). In the case of Raf-1, GA promotes its degradation
via a proteasome-mediated pathway (33-35). Treatment of quiescent
splenic B cells with 2 µM GA for 4 h led to a nearly
complete depletion of cellular Raf-1, whereas MEK1/2, p44/p42 ERK, and
hsp90 levels were not decreased (Fig.
3A). We observed protection of
Raf-1 depletion from GA treated B cells by the proteasome inhibitor, MG-132 (Fig. 3B). Moreover, GA treatment led to a rapid
disruption of Raf-1-hsp90 complexes, which preceded Raf-1 depletion, as
evidenced by a reduced amount of coprecipitated hsp90 in Raf-1 immune
complexes (Fig. 3C).
We next tested whether GA blocks downstream activation of MEK1/2 and
ERK1/2 in response to BCR cross-linking. BCR signals phosphorylation of
MEK1/2 within 1-5 min, with phosphorylation decreasing to control
levels by 20 min (Fig. 4A).
Treatment with 2 or 0.2 µM GA blocked BCR-induced
phosphorylation of MEK1/2 on activation residues
Ser217/Ser221 (Fig. 4B). BCR-induced
phosphorylation of ERK1/2 on activation residues
Thr201/Tyr204 was also abrogated (Fig.
4B). To test whether the inhibitory action of GA was
specific for the Raf-1-MEK1/2-ERK pathway, phosphorylation of Akt on
Ser473 and CREB on Ser133 was monitored as
downstream readouts for phosphatidylinositol 3-kinase and
PLC Cyclin D2 Accumulation and Proliferation in Response to Anti-Ig
Stimulation of Splenic B Lymphocytes Is Blocked by GA--
We observed
that GA treatment of splenic B cells led to a nearly complete block in
cyclin D2 mRNA accumulation at 4 h, whereas cyclin D2 mRNA
accumulation was completely blocked at 18 h following stimulation
with 10 µg/ml anti-Ig (Fig.
5A). This was accompanied by a
suppression of cyclin D2 protein accumulation in response to BCR
cross-linking at several time points examined (Fig. 5B). By
contrast, the accumulation of CDK4 protein in response to BCR cross-linking was reduced ~40% by GA, as determined by analyzing the
resulting autoradiogram by densitometry (Fig. 5B). Since
cyclin D2 accumulation is rate-limiting for G1-to-S phase
progression in splenic B cells stimulated with 10 µg/ml anti-Ig, we
tested the functional consequences of GA-dependent
inhibition of cyclin D2 accumulation on proliferation. As shown in Fig.
5C, splenic B cells undergo cellular proliferation upon BCR
cross-linking with 10 µg/ml anti-Ig. In comparison, treatment with 2 or 0.2 µM GA completely blocked anti-Ig-induced
proliferation of splenic B cells as monitored by
[3H]thymidine incorporation.
Mature B cells activate a Raf-1-dependent MEK1/2 and
ERK2 signaling module upon BCR cross-linking (2, 36). A recent report by DeFranco and co-workers (20) provided evidence that BCR-induced proliferation of mature splenic B cells requires signaling through MEK1/2 and ERK; however, the mechanism by which this pathway mediates BCR-induced proliferation remains unknown. Given that the principal initial determinant to G1-to-S phase progression lies in
the sustained accumulation of cyclin D2, we sought herein to examine
whether components of the MEK1/2-ERK pathway are required for induction of cyclin D2 expression by the BCR (13, 15, 28). We show that the
MEK1/2 inhibitors, PD98059 and U0126, inhibit BCR-mediated activation
of ERK. Although certain caveats exist with regard to the specificity
of such reagents in vivo, neither reagent measurably inhibited BCR-induced activation of SAPK/JNK or p38 MAPK. Importantly, treatment with U0126 and, to a lesser degree, PD98059 inhibits BCR-induced expression of cyclin D2 at the mRNA and protein levels. The block in cyclin D2 expression is accompanied by impaired
phosphorylation of endogenous pRb on CDK4/6-targeted
Ser807/Ser811 residues. These findings provide
a molecular explanation for the block in BCR-induced proliferation of
splenic B cells in which MEK1/2 activity has been abrogated, as
recently reported by DeFranco and co-workers (20).
Experiments in which hsp90 function was disrupted provide additional
support for the role of MEK1/2 and ERK in BCR-induced cyclin D2
mRNA expression. hsp90 functions as a general protein chaperone,
maintaining the functional state of a select group of protein kinases
(e.g. Raf-1 and CDK4) involved in signal transduction and
cell cycle control (34, 35, 38). The ansamycin antibiotic GA disrupts
the association of hsp90 with its client proteins (33-35). Only
recently was it discovered that GA binds with high specificity within
the ADP/ATP binding pocket of the hsp90, thereby inhibiting the
function of hsp90 (31). GA also targets the hsp90 homolog, GP96,
localized in the endoplasmic reticulum (32). Interestingly, ansamycins
cause a pRb-dependent G1 phase arrest in tumor
cells (39). We find that treatment of normal splenic B cells with GA
inhibits BCR-induced activation of MEK1/2 and ERK and that this occurs
via a mechanism involving proteasome-mediated depletion of endogenous
Raf-1 but not MEK1/2 or ERK. Perhaps most importantly, GA-mediated
disruption of BCR-coupled signaling through Raf-1-MEK1/2-ERK prevents
downstream induction of cyclin D2 mRNA expression. Consistent with
the role of cyclin D2 in mediating G1-to-S phase
progression, we find that GA blocks anti-Ig-stimulated splenic B cell proliferation.
The findings herein also indicate that cyclin D2 protein accumulation
is blocked following inhibition of BCR-coupled Raf-1-MEK1/2-ERK signaling. This is probably secondary to the block in cyclin D2 mRNA expression, in part because up-regulation of cyclin D2 protein in quiescent B cells stimulated with anti-Ig requires de
novo mRNA and cyclin D2 protein synthesis (13, 40). We cannot
rule out the possibility that components of the Raf-1-MEK1/2-ERK
pathway may act concurrently to control the magnitude and duration of cyclin D2 protein availability via post-transcriptional mechanisms (4,
18, 19, 41). In this view, it may be significant that a recent report
by Lam and co-workers (42) suggests that inhibition of
phosphatidylinositol 3-kinase with LY294002 down-regulates cyclin D2
steady-state levels in immature WEHI-231 B-cell lymphomas.
Our finding that the MEK1/2-ERK pathway is necessary for BCR-mediated
cyclin D2 gene expression in normal splenic B cells is supported,
albeit indirectly, by studies in mice containing mutant Btk. B cells
from Btk-null and xid mutant mice exhibit defective cyclin
D2 expression and fail to proliferate in response to BCR cross-linking
(43, 44). BCR-mediated ERK activity in splenic B cells from
xid mutant mice is severely attenuated in comparison with
wild-type littermates, suggesting that Btk is important for
BCR-mediated ERK activation in murine splenic B cells (45). These
findings are also supported by studies in DT40 chicken B cells
expressing a conditional Btk-estrogen receptor (Btk:ER) fusion protein
and Btk-null DT40 B cells, for which BCR-induced ERK2 activation is
dependent on Btk (46, 47).
xid B cells also exhibit impaired PLC The above findings seem to contradict the studies herein with normal
splenic B cells. The discrepancy between these data may reflect the
readouts for cyclin D2 expression used, namely that the BLNK- and
Vav-deficient studies examined cyclin D2 protein levels, whereas our
study has identified a role for MEK1/2-ERK in cyclin D2 gene
expression. With this in mind, it can be envisaged that cyclin D2
protein availability may be controlled at the translational and/or
post-translational levels through PLC The requirement for c-myc in colony-stimulating factor-1
receptor induced cyclin D2 gene expression may be significant to BCR
signaling, given a recent report demonstrating induction of cyclin D2
mRNA after expression of a conditional Myc-estrogen receptor
(Myc-ER) fusion protein (50). Furthermore, Myc, as part of a
heterodimeric complex with Max, has been shown to activate the murine
cyclin D2 gene promoter via a high affinity E-box in Rat1 and NIH-3T3
cells (51, 52). In this view, the established role for NF- In summary, this is the first study to provide direct evidence for a
role for active Raf-1-MEK1/2-ERK in BCR-induced cyclin D2 expression in
normal ex vivo splenic B cells. These findings also provide
a molecular explanation for the requirement of MEK1/2 in BCR-induced
proliferation of splenic B cells.
We thank Drs. Joseph R. Tumang and Thomas L. Rothstein (Boston University Medical Center) for assistance with
developing the RT-PCR in the laboratory. We also thank Ms. Fay Dufort
for assistance with the proliferation assays.
*
This work was supported by United States Public Health
Service National Institutes of Health Grants AI34586 and AI49994.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.
Published, JBC Papers in Press, January 31, 2002, DOI 10.1074/jbc.M200102200
2
F. Dufort and T. C. Chiles, unpublished observations.
The abbreviations used are:
BCR, B cell antigen
receptor;
Ab, antibody;
anti-Ig, F(ab')2 fragments of
anti-mouse IgM;
Btk, Bruton's tyrosine kinase;
CREB, cAMP-response
element-binding protein;
CDK, cyclin-dependent kinase;
ERK, extracellular signal-regulated kinase;
GA, geldanamycin;
hsp90, heat
shock protein 90;
JNK, c-Jun NH2-terminal kinase;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
mAb, monoclonal
antibody;
PLC
Requirement for a hsp90 Chaperone-dependent
MEK1/2-ERK Pathway for B Cell Antigen Receptor-induced Cyclin D2
Expression in Mature B Lymphocytes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and -2,
phosphatidylinositol 3-kinase, the Vav/Rho family pathway, and
mitogen-activated protein kinase (MAPK) pathways (1, 3). Recent efforts
have focused on understanding how individual signaling pathways lead to
distinct cell fates (1). In particular, the nature of signal
transduction that controls BCR-induced proliferation in mature B cells
remains incomplete. In mammalian cells, the D-type cyclins are
considered end point targets of mitogenic signaling pathways and
function as growth factor sensors (4, 5). The requirement of D-type
cyclins in G1 phase progression has been definitively
established in mammalian cells (5-8). D-type cyclins function as
positive regulatory subunits for a subset of
cyclin-dependent kinases (CDKs) 4 and 6 (9, 10).
Several findings contribute to the emerging view that CDK4/6, together
with CDK2, represent the primary protein kinases that drive cells
through G1 phase, presumably through the phosphorylation of
the retinoblastoma gene product (pRb) (11, 12). A current model holds
that sequential phosphorylation of pRb by CDK4/6 and CDK2 disrupts its
association with E2F family proteins, leading to the coordinated
transcription of E2F-responsive genes directly involved in
G1/S transition and DNA metabolism (11).
2 activation and Ca2+
mobilization, whereas activation of the MEK1/2-ERK pathway is normal.
In the present study, we sought to determine whether activation of
MEK1/2 and ERK is required for BCR-induced cyclin D2 expression in
ex vivo cultures of splenic B cells. Results of this study indicate that Raf-1, MEK1/2, and ERK are necessary for BCR-induced expression of cyclin D2. Our findings also suggest that the
Raf-1/MEK1/2-ERK pathway requires functional heat shock protein 90 (hsp90) in order to signal induction of cyclin D2 gene expression by
BCR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 M
2-ME, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10%
heat-inactivated fetal calf serum (BioWhittaker). B cells were
maintained in a 37 °C humidified incubator at 5%
CO2.
2-microglobulin gene (2-microglobulin sense primer corresponds to 5'-cggtcgcttcagtcgtcagc-3'; antisense primer corresponds to 5'-cccagtagacggtcttggg-3'). The PCRs were performed in 25-µl final reaction volumes, each consisting of 5 µl
of template cDNA using reagents from GeneAmpR PCR kit
(Roche Molecular Systems, Inc., Branchburg, NJ) supplemented with 10 µCi of 
-[32P]dCTP (6000 Ci/mmol; PerkinElmer Life
Sciences). Amplification was conducted with an Eppendorf Mastercycler
gradient (Brinkmann Instrument, Inc. Westbury, NY). Temperature cycling
parameters for the 2-microglobulin gene were as follows:
95 °C for 3 min, followed by 17 cycles of 95 °C for 30 s,
55 °C for 1 min for annealing, and 72 °C for 1 min. A 72 °C
final elongation step for 10 min was included following the last cycle.
PCRs were optimized with respect to Mg2+ concentration, pH,
annealing temperature, and the number of cycles as described (24). PCR
products (20 µl) were electrophoresed on a 8% polyacrylamide gel and
then visualized by autoradiography. The identity of each PCR product
was determined based on its expected size. Autoradiographic bands were
quantitated with the aid of a Molecular Dynamics PhosphorImager. Under
these conditions, the number of PCR cycles and the amount of cDNA
used were such that increasing amounts of cDNA template yielded
proportionately increasing amounts of
2-microglobulin-specific PCR product (data not shown). The variation of 2-microglobulin PCR product between
adjusted samples was ±5%, as judged by densitometric analysis of the
resulting autoradiograms. The number of cycles and amount of cDNA
used were then adjusted accordingly to yield the same signal output.
Cyclin D2 cDNA was then amplified from the normalized
2-microglobulin cDNA samples using the identical PCR
and temperature cycling parameters. The sequence of the cyclin D2
primer set corresponds cyclin D2 sense primer
5'-agctgtccctgatccgcaag-3' and antisense primer
5'-gtcaacatcccgcacgtctg-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin mAb to verify that an
equal amount of cellular protein was being compared between conditions
(Fig. 1A). It is noteworthy that basal and BCR-induced phosphorylation of individual MAPKs were not affected by pretreatment of parallel B cells with the corresponding solvent controls (data not
shown). Collectively, these data indicate that PD98059 and U0126
selectively inhibit ERK activation in response to BCR cross-linking. These results are consistent with a more detailed report confirming the
selectively of these reagents as inhibitors of the MEK1/2-ERK pathway
in splenic B cells (20).
View larger version (24K):
[in a new window]
Fig. 1.
U0126 and PD98059 inhibit BCR-induced ERK
activation and downstream induction of cyclin D2 mRNA accumulation
in splenic B lymphocytes. A, quiescent splenic B cells
(M) were incubated for 20 min with 10 µg/ml anti-Ig
(aIg) in the presence or absence of a 60-min pretreatment
with 10 µM U0126 (U) or 10 µM
PD98059 (P). Activation of p44/p42 ERK was monitored by
immunoblotting of whole cell lysates with anti-phospho-p44/42MAPK Ab.
Total ERK1/2 was evaluated with an anti-ERK1/2 Ab. Immunoblotting with
anti-phospho-p38 MAPK and anti-phospho-SAPK/JNK Abs was carried out in
parallel to monitor p38 MAPK and JNK activation, respectively.
Expression of -actin was examined in parallel to confirm that equal
amounts of cellular protein were being analyzed for each condition. The
arrows indicate the positions of individually phosphorylated
MAPKs. B, quiescent splenic B cells (time 0) were incubated
for 4 and 18 h with 10 µg/ml anti-Ig (aIg).
C and D, parallel cultures were incubated for 4 and 18 h with 10 µg/ml anti-Ig in the presence or absence of a
60-min pretreatment with 10 µM U0126 or 10 µM PD98059. Quiescent and anti-Ig stimulated B cell
cultures contained an equal volume of the corresponding solvent
vehicle. Note that the solvent had no effect on cyclin D2 gene
expression in comparison with parallel cultures of B cells incubated in
the absence of solvent (data not shown). RT-PCR was carried out as
described under "Experimental Procedures." Cyclin D2 expression is
reported as a proportion (-fold induction) of expression present in
unstimulated (time 0) B cell samples. The insets show the corresponding
2-microglobulin (2 MG) and
cyclin D2 RT-PCR analysis for the 18-h cultures treated with U0126
(C) or PD98059 (D). These data are representative
of three independent experiments.
-actin expression (Fig. 2, -actin).
View larger version (24K):
[in a new window]
Fig. 2.
MEK1/2-ERK activity is required for cyclin D2
protein accumulation and pRb phosphorylation. Quiescent splenic B
cells (M) were incubated for 18 h with 10 µg/ml
anti-Ig (aIg) in the presence or absence of a 60-min
pretreatment with 10 µM U0126 (A and
C; denoted U) or 10 µM PD98059
(B; denoted P) or an equal volume of the
corresponding solvent vehicle (S). Note that the
corresponding solvent vehicles had no measurable effect on cyclin D2 or
CDK4 expression in quiescent B cells (data not shown). Cyclin D2 and
CDK4 protein levels in whole cell extracts were measured by
immunoblotting with anti-cyclin D2 (A and B) and
anti-CDK4 (C) Abs. D, quiescent splenic B cells
(M) were incubated for 24 h with 10 µg/ml anti-Ig
(aIg) or anti-Ig following a 60-min pretreatment with 10 µM U0126 (U). Note that quiescent and anti-Ig
stimulated B cell cultures contained an equal volume of the solvent
vehicle. Immunoblot analysis of cell lysates was conducted with an
anti-pRb mAb that recognizes both hypo- and hyperphosphorylated Rb or
with anti- 
actin mAb to monitor -actin levels. E,
immunoblot analysis with an
anti-phospho-pRb(Ser807/Ser811) Ab was also
carried out to detect pRb phosphorylation on
Ser807/Ser811. These data are representative of
at least two independent experiments.
View larger version (24K):
[in a new window]
Fig. 3.
GA decreases the steady-state level of Raf-1,
but not MEK1/2, p44/p42 ERK, or hsp90. A, quiescent
splenic B cells (M) were stimulated with 10 µg/ml anti-Ig
for 2 or 4 h following a 60-min pretreatment with 2 µM GA. Whole cell extracts were prepared and
immunoblotted with Abs specific for Raf-1, MEK1/2, p44/p42 ERK, hsp90,
or -actin as indicated by the arrows. B, B
cells treated with 2 µM GA in the absence () and
presence (+) of 25 µM MG-132 were also evaluated at
6 h. C, B cells were pretreated for 60 min in the
absence (C) or presence of 2 µM GA
(GA). Cellular lysates were immunoprecipitated
(IP) with nonimmune (NI) or anti-Raf-1 Abs as
described under "Experimental Procedures." The immune complexes
were then analyzed by SDS-PAGE and immunoblotted with anti-hsp90
Ab.
2-protein kinase C pathway activation, respectively (36, 37). As
shown in Fig. 4C, GA had no measurable inhibitory effect on
anti-Ig-induced phosphorylation of CREB on Ser133.
Similarly, the rapid phosphorylation of Akt in response to BCR cross-linking was not inhibited by GA at the time points examined (Fig.
4C). Collectively, these results suggest that GA does not impair general BCR-mediated signaling but rather disrupts
Raf-1-dependent signaling through MEK1/2 and ERK.
View larger version (24K):
[in a new window]
Fig. 4.
GA blocks BCR-dependent
activation of MEK1/2 and ERK1/2 but not anti-Ig-induced phosphorylation
of Akt or CREB. A, splenic B lymphocytes (M)
were stimulated with 10 µg/ml anti-Ig (aIg) for the
indicated times. Whole cell extracts were prepared and immunoblotted
with anti-phospho-MEK1/2(Ser217/Ser221) Ab.
B, splenic B cells (M) were stimulated with 10 µg/ml anti-Ig (aIg) for 5 min, following a 60-min
pretreatment with either 2 or 0.2 µM GA. Whole cell
extracts were immunoblotted with anti-phospho-MEK1/2 or
anti-phospho-p44/42 MAPK Abs. C, splenic B cells
(M) were stimulated with 10 µg/ml anti-Ig (aIg)
for the indicated times, following a 60-min pretreatment with 2 µM GA. Whole cell extracts were isolated and
immunoblotted with anti-phospho-CREB(Ser133) or
anti-phospho-Akt(Ser473) Abs. It should be noted that the
anti-phospho-CREB(Ser133) Ab cross-reacts with ATF-1
phosphorylated on serine 63, which exhibits a faster migrating
immunoreactive band in comparison with phospho-CREB.
View larger version (19K):
[in a new window]
Fig. 5.
GA blocks anti-Ig-induced cyclin D2
expression and proliferation in splenic B lymphocytes.
A, quiescent B cells (0 h) were incubated for 4 and 18 h with 10 µg/ml anti-Ig (aIg) in the presence or absence
of a 60-min pretreatment with 2 µM GA. RNA was isolated,
and RT-PCR was carried out as described under "Experimental
Procedures." Cyclin D2 expression is reported as a proportion (-fold
induction) of expression present in unstimulated (time 0) B cell
samples. B, quiescent splenic B lymphocytes (M)
were stimulated with 10 µg/ml anti-Ig (aIg) for 9 or
12 h, following a 60-min pretreatment with 2 µM GA.
Whole cell extracts were prepared and immunoblotted with either
anti-cyclin D2, anti-CDK4, or anti- -actin Abs. C, splenic
B cells were cultured in media alone (M) or
stimulated with 10 µg/ml anti-Ig (aIg), following a 60-min
preincubation with GA at the indicated concentrations. After 42 h,
[3H]thymidine was added, and proliferation was assayed
after an additional 6 h as described (13). These data are
representative of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 activation and
attenuated Ca2+ mobilization (48). The significance of this
signaling phenotype to the defect in BCR-induced cyclin D2 expression
is not known for the xid mouse; however, a similar mutant
phenotype has been demonstrated in BLNK-deficient mice (21). BLNK is an
Src homology 2 and 3 domain-containing adaptor protein that is
phosphorylated upon BCR ligation and links Syk activation to Vav, Nck,
Grb2, and PLC2 (3). Anti-Ig-stimulated B cells from BLNK-deficient mice do not proliferate due to an inability to induce cyclin D2 (and
CDK4) expression (21). These cells exhibit impaired PLC2 activation
and downstream activation of NF-
B in response to BCR cross-linking;
however, in contrast to xid mutant mice, BCR-induced activation of ERK, SAPK/JNK, and p38 MAPK appears normal. The phenotype
of splenic B cells from mice null for the vav
proto-oncogene, a Rho family guanine-nucleotide exchange factor,
resembles the BLNK/ mice (22). Vav-deficient splenic B
cells are capable of activating ERK and SAPK/JNK in response to BCR
cross-linking, yet these cells exhibit a defective [Ca2+]
response, impaired cyclin D2 expression, and fail to proliferate (22).
Taken together, these observations suggest that cyclin D2 expression
depends upon [Ca2+] mobilization triggered by PLC2
activity. Moreover, the BLNK/ and
Vav/ mutant phenotypes imply that expression of cyclin
D2 is impaired despite the presence of active ERK.
2 and/or
Ca2+-dependent pathways, whereas cyclin D2 gene
expression is regulated by the MEK1/2-ERK pathway. Equal is the
consideration that full induction of cyclin D2 gene expression is
perhaps achieved by several distinct signaling pathways
(i.e. Raf-1-MEK1/2-ERK, PLC2, and/or Ca2+
mobilization). We base this conclusion on a recent study involving the
regulation of cyclin D2 gene transcription by the colony-stimulating factor-1 receptor. Dey et al. (49) found that in BAC1.2F5
macrophages colony-stimulating factor-1 receptor signaling to cyclin D2
gene promoter activation required three distinct pathways, a
rottlerin-sensitive protein kinase C
-c-myc pathway, ERK,
and a Src-dependent pathway. Additional work is needed to
determine whether a similar scenario exists in mature B lymphocytes and
specifically whether PLC2- and/or
Ca2+-dependent pathways regulate cyclin D2 gene expression.
B/Rel in
regulating c-myc expression in murine B cells may provide a
mechanism whereby PLC2, and hence Ca2+ mobilization
could regulate cyclin D2 gene expression (53). By contrast, the
mechanism by which the Raf-1-MEK1/2-ERK pathway couples BCR to cyclin
D2 mRNA accumulation in mature B cells is not yet defined. The
5'-flanking region of the murine cyclin D2 gene promoter contains
several transcriptional factor binding sites that are targets for ERK;
among these, PEA3, AP-1, CCAAT/enhancer-binding protein, CREB, and Sp1
have been shown to be directly phosphorylated by ERK or regulated via
an intermediate downstream ERK-dependent kinase (54).
Clearly, more work is warranted to identify the nuclear
trans-acting factors that contribute to the full induction of cyclin D2 gene expression in response to BCR ligation. A schematic representation of the signaling pathways by which the BCR may induce
cyclin D2 gene expression is shown in Fig.
6. It should be noted that additional
signaling pathway(s) may also be involved in the induction of cyclin D2
gene expression.
View larger version (14K):
[in a new window]
Fig. 6.
A model representing BCR signaling to cyclin
D2 gene in mature B lymphocytes. The findings herein support a
role for a Raf-1 
MEK1/2 ERK1/2 signaling module that couples
BCR to cyclin D2 gene expression. GA targets for depletion by the
proteasome, Raf-1-hsp90 complexes, whereas U0126 and PD98059
inhibit MEK1/2 activity and block downstream ERK1/2. The mechanism by
which ERK1/2 activates the cyclin D2 gene promoter remains to be
defined. Also shown is a second pathway that may be necessary for BCR
signaling to cyclin D2 gene, as identified by deletional mutations in
components of the B cell signalosome. Splenic B cells deficient in Btk
and BLNK exhibit impaired PLC2 activation; Vav/ B
cells exhibit impaired intracellular Ca2+ mobilization (21,
22, 43, 44). Taken together, these studies support a model in which
PLC2 Ca2+ couples the BCR to cyclin D2 gene
expression via a pathway that may involve activation of NF-B and
c-myc gene expression (51-53). PM and
NM, the plasma membrane and nuclear membrane,
respectively.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
Boston College, 414 Higgins Hall, Chestnut Hill, MA 02467. Tel.: 617-552-0840; Fax: 617-552-2011; E-mail: ChilesT@bc.edu.
ABBREVIATIONS
, phospholipase C;
PTK, protein-tyrosine kinase;
pRb, retinoblastoma gene product;
xid, X-linked
immunodeficiency;
SAPK, stress-activated protein kinase;
RT, reverse
transcription;
BLNK, B cell linker.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Craxton, A.,
Otipoby, K. L.,
Jiang, A.,
and Clark, E. A.
(1999)
Adv. Immunol.
73,
79-152[Medline]
[Order article via Infotrieve] 2.
Sefton, B. M.,
and Taddie, J. A.
(1994)
Curr. Opin. Immunol.
6,
372-379[CrossRef][Medline]
[Order article via Infotrieve] 3.
Campbell, K. S.
(1999)
Curr. Opin. Immunol.
11,
256-264[CrossRef][Medline]
[Order article via Infotrieve] 4.
Sherr, C. J.,
and Roberts, J. M.
(1999)
Genes Dev.
13,
1501-1512 5.
Ekholm, S.,
and Reed, S. I.
(2000)
Curr. Opin. Cell Biol.
12,
676-684[CrossRef][Medline]
[Order article via Infotrieve] 6.
Sicinski, P.,
Donaher, J. L.,
Parker, S. B., Li, T.,
Fazeli, A.,
Gardner, H.,
Haslam, S. Z.,
Bronson, R. T.,
Elledge, S. J.,
and Weinberg, R. A.
(1995)
Cell
82,
621-630[CrossRef][Medline]
[Order article via Infotrieve] 7.
Resnitzky, D.,
Gossen, M.,
Bujard, H.,
and Reed, S. I.
(1994)
Mol. Cell. Biol.
14,
1669-1679 8.
Baldin, V.,
Lukas, J.,
Marcote, M. J.,
Pagano, M.,
and Draetta, G.
(1993)
Genes Dev.
7,
812-821 9.
Matsushime, H.,
Quelle, D. E.,
Shurtleff, S. A.,
Shibuya, M.,
Sherr, C. J.,
and Kato, J.-Y.
(1994)
Mol. Cell. Biol.
14,
2066-2076 10.
Sherr, C. J.
(1994)
Cell
79,
551-555[CrossRef][Medline]
[Order article via Infotrieve] 11.
Lundberg, A. S.,
and Weinberg, R. A.
(1998)
Mol. Cell. Biol.
18,
753-761 12.
Harbour, J. W.,
Luo, R. X.,
Dei Santi, A.,
Postigo, A. A.,
and Dean, D. C.
(1999)
Cell
98,
859-869[CrossRef][Medline]
[Order article via Infotrieve] 13.
Tanguay, D. A.,
and Chiles, T. C.
(1996)
J. Immunol.
156,
539-548[Abstract] 14.
Solvason, N., Wu, W. W.,
Kabra, N., Wu, X.,
Lees, E.,
and Howard, M.
(1996)
J. Exp. Med.
184,
407-417 15.
Solvason, N., Wu, W. W.,
Parry, D.,
Mahony, D.,
Lam, E.,
Glassford, J.,
Klaus, G.,
Sicinski, P.,
Weinberg, R.,
Liu, Y. J.,
Howard, M.,
and Lees, E.
(2000)
Int. Immunol.
12,
631-638 16.
Weber, J. D.,
Raben, D. M.,
Phillips, P. J.,
and Baldassare, J. J.
(1997)
Biochem. J.
326,
61-68[Medline]
[Order article via Infotrieve] 17.
Cheng, M.,
Sexl, V.,
Sherr, C. J.,
and Roussel, M. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1091-1096 18.
Diehl, J. A.,
Cheng, M.,
Roussel, M. F.,
and Sherr, C. J.
(1998)
Genes Dev.
12,
3499-3511 19.
Muise-Helmericks, R. C.,
Grimes, H. L.,
Bellacosa, A.,
Malstrom, S. E.,
Tsichlis, P. N.,
and Rosen, N.
(1998)
J. Biol. Chem.
273,
29864-29872 20.
Richards, J. D.,
Dave, S. H.,
Chou, C.-H. G.,
Mamchak, A. A.,
and DeFranco, A. L.
(2001)
J. Immunol.
166,
3855-3864 21.
Tan, J. E.-L.,
Wong, S.-W.,
Gan, S. K.-E., Xu, S.,
and Lam, K.-P.
(2001)
J. Biol. Chem.
276,
20055-20063 22.
Glassford, J.,
Holman, M.,
Banerji, L.,
Clayton, E.,
Klaus, G. G. B.,
Turner, M.,
and Lam, E. W.-F.
(2001)
J. Biol. Chem.
276,
41040-41048 23.
Zhang, H.,
Hannon, G. J.,
and Beach, D.
(1994)
Genes Dev.
8,
1750-1758 24.
Huo, L.,
and Rothstein, T. L.
(1995)
J. Immunol.
154,
3300-3309[Abstract] 25.
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494 26.
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. L.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyk, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632 27.
Sutherland, C. L.,
Heath, A. W.,
Pelech, S. L.,
Young, P. R.,
and Gold, M. R.
(1996)
J. Immunol.
157,
3381-3390[Abstract] 28.
Reid, S.,
and Snow, E. C.
(1996)
Mol. Immunol.
33,
1139-1151[CrossRef][Medline]
[Order article via Infotrieve] 29.
Zarkowska, T.,
and Mittnacht, S.
(1997)
J. Biol. Chem.
272,
12738-12746 30.
Boylan, J. F.,
Sharp, D. M.,
Leffet, L.,
Bowers, A.,
and Pan, W.
(1999)
Exp. Cell Res.
248,
110-114[CrossRef][Medline]
[Order article via Infotrieve] 31.
Stebbins, C. E.,
Russo, A. A.,
Schneider, C.,
Rosen, N.,
Hartl, F. U.,
and Pavletich, N. P.
(1997)
Cell
89,
239-250[CrossRef][Medline]
[Order article via Infotrieve] 32.
Chavany, C.,
Mimnaugh, E.,
Miller, P.,
Bitton, R.,
Nguyen, P.,
Trepel, J.,
Whitesell, L.,
Schnur, R.,
Moyer, J. D.,
and Neckers, L.
(1996)
J. Biol. Chem.
271,
4974-4977 33.
Schulte, T. W.,
Blagosklonnova, M. V.,
Romanova, L.,
Mushinski, J. F.,
Monia, B. P.,
Johnston, J. F.,
Nguyen, P.,
Trepel, J.,
and Neckers, L. M.
(1996)
Mol. Cell. Biol.
16,
5839-5845[Abstract] 34.
Schulte, T. W., An, W. G.,
and Neckers, L. M.
(1997)
Biochem. Biophys. Res. Commun.
239,
655-659[CrossRef][Medline]
[Order article via Infotrieve] 35.
Schulte, T. W.,
Blagosklonny, M. V.,
Ingui, C.,
and Neckers, L.
(1995)
J. Biol. Chem.
270,
24585-24588 36.
Gold, M. R.,
Scheid, M. P.,
Santos, L.,
Dang-Lawson, M.,
Roth, R. A.,
Matsuuchi, L.,
Duronio, V.,
and Krebs, D. L.
(1999)
J. Immunol.
163,
1894-1905 37.
Xie, H.,
and Rothstein, T. L.
(1995)
J. Immunol.
154,
1717-1723[Abstract] 38.
Grammatikakis, N.,
Lin, J.-H.,
Grammatikakis, A.,
Tsichlis, P. N.,
and Cochran, B. H.
(1999)
Mol. Cell. Biol.
19,
1661-1672 39.
Srethapakdi, M.,
Liu, F.,
Tavorath, R.,
and Rosen, N.
(2000)
Cancer Res.
60,
3940-3946 40.
Tanguay, D. A.,
Colarusso, T. P.,
Pavlovic, S.,
Irigoyen, M.,
Howard, R. G.,
Bartek, J.,
Chiles, T. C.,
and Rothstein, T. L.
(1999)
J. Exp. Med.
189,
1685-1690 41.
Gille, H.,
and Downward, J.
(1999)
J. Biol. Chem.
274,
22033-22040 42.
Banerji, L.,
Glassford, J.,
Lea, N. C.,
Thomas, S. B.,
Klaus, G. G. B.,
and Lam, E. W.-F.
(2001)
Oncogene
20,
3855-3864 43.
Brorson, K.,
Brunswick, M.,
Ezhevsky, S.,
Wei, D. G.,
Berg, R.,
Scott, D.,
and Stein, K. E.
(1997)
J. Immunol.
59,
135-143 44.
Forssell, J.,
Nilsson, A.,
and Sideras, P.
(1999)
Scand. J. Immunol.
49,
155-161[CrossRef][Medline]
[Order article via Infotrieve] 45.
Wu, H.-J.,
Venkataraman, C.,
Estus, S.,
Dong, C.,
Davis, R. J.,
Flavell, R. A.,
and Bondada, S.
(2001)
J. Immunol.
167,
1263-1273 46.
Tomlinson, M. G.,
Woods, D. B.,
McMahon, M.,
Wahl, M. I.,
Witte, O. W.,
Kurosaki, T.,
Bolen, J. B.,
and Johnston, J. A.
(2001)
BMC Immunol.
2,
4-15[CrossRef][Medline]
[Order article via Infotrieve] 47.
Jiang, A.,
Craxton, A.,
Kurosaki, T.,
and Clark, E. A.
(1998)
J. Exp. Med.
188,
1297-1306 48.
Fruman, D. A.,
Satterthwaite, A. B.,
and Witte, O. N.
(2000)
Immunity
13,
1-3[CrossRef][Medline]
[Order article via Infotrieve] 49.
Dey, A.,
She, H.,
Kim, L.,
Boruch, A.,
Guris, D. L.,
Carlberg, K.,
Sebti, S. M.,
Woodley, D. T.,
Imamoto, A.,
and Wei, L.
(2000)
Mol. Biol. Cell
11,
3835-3848 50.
Coller, H. A.,
Grandori, C.,
Tamayo, P.,
Colbert, T.,
Lander, E. S.,
Eisenman, R. N.,
and Golub, T. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3260-3265 51.
Perez-Roger, I.,
Kim, S. H.,
Griffiths, B.,
Sewing, A.,
and Land, H.
(1999)
EMBO J.
18,
5310-5320[CrossRef][Medline]
[Order article via Infotrieve] 52.
Bouchard, C.,
Thieke, K.,
Mairer, A.,
Saffrich, R.,
Hanley-Hyde, J.,
Ansorge, W.,
Reed, S.,
Sicinski, P.,
Bartek, J.,
and Eilers, M.
(1999)
EMBO J.
18,
5321-5333[CrossRef][Medline]
[Order article via Infotrieve] 53.
Lee, H., Wu, M., La,
Rosa, F. A.,
Duyao, M. P.,
Buckler, A. J.,
and Sonenshein, G. E.
(1995)
Curr. Top. Microbiol. Immunol.
194,
247-255[Medline]
[Order article via Infotrieve] 54.
Jun, D. Y.,
Kim, M.-K.,
Gwon, I.,
and Kim, Y. H.
(1997)
Mol. Cells
7,
537-543[Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. MOSER, S. A. LANG, and O. STOELTZING Heat-shock Protein 90 (Hsp90) as a Molecular Target for Therapy of Gastrointestinal Cancer Anticancer Res, June 1, 2009; 29(6): 2031 - 2042. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Matsumiya, T. Imaizumi, H. Yoshida, K. Satoh, M. K. Topham, and D. M. Stafforini The Levels of Retinoic Acid-Inducible Gene I Are Regulated by Heat Shock Protein 90-{alpha} J. Immunol., March 1, 2009; 182(5): 2717 - 2725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Tucker, M. A. Gijon, D. M. Spencer, Z.-H. Qiu, M. H. Gelb, and C. C. Leslie Regulation of cytosolic phospholipase A2{alpha} by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages J. Leukoc. Biol., September 1, 2008; 84(3): 798 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. MacLeod, N. Bhasin, and L. M. Wetzler Role of Protein Tyrosine Kinase and Erk1/2 Activities in the Toll-Like Receptor 2-Induced Cellular Activation of Murine B Cells by Neisserial Porin Clin. Vaccine Immunol., April 1, 2008; 15(4): 630 - 637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-D. Ha, D. Ng, S. L. Pelech, and S. O. Kim Critical Role of the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase-3 Signaling Pathway in Recovery from Anthrax Lethal Toxin-induced Cell Cycle Arrest and MEK Cleavage in Macrophages J. Biol. Chem., December 14, 2007; 282(50): 36230 - 36239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Dye, A. Palvanov, B. Guo, and T. L. Rothstein B Cell Receptor Cross-Talk: Exposure to Lipopolysaccharide Induces an Alternate Pathway for B Cell Receptor-Induced ERK Phosphorylation and NF-{kappa}B Activation J. Immunol., July 1, 2007; 179(1): 229 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Barnett, D. Tomic, R. K. Gupta, K. P. Miller, S. Meachum, T. Paulose, and J. A. Flaws The Aryl Hydrocarbon Receptor Affects Mouse Ovarian Follicle Growth via Mechanisms Involving Estradiol Regulation and Responsiveness Biol Reprod, June 1, 2007; 76(6): 1062 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Villanueva, Y. Yung, J. L. Walker, and R. K. Assoian ERK Activity and G1 Phase Progression: Identifying Dispensable Versus Essential Activities and Primary Versus Secondary Targets Mol. Biol. Cell, April 1, 2007; 18(4): 1457 - 1463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Hinman, J. N. Bushanam, W. A. Nichols, and A. B. Satterthwaite B Cell Receptor Signaling Down-Regulates Forkhead Box Transcription Factor Class O 1 mRNA Expression via Phosphatidylinositol 3-Kinase and Bruton's Tyrosine Kinase J. Immunol., January 15, 2007; 178(2): 740 - 747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. de la Fuente, L. Kumar, B. Lu, and R. S. Geha 3BP2 Deficiency Impairs the Response of B Cells, but Not T Cells, to Antigen Receptor Ligation. Mol. Cell. Biol., July 1, 2006; 26(14): 5214 - 5225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fang, L. Xu, T. Y. Chen, J. M. Cyr, and D. M. Frucht Anthrax Lethal Toxin Has Direct and Potent Inhibitory Effects on B Cell Proliferation and Immunoglobulin Production J. Immunol., May 15, 2006; 176(10): 6155 - 6161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lipfert, J. E. Fisher, N. Wei, A. Scafonas, Q. Su, J. Yudkovitz, F. Chen, S. Warrier, E. T. Birzin, S. Kim, et al. Antagonist-Induced, Activation Function-2-Independent Estrogen Receptor {alpha} Phosphorylation Mol. Endocrinol., March 1, 2006; 20(3): 516 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Cohen-Saidon, I. Carmi, A. Keren, and E. Razin Antiapoptotic function of Bcl-2 in mast cells is dependent on its association with heat shock protein 90beta Blood, February 15, 2006; 107(4): 1413 - 1420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. V. Georgakis, Y. Li, G. Z. Rassidakis, H. Martinez-Valdez, L. J. Medeiros, and A. Younes Inhibition of Heat Shock Protein 90 Function by 17-Allylamino-17-Demethoxy-Geldanamycin in Hodgkin's Lymphoma Cells Down-Regulates Akt Kinase, Dephosphorylates Extracellular Signal-Regulated Kinase, and Induces Cell Cycle Arrest and Cell Death Clin. Cancer Res., January 15, 2006; 12(2): 584 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-J. Hung, C.-Y. Wu, P.-C. Liao, and W.-C. Chang Hsp90{alpha} Recruited by Sp1 Is Important for Transcription of 12(S)-Lipoxygenase in A431 Cells J. Biol. Chem., October 28, 2005; 280(43): 36283 - 36292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Castro, C. E. Prada, O. Loria, A. Kamal, L. Chen, F. J. Burrows, and T. J. Kipps ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia Blood, October 1, 2005; 106(7): 2506 - 2512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Guo and T. L. Rothstein B Cell Receptor (BCR) Cross-Talk: IL-4 Creates an Alternate Pathway for BCR-Induced ERK Activation That Is Phosphatidylinositol 3-Kinase Independent J. Immunol., May 1, 2005; 174(9): 5375 - 5381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Mizuno and T. L. Rothstein B Cell Receptor (BCR) Cross-Talk: CD40 Engagement Enhances BCR-Induced ERK Activation J. Immunol., March 15, 2005; 174(6): 3369 - 3376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Symonds, D. Tomic, K. P. Miller, and J. A. Flaws Methoxychlor Induces Proliferation of the Mouse Ovarian Surface Epithelium Toxicol. Sci., February 1, 2005; 83(2): 355 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Tomic, K. P. Miller, H. A. Kenny, T. K. Woodruff, P. Hoyer, and J. A. Flaws Ovarian Follicle Development Requires Smad3 Mol. Endocrinol., September 1, 2004; 18(9): 2224 - 2240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Chiles Regulation and Function of Cyclin D2 in B Lymphocyte Subsets J. Immunol., September 1, 2004; 173(5): 2901 - 2907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Braga-Basaria, E. Hardy, R. Gottfried, K. D. Burman, M. Saji, and M. D. Ringel 17-Allylamino-17-Demethoxygeldanamycin Activity against Thyroid Cancer Cell Lines Correlates with Heat Shock Protein 90 Levels J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2982 - 2988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Piatelli, C. Wardle, J. Blois, C. Doughty, B. R. Schram, T. L. Rothstein, and T. C. Chiles Phosphatidylinositol 3-Kinase-Dependent Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1/2 and NF-{kappa}B Signaling Pathways Are Required for B Cell Antigen Receptor-Mediated Cyclin D2 Induction in Mature B Cells J. Immunol., March 1, 2004; 172(5): 2753 - 2762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Calabrese, A. Frank, K. Maclean, and R. Gilbertson Medulloblastoma Sensitivity to 17-Allylamino-17-demethoxygeldanamycin Requires MEK/ERK J. Biol. Chem., July 4, 2003; 278(27): 24951 - 24959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Yankee, S. A. Solow, K. D. Draves, and E. A. Clark Expression of the Grb2-Related Protein of the Lymphoid System in B Cell Subsets Enhances B Cell Antigen Receptor Signaling Through Mitogen-Activated Protein Kinase Pathways J. Immunol., January 1, 2003; 170(1): 349 - 355. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |