From the Division of Biology and UCSD Cancer Center, University of California, San Diego, La Jolla, California 92093-0322
Received for publication, May 9, 2000, and in revised form, October 16, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD19 is rapidly phosphorylated upon B-cell
antigen receptor (BCR) cross-linking, leading to the recruitment of
downstream signaling intermediates. A prominent feature of CD19
signaling is the binding and activation of phosphoinositide 3-kinase
(P13K), which accounts for the majority of PI3K activity
induced by BCR ligation. Recent findings have implicated activation of
the serine/threonine kinase Akt as imparting survival signals in a
PI3K-dependent fashion. Using CD19-deficient
B-lymphoma cells and mouse splenic B-cells, we show that CD19 is
necessary for efficient activation of Akt following cross-linking of
surface immunoglobulin or Ig Signaling through the B-cell receptor
(BCR)1 complex effects
differing cellular fates depending upon the stage of differentiation, nature of the antigen, and the contribution of surface coreceptors and
accessory molecules. During early B-cell maturation, signaling through
the pre-BCR induces proliferation and differentiation, tantamount to
the production of IgM-positive immature cells. However, as newly formed
B-cells transit from the bone marrow to become mature peripheral
B-cells they become subject to regulation by additional surface markers
that can modulate the nature or degree of signaling through the BCR.
Collectively, these inductive signals act to direct further B-cell
differentiation and antibody production.
Prominent among the B-cell coreceptors is CD19, a 95-kD transmembrane
protein expressed throughout B-cell development. CD19 is a proximal
substrate for tyrosine phosphorylation following surface immunoglobulin
(sIg) cross-linking and is thought to be the primary signaling
component of the CD19/CD21/CD81/Leu13 B-cell coreceptor complex.
CD19 has also been implicated as a signaling partner for several other
surface receptors including CD40 (1), CD38 (2), CD72 (1), VLA-4 (3) and
Fc One of the key functional attributes of CD19 signaling is the
recruitment and activation of phosphoinositide 3-kinase (PI3K) following sIg cross-linking (8, 9). CD19 is recruited to the BCR
complex via its membrane-proximal cytoplasmic domain and binds the p85
subunit of PI3K following phosphorylation of dual YXXM motifs in the cytoplasmic tail of CD19 (9, 10).
Through the generation of 3'-phosphorylated inositides, PI3K can
regulate the membrane localization and activation of numerous
downstream effector molecules. Recent work has lead to the elaboration
of a key cell survival pathway activated by the PI3K products
phosphatidylinositol-3,4 bisphosphate and -3,4,5 triphosphate. Newly
generated phosphatidylinositol-3,4 bisphosphate and
phosphatidylinositol-3,4,5 triphosphate bind and activate
3'-phosphoinositide-dependent protein kinase-1 and -2 (11,
12), which in turn phosphorylate the Ser/Thr kinase, Akt (13), on
residues Thr308 and Ser473, respectively. Once
activated, Akt can inhibit apoptosis by phosphorylating the
proapoptotic factor Bad (14), caspase-9 (15), or other uncharacterized
substrates. Most recently, it has been shown that BCR engagement leads
to activation of Akt that can be terminated by coligation of the
Fc Cell Lines and Isolation of CD19-deficient A20 Variant--
The
A20 B-lymphoma cell line and A20.4 CD19-deficient A20 variant were
grown in complete media (RPMI supplemented with 10% fetal
bovine serum, penicillin, streptomycin, L-glutamine, sodium pyruvate, nonessential amino acids (Cellgro; Mediatech, Herndon, VA),
and 50 mM 2-mercaptoethanol (Life Technologies,
Inc.)). CD19-deficient A20 variants were isolated by depletion
of CD19-positive A20 cells using rat anti-mouse CD19 (1D3)
antibody coupled to Magna Bind goat anti-rat IgG magnetic beads
(Pierce). Clones were expanded and stained for surface CD19, IgG, and
CD21 levels and were analyzed by fluorescence-activated cell sorting
(anti-CD19-phosphatidylethanolamine, anti-CD21-fluorescein
isothiocyanate, biotinylated anti-IgG, streptavidin-Cychrome; Pharmingen, San Diego, CA). A20.4 is representative of several clones
isolated in this manner.
Purification of Primary Splenic B-cells--
Splenic B-cells
from 6-12-week-old wild-type BALB/c and CD19 null mice (BALB/c
background) were isolated by depletion using the MidiMACS system with
anti-CD43 (Ly-48) microbeads (Miltenyi Biotech, Auburn, CA) as
per the manufacturer's instructions. Isolated B-cells were greater
than 97% pure by fluorescence-activated cell sorting analysis
(anti-B220-phosphatidylethanolamine, anti-CD3-fluorescein isothiocyanate; Pharmingen). Cells were kept at room temperature at all
times with the exception of the antibody/magnetic bead binding step,
which was done at 4 °C for 15 min.
Stimulations and Immunoblotting--
A20 B-lymphoma cells were
grown overnight in complete RPMI supplemented with 1% fetal bovine
serum followed by 3-5 h of serum starvation before stimulations.
Cells were adjusted to 1-2 × 107 cells/ml in PBS at
room temperature. Stimulations were allowed to proceed for the
indicated times and were stopped by the addition of an equal volume of
2× Nonidet P-40 lysis buffer (1× = 1% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 10 mM sodium fluoride, 1 mM pyrophosphate,
1 mM EDTA, 1 mM orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, and 1 mM microcystin). Antibodies used for
stimulations were goat anti-mouse IgG F(ab')2, minimal
cross-reactive rat anti-mouse IgG F(ab')2 (Jackson
ImmunoResearch, West Grove, PA), and hamster anti-mouse CD79b (HM79b;
Pharmingen). 15 µg of total cell lysate were loaded onto 7.5% SDS
polyacrylamide gels, and separated proteins were transferred to PVDF
(Millipore, Bedford, MA). Primary antibodies for Western blots were
rabbit anti-phospho Akt (Thr308 and
Ser473), rabbit anti-Akt (New England Biolabs,
Beverly, MA), and rabbit anti-mouse CD19 (a generous gift from
Dr. John Cambier, National Jewish Center, Denver, CO). Western blots
were probed with horseradish peroxidase-conjugated secondary antibodies
(either goat anti-mouse IgG or goat anti-rabbit IgG) for 1 h at
room temperature and developed with ECL+ (Amersham Pharmacia
Biotech). Quantitation was performed by comparing densitometry
readings using NIHimage software; numbers (arbitrary
units) represent values corrected for loading (reprobes for total Akt).
Purified splenic B-cells were stimulated, and protein lysates were
prepared and analyzed as described above for A20 cells. Stimulations
were performed with biotinylated goat anti-mouse IgM
F(ab')2 (Jackson ImmunoResearch), biotinylated rat
anti-mouse CD19 (1D3), and hamster anti-mouse CD79b (HM79b)
(Pharmingen). For synergistic activation of purified splenic B-cells,
cells at 2 × 107 cells/ml in PBS were preincubated
with 5 µg/ml FcBlock (anti-CD16/CD32; Pharmingen) for 10 min at room
temperature. Cells were washed once with PBS before being resuspended
in PBS at 3 × 107 cells/ml containing varying amounts
of biotinylated goat anti-mouse IgM F(ab')2 fragments with
or without varying amounts of biotinylated rat anti-mouse CD19.
Antibodies were allowed to bind for 10 min at room temperature, after
which 10 µg/ml avidin was added, and cells were stimulated at
37 °C for 2 min. Cell lysates were prepared and analyzed as
described above.
Akt in Vitro Kinase Assay--
In vitro kinase assays
were carried out with A20 or A20.4 cells using the Akt kinase assay kit
from New England Biolabs as per the manufacturer's instructions. In
brief, Akt was immunoprecipitated from 0.5-1 × 107
unstimulated or stimulated cells, and kinase assays using glutathione S-transferase-GSK-3 Akt Kinase Activity Is Reduced in CD19-deficient
Cells--
Several groups, including ours, have found induced Akt
kinase activity following sIg cross-linking (16-19). This occurs in a
PI3K-dependent manner; hence we were interested in
determining how Akt activity is induced by the BCR complex. Although
several BCR-associated proteins, including Ig
To assess BCR-induced activation of Akt in the presence or absence of
CD19, A20 and A20.4 cells were stimulated with anti-IgG F(ab')2 fragments for the indicated time points, and kinase
activity was determined by a cold kinase assay to specifically measure Akt-induced phosphorylation of GSK-3 in vitro on position
Ser21 as revealed by blotting with a phosphospecific
antibody (Fig. 2A). In
parallel, [ CD19 Is Necessary to Promote Dual Phosphorylation of Akt--
Full
activation of Akt is dependent upon dual phosphorylation at residues
Thr308 and Ser473 by
3'-phosphoinositide-dependent protein kinase-1 and
3'-phosphoinositide-dependent protein kinase-2,
respectively (11, 12). To explore the enzyme kinetics, activation
threshold, and regulation of Akt phosphorylation, we employed
phosphospecific antibodies directed against each of these sites. We
found that Akt was rapidly phosphorylated at both positions in A20
cells (Fig. 3A). Peak
phosphorylation was observed at 1-2 min for Thr308 and
2-5 min for Ser473 post-stimulation and was sustained
above background levels for 20-60 min (Fig. 2A and data not
shown). In striking contrast, Akt phosphorylation in A20.4 cells showed
reduced levels of phosphorylation on both Thr308 and
Ser473 residues, which was not sustained beyond 10-20 min.
These results are consistent with the differential kinase activity
observed in CD19-sufficient and -deficient cells.
To address whether CD19 was promoting the activation of Akt or
preventing its inactivation, we performed pulse-chase experiments on
activated Akt. A20 and A20.4 cells were stimulated with anti-IgG F(ab')2 fragments for 2 min; further PI3K-mediated
activation of Akt was blocked with wortmannin, and the amount of Akt
phosphorylation at positions Thr308 and Ser473
was measured at the indicated time points (Fig. 3B).
Although BCR-induced Akt phosphorylation on Thr308 and
Ser473 was reduced in A20.4 cells, pulse-chase studies
showed comparable rates of dephosphorylation in both cell lines (Fig.
3B). The similar rates of dephosphorylation in A20 and A20.4
cells suggest that CD19 acts to promote Akt kinase activity by induced
phosphorylation of Akt, as opposed to preventing dephosphorylation or
degradation. To show that reduced Akt phosphorylation in the A20.4
cells was due soley to the absence of CD19, A20.4 cells were
transfected with a murine CD19 cDNA construct and stimulated as
described above. Phosphorylation of Akt on both sites was
restored in A20.4 cells expressing CD19 (Fig. 3C).
Reduced Phosphorylation of Akt in CD19 CD19 Coligation with the BCR Augments Akt
Phosphorylation--
Much of the attention to CD19 has focused on its
ability to augment signaling through the BCR (22). As CD19 is also
thought to be the primary signal transducing component for the
CD21(CR2)/CD19/CD81 coreceptor complex, this effect may be attributed
to the recruitment of downstream signaling intermediates following
B-cell recognition of C3d-bearing antigen (23). In addition, CD19
appears to be a nominal component of the BCR complex as it is rapidly
phosphorylated upon cross-linking of sIg alone. To address whether CD19
can synergize with sIg in promoting Akt activation, splenic B-cells
were stimulated with suboptimal doses of sIg cross-linking in the
presence or absence of CD19 coligation. We observed a strong synergy in
Akt phosphorylation on Thr308/Ser473 upon CD19
coengagment with sIg (Fig. 4B). This effect was clearly elevated over the induction observed with sIg or CD19 alone (Fig. 4B, lanes 2, 3, and
10-12). Interestingly, Akt phosphorylation at position
Ser473 is induced at a lower threshold of sIg cross-linking
than phosphorylation on Thr308 (Fig. 4B,
lane 2).
The Ig-associated Ig
In summary, our findings support a prominent role for the B-cell
coreceptor, CD19, in the regulation of BCR-induced Akt kinase activity
in normal B-cells. As a group, the B-cell coreceptors (e.g. CD19, CD21, CD22, and Fc. In the absence of CD19, Akt kinase
activity is reduced and transient. In addition, coligation of CD19 with
surface immunoglobulin leads to augmented Akt activity in a
dose-dependent manner. Thus, CD19 is a key regulator of Akt
activity in B-cells; as such it may contribute to pre-BCR or
BCR-mediated cell survival in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RIIB (4, 5). Mice deficient for CD19 show reduced B-cell
lymphopoiesis and antibody responses to T-cell-dependent
antigens (6, 7). Hence, it appears that CD19 may participate in B-cell
activation and selection at several stages of development. We sought to
determine the biochemical bases of these phenotypes as they may relate
to in vivo proliferation and/or survival of
B-lymphocytes.
RIIB receptor (16). Here we investigate the regulation of Akt
activity in primary and transformed mature B-cells. Our findings
demonstrate that CD19 is critical for BCR-induced Akt activity,
suggesting that the prosurvival capacity of Akt in B-cells is regulated
by CD19 in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
fusion protein as substrate were
performed. Akt-mediated phosphorylation of GSK-3
on
Ser21 was determined by Western blot using an antibody that
specifically recognizes phospho-Ser21 on GSK-3
. To
quantitatively measure differences in Akt activity, 5 µCi of
32P
-ATP and 50 µM of unlabeled ATP
were used in the Akt kinase assay. After transfer to PVDF,
incorporation of labeled phosphate was quantitated on a
PhosphorImager:SI (Molecular Dynamics, Sunnyvale, CA).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and Ig
, have been
implicated as associating with PI3K (20), CD19 is the only coreceptor
that has been functionally linked to PI3K activation (8, 9). To assess
the role of CD19 in Akt activation, we isolated CD19-deficient variants
of A20 murine B-lymphoma cells by immunodepletion of CD19-positive
cells. One such line, designated A20.4, was selected for further
characterization and was found to be devoid of cell surface and
intracellular expression of CD19 protein, yet it retained similar
levels of sIg (Fig. 1).
View larger version (21K):
[in a new window]
Fig. 1.
Characterization of CD19-deficient
B-cells. A20 (thin line) and A20.4 (thick
line) cells were examined for expression of surface CD19
(upper histogram) and IgG (lower histogram;
secondary alone is indicated by the dotted line) by flow
cytometry. Whole cell lysates from A20 and A20.4 were separated on a
7.5% SDS polyacrylamide gel, transferred to PVDF, and immunoblotted
with anti-CD19 or anti-Akt rabbit antisera. PE,
phosphatidylethanolamine.
-32P]-ATP was incorporated into the kinase
assay to directly measure kinase activity levels with the natural
substrate GSK-3 (Fig. 2B). A20 cells showed rapid induction
of Akt kinase activity, which began to diminish by 15 min. In the
absence of CD19, BCR-induced Akt activity is not only reduced (Fig.
2B) but is also more rapidly attenuated (Fig. 2A,
compare lanes 4 and 5 with lanes 10 and 11). Remaining Akt kinase activity in the CD19-deficient
A20.4 is still PI3K-dependent as seen by sensitivity to
Wortmannin treatment (Fig. 2A, lane 12). To
assess the effects of reduced Akt activity on downstream substrates in
primary cells, phosphorylation of endogenous GSK-3 was measured by
Western blots of lysates from nonstimulated and stimulated splenic
B-cells from wild-type and CD19-deficient mice (Fig. 2C).
Although phosphorylation of GSK-3 was similar at earlier time points
(lanes 2 and 8), CD19-deficient splenic B-cells
had reduced GSK-3 phosphorylation at later time points (Fig.
2C, compare lanes 4 and 5 with
lanes 10 and 11). Hence, CD19 is involved in
promoting and sustaining Akt function.
View larger version (23K):
[in a new window]
Fig. 2.
Reduced Akt kinase activity in CD19-deficient
cells. A, A20 and A20.4 cells were stimulated with 10 µg/ml goat anti-mouse IgG F(ab')2 fragments for the
indicated periods of time. In vitro phosphorylation of the
exogenous Akt substrate GSK-3 was measured by Western blot using an
antibody (p-GSK-3) that specifically recognizes
phospho-Ser21 on GSK-3
. The lower panel shows
relative amounts of immunoprecipitated Akt. The degree of induced
phosphorylation was quantitated by densitometry and was corrected for
immunoprecipitated Akt. These data are representative of at least five
experiments. B, Akt was immunoprecipitated from cell lysates
following stimulation as in A and was subjected to an
in vitro kinase assay using [
-32P]-ATP and
GSK-3
as an exogenous substrate. After transfer to PVDF,
incorporation of labeled phosphate into GSK-3
was quantitated by
phosphorimager analysis, and standard deviations from three experiments
are shown. p values are shown as a measurement of the
significance of the difference in Akt activity observed in A20 and
A20.4 cells at the indicated time points. C, induced
phosphorylation of the Akt substrate GSK-3
was measured as in
A. Here, whole cell lysates were probed for phosphorylation
of endogenous GSK-3
in primary B-cells.
View larger version (35K):
[in a new window]
Fig. 3.
A, CD19-deficient cells show reduced and
transient Akt phosphorylation on residues Thr308 and
Ser473. A20 and A20.4 cells were stimulated with 1 µg/ml
goat anti-mouse IgG F(ab')2 for the indicated periods of
time. Cell lysates were separated on a 7.5% SDS polyacrylamide gel,
transferred to PVDF, and immunoblotted with antibodies that
specifically recognize Akt phosphorylated on residues
Thr308 (p-T308 Akt) or Ser473
(p-S473 Akt) as indicated by arrows. The
lower panel shows relative amounts of Akt in cell lysates.
The graph shows levels of induced phosphorylation as
measured by densitometry and corrected for loading. Data are
representative of at least five experiments. B, A20 and
A20.4 cells were stimulated with 1 µg/ml goat anti-mouse IgG
F(ab')2 for 2 min, and further activation of Akt was halted
by the addition of 100 nM wortmannin (wort) and
5 µM LY294002 (LY). Thereafter, kinetics of
Akt dephosphorylation on Thr308 and Ser473 was
monitored with phosphospecific antibodies as in A. The
lower panel shows relative amounts of Akt in cell lysates.
The graph shows levels of induced phosphorylation as
measured by densitometry and corrected for loading. These data
represent one of three separate experiments. C, A20.4 cells
were transfected with a murine CD19 expression construct, and stable
lines were generated. Transfected A20.4, as well as parent A20.4 and
A20 cells, were stimulated with 0.5 µg/ml anti-IgG
F(ab')2 for the indicated time points. Akt phosphorylation
on Thr308 and Ser473 was monitored as in
A. The graph shows levels of induced
phosphorylation as measured by densitometry and corrected for loading.
These data represent one of three separate experiments.
/
Splenic
B-cells--
Akt phosphorylation and activity varies widely in
transformed B-cell lines representing various stages of B-cell
differentiation (21) and may in fact be a contributor to, or a
consequence of, the transformed state. We therefore examined regulation
of Akt activity in primary splenic B-cells from wild-type and
CD19
/
mice. Wild-type splenic B-cells showed rapid
induction of Akt phosphorylation on Thr308 and
Ser473 at both optimal (10 µg/ml) and suboptimal (0.1 µg/ml) levels of sIg cross-linking, whereas CD19-deficient cells
exhibited minimal Akt phosphorylation at either Thr308 or
Ser473 even at the higher level of sIg cross-linking (Fig.
4A). Moreover, whereas
wild-type B-cells achieved a peak state of phosphorylation within 1 min
of induction, CD19
/
cells showed delayed kinetics of
2-5 min. This was not attributed to inefficient engagement of the BCR
as both cell populations displayed similar extracellular
signal-regulated kinase 1/2 phosphorylation at 2 min post-stimulation
(data not shown). These data correlate with the loss of
Akt-dependent GSK-3 phosphorylation seen at later time
points in CD19
/
splenic B-cells (Fig.
2C).
View larger version (35K):
[in a new window]
Fig. 4.
CD19 promotes Akt activation in splenic
B-cells. A, purified splenic B-cells from wild-type
(wt) or CD19-deficient (CD19 /
)
mice were stimulated with 1 or 10 µg/ml goat anti-mouse IgG
F(ab')2 fragments in the presence or absence of 100 nM wortmannin (wort) for the indicated periods
of time. Cell lysates from CD19
/
and wild-type B-cells
were resolved on separate 7.5% SDS polyacrylamide gels, cotransferred
to PVDF, and coincubated with antibodies that specifically recognize
Akt phosphorylated on residues Thr308 (p-T308
Akt) or Ser473 (p-S473 Akt) as indicated by
arrows. The lower panel shows relative amounts of
Akt in cell lysates. Western blots from CD19
/
and
wild-type mice were developed by enhanced chemiluminescence and were
coexposed for identical time periods. Densitometry readings were made
with NIHimage software and are corrected for loading controls. These
data represent one of four separate experiments. B,
synergistic activation of Akt by CD19 and sIg was assessed using
suboptimal (0.01 or 0.05 µg/ml) doses of biotinylated goat anti-mouse
IgM F(ab')2 fragments in the presence of varying levels of
biotinylated anti-CD19 antibody and 10 µg/ml avidin. sIgM/CD19
coligation-induced phosphorylation on Thr308 and
Ser473 is compared with sIgM or CD19 cross-linking alone.
The lower panel shows relative amounts of Akt in cell
lysates. The graph displays densitometry readings of
p-Ser473 (solid line) and
p-Thr308 (dotted line) of anti-CD19
stimulations without (open symbols) or with (solid
symbols) 0.01 µg/ml anti-IgM. These data represent one of three
experiments.
/Ig
heterodimer becomes rapidly
phosphorylated upon BCR cross-linking and is crucial for initiating subsequent signaling events. However, Carter et al. (10)
have provided evidence that CD19 may associate with sIg directly.
Insofar as the Ig
/Ig
heterodimer and CD19 can both bind PI3K
directly (20), we sought to address whether CD19 played a downstream role in Ig
/Ig
-induced Akt activation. As shown in Fig.
5 for both primary B-cell and A20
lymphoma cells, Ig
cross-linking induced high levels of Akt
phosphorylation on Thr308 and Ser473. This was
greatly diminished in CD19-deficient cells, supporting the primary role
of CD19 in recruiting PI3K to the BCR complex. In the absence of CD19,
Ig
/Ig
can support some PI3K activation and likely accounts for
the residual Akt activity we observe in CD19-deficient cells.
View larger version (42K):
[in a new window]
Fig. 5.
Ig -induced
activation of Akt is CD19-dependent. A,
purified splenic B-cells from wild-type (wt) or
CD19-deficient (CD19
/
) mice were stimulated
with 1 µg/ml of anti-Ig
antibody for the indicated periods of
time. Cell lysates were separated on a 7.5% SDS polyacrylamide gel,
transferred to PVDF, and immunoblotted with antibodies that
specifically recognize Akt phosphorylated on residues
Thr308 (p-T308 Akt) or Ser473
(p-S473 Akt) as indicated by arrows. The
lower panel shows relative amounts of Akt in cell lysates.
Densitometry readings were analyzed using the NIHimage software and
were corrected for loading controls. B, A20 and A20.4 cells
were stimulated with anti-Ig
and were assessed for Akt
phosphorylation as in A. The lower panel shows
relative amounts of Akt in cell lysates. These data represent one of
three experiments.
RIIB) are
receiving increased attention because of their pivotal roles as
contextual molecules involved in determining cell fate decisions at
distinct stages of B-cell differentiation. CD19 has been implicated as
a proximal downstream target to effect both positive and negative
regulation of BCR-mediated signals. In the latter context, several
groups have noted decreased tyrosine phosphorylation of CD19 following
coligation of sIg and Fc
RIIB (4, 16, 18). This is likely because of
Fc
RIIB-mediated recruitment and activation of SHP-1, resulting in
impaired SH2-mediated binding of the p85 subunit of PI3K to the
cytoplasmic tail of CD19. Recent reports have also shown that Fc
RIIB
coligation with sIg results in reduced Akt activation (16-18, 24),
most probably through the activation of the SH2-containing
inositol-5-phosphtase, which acts on phosphatidylinositol-3,4,5
triphosphate. Thus, CD19 is likely a central target for inducing and
later down-regulating Akt activity. This function may contribute to its
key role in promoting B-cell lymphopoiesis and antibody responses
in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael David and Jean Wang for insightful discussions and helpful suggestions. We also thank Dr. John Cambier for providing the CD19 antisera and Bob Carter for the mCD19 expression vector.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI41649 (to R. R.) and by a biomedical science grant from the Arthritis Foundation (to R. R.).
Supported by National Cancer Institute National Research Service
Award Training Grant 5T32 CA09345.
§ To whom correspondence should be addressed: 9500 Gilman Dr., 0322, La Jolla, CA 92093. Tel.: 858-822-1271; Fax: 858-822-1241; E-mail: rrickert@ucsd.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M003918200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: BCR, B-cell receptor; PI3K, phosphoinositide 3-kinase; GSK, glycogen synthase kinase; sIg, surface immunoglobulin; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Uckun, F. M.,
Burkhardt, A. L.,
Jarvis, L.,
Jun, X.,
Stealey, B.,
Dibirdik, I.,
Myers, D. E.,
Tuel-Ahlgren, L.,
and Bolen, J. B.
(1993)
J. Biol. Chem.
268,
21172-21184 |
2. | Kitanaka, A., Ito, C., Coustan-Smith, E., and Campana, D. (1997) J. Immunol. 159, 184-192[Abstract] |
3. |
Xiao, J.,
Messinger, Y.,
Jin, J.,
Myers, D. E.,
Bolen, J. B.,
and Uckun, F. M.
(1996)
J. Biol. Chem.
271,
7659-7664 |
4. |
Kiener, P. A.,
Lioubin, M. N.,
Rohrschneider, L. R.,
Ledbetter, J. A.,
Nadler, S. G.,
and Diegel, M. L.
(1997)
J. Biol. Chem.
272,
3838-3844 |
5. | Hippen, K. L., Buhl, A. M., D'Ambrosio, D., Nakamura, K., Persin, C., and Cambier, J. C. (1997) Immunity 7, 49-58[Medline] [Order article via Infotrieve] |
6. | Rickert, R. C., Rajewsky, K., and Roes, J. (1995) Nature 376, 352-355[CrossRef][Medline] [Order article via Infotrieve] |
7. | Engel, P., Zhou, L. J., Ord, D. C., Sato, S., Koller, B., and Tedder, T. F. (1995) Immunity 3, 39-50[Medline] [Order article via Infotrieve] |
8. |
Buhl, A. M.,
Pleiman, C. M.,
Rickert, R. C.,
and Cambier, J. C.
(1997)
J. Exp. Med.
186,
1897-1910 |
9. | Tuveson, D. A., Carter, R. H., Soltoff, S. P., and Fearon, D. T. (1993) Science 260, 986-989[Medline] [Order article via Infotrieve] |
10. | Carter, R. H., Doody, G. M., Bolen, J. B., and Fearon, D. T. (1997) J. Immunol. 158, 3062-3069[Abstract] |
11. |
Delcommenne, M.,
Tan, C.,
Gray, V.,
Rue, L.,
Woodgett, J.,
and Dedhar, S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11211-11216 |
12. | Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997) Curr. Biol. 7, 261-269[Medline] [Order article via Infotrieve] |
13. | Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727-736[Medline] [Order article via Infotrieve] |
14. |
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
15. |
Cardone, M. H.,
Roy, N.,
Stennicke, H. R.,
Salvesen, G. S.,
Franke, T. F.,
Stanbridge, E.,
Frisch, S.,
and Reed, J. C.
(1998)
Science
282,
1318-1321 |
16. |
Aman, M. J.,
Lamkin, T. D.,
Okada, H.,
Kurosaki, T.,
and Ravichandran, K. S.
(1998)
J. Biol. Chem.
273,
33922-33928 |
17. |
Gupta, N.,
Scharenberg, A. M.,
Fruman, D. A.,
Cantley, L. C.,
Kinet, J. P.,
and Long, E. O.
(1999)
J. Biol. Chem.
274,
7489-7494 |
18. |
Jacob, A.,
Cooney, D.,
Tridandapani, S.,
Kelley, T.,
and Coggeshall, K. M.
(1999)
J. Biol. Chem.
274,
13704-13710 |
19. |
Li, H. L.,
Davis, W. W.,
Whiteman, E. L.,
Birnbaum, M. J.,
and Pur, E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6890-6895 |
20. | Clark, M. R., Campbell, K. S., Kazlauskas, A., Johnson, S. A., Hertz, M., Potter, T. A., Pleiman, C., and Cambier, J. C. (1992) Science 258, 123-126[Medline] [Order article via Infotrieve] |
21. |
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 |
22. | Carter, R. H., and Fearon, D. T. (1992) Science 256, 105-107[Medline] [Order article via Infotrieve] |
23. | Dempsey, P. W., Allison, M. E., Akkaraju, S., Goodnow, C. C., and Fearon, D. T. (1996) Science 271, 348-350[Abstract] |
24. |
Astoul, E.,
Watton, S.,
and Cantrell, D.
(1999)
J. Cell Biol.
145,
1511-1520 |