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*
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710;
Department of Analytical Chemistry, Glaxo Wellcome, Research Triangle Park, NC 27709; and
Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two strains of c-Abl-deficient mice have been generated. The abl2 mice have a targeted deletion of the c-abl gene such that the animal is a complete c-abl null, whereas the ablm1 mice have a targeted carboxyl-terminal truncation that produces a kinase-active protein, lacking binding sites for SH3-containing adaptors and DNA and actin binding domains (5, 6). Interestingly, both lines of mice exhibit a variety of similar phenotypes, including high perinatal lethality, runting, abnormal cranial and splenic architecture, and an increased susceptibility to infection (3, 5, 6). Both lines of c-Abl-deficient mice exhibit splenic and thymic atrophy, and are lymphopenic (5, 6). Interestingly, B cells derived from the spleen, but not the bone marrow, of the ablm1 mice exhibit a severe reduction in the ability to form colonies in agar in response to LPS compared with wild-type controls (7). However, this reduction was not observed in liquid culture assays (7). Similarly, the proliferative response to anti-IgM stimulation in liquid culture was reduced in splenocytes from ablm1 mice an average of 50% compared with that from wild-type controls (7). Additionally, progenitor B cell lines derived from the ablm1 mice are more sensitive to apoptosis induced by IL-7 deprivation than lines isolated from wild-type littermates (8). Overall, the phenotype of the c-Abl mutant mice suggests a role for c-Abl in transducing signals in B lymphocytes. However, the role of c-Abl in mediating signaling events in B cells has not been examined.
Various transmembrane molecules regulate B cell activation and
signaling. Among these is the B cell Ag receptor (BCR), a multimeric
complex composed of the surface Ig and associated CD79 
and ß
subunits (9). Upon clustering of the BCR, Lyn and other
Src family kinases are activated to initiate downstream signaling
events (9, 10). Numerous signaling events then follow,
including amplification of Src family kinase activation, stimulation of
the Syk and Btk tyrosine kinases, activation of the
Ras/mitogen-activated protein kinase (MAPK) pathway, and increased
calcium flux (9, 10, 11). These events lead to gene
transcription and regulation of B cell proliferation and
differentiation.
In addition to the core BCR complex, a number of other cell surface proteins regulate BCR signal transduction. One such protein is CD19, a 95 kDa membrane-spanning glycoprotein expressed only by B cells, from the pre-B cell stage until plasma cell differentiation, and by follicular dendritic cells (12, 13). Aggregation of CD19 alone can lead to signal generation (12, 13). Coaggregation of CD19 with the BCR reduces the threshold for B cell activation by the BCR, indicating that CD19 is a costimulatory molecule in this context (12, 14). Furthermore, CD19 expression is critical for regulating B cell-signaling events. B cells from CD19-deficient mice exhibit reduced tyrosine phosphorylation, phosphoinositide turnover, and proliferation following BCR activation (11, 12, 15, 16). By contrast, overexpression of CD19 in transgenic mice results in hyperresponsiveness to transmembrane signals (12). Thus, CD19 is an important regulator of signaling in B cells.
The CD19 protein contains two C2 Ig-like domains and a 
240 amino
acid cytoplasmic tail that is tyrosine phosphorylated at multiple sites
in response to BCR activation (12, 13). Phosphorylation of
these sites regulates protein interactions that are critical for BCR
signaling. However, the protein tyrosine kinases phosphorylating CD19
have not been clearly identified. Recently, Lyn was shown to directly
phosphorylate CD19 in vitro and in vivo (11, 17). However,
CD19 contains nine distinct potential sites of tyrosine
phosphorylation, so it is possible that multiple protein tyrosine
kinases phosphorylate CD19. Interestingly, CD19 contains a consensus
c-Abl phosphorylation site and two potential binding sites for the
c-Abl SH2 domain (18, 19). Herein, we show that CD19 is
both a substrate and binding partner for the c-Abl kinase. Furthermore,
we demonstrate that the levels of c-Abl tyrosine kinase are elevated in
the cytosol following BCR engagement. Significantly, B cells derived
from abl2 knockout mice exhibit an
impaired proliferative response following BCR engagement compared with
cells derived from wild-type mice. Our results establish that c-Abl is
a downstream target of the BCR complex and may modulate BCR signaling
events via interactions with CD19.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-abl2 mice (C57BL6/129Sv/Ev hybrid background) heterozygous for targeted deletion of the c-abl gene were the generous gift of Stephen Goff (Columbia University, New York, NY) (6). Mice were bred and housed in the Duke University vivarium barrier facility. CD-1 outbred mice were obtained from Charles River Breeding Laboratories (Wilmington, MA). Mice were genotyped by PCR.
Ramos and Raji Burkitt lymphoma B cell lines were cultured in RPMI 1640
medium (Life Technologies, Grand Island, NY) containing 10% FBS (Life
Technologies). 293T and Bosc23 cells were maintained and transfected as
previously described (20, 21). Plasmids encoding the GST,
GST-Crk, and GST-Abl SH2 domain proteins have been previously described
(4, 22). pSR-c-Abl and
pSR-c-AblK290R plasmids, encoding wild-type
and kinase-inactive c-Abl, respectively, and pMT2CD19 have
been previously described (20, 23). GST-CD19 constructs
were produced by cloning into the pGex3X vector (Promega, Madison,
WI).
Polyclonal antisera 5381 and 5382 against CD19 were generated in New Zealand White rabbits with GST fusions of CD19 amino acids 362–468 and 457–540 (amino acid composition based on human CD19 amino acid sequence reviewed in Ref. 12) as immunogens, respectively. Anti-protein kinase Cß1 mouse mAb was purchased from Transduction Laboratories (Lexington, KY).
Isolation of primary B lymphocytes and proliferation assays
For preparation of B cells, spleens were removed from wild-type and abl2 knockout mice (6–10 wk of age) and made into single-cell suspensions. RBC were lysed by washing with 17 mM Tris-HCl, pH 7.4, with 144 mM NH4Cl, and the remaining cells were resuspended in RPMI 1640 medium containing 5% FBS and 2.75 x 10-5 M 2-ME. T cells were depleted using anti-Thy1.2 Ab-coupled magnetic beads (Dynal, Great Neck, NY), and splenocytes were plated in 24-well dishes (1 x 105/well) and incubated with increasing amounts of goat anti-mouse IgM F(ab')2 Ab fragments (Cappel ICN, Costa Mesa, CA) or LPS (Sigma, St. Louis, MO) for 72 h. [3H]Thymidine (1 µCi/ml, 6.7 Ci/mmol; New England Nuclear, Boston, MA) was added to the cultures for the last 17 h. Cells were collected using a PHD cell harvester, and [3H]thymidine incorporation was measured by scintillation counting in a Beckman scintillation counter.
GST fusion proteins and pulldown assays
GST fusion proteins were prepared from bacteria as previously described except that GST-CD19 tail (amino acids 362–540) was prepared in buffers lacking Triton X-100 but containing 0.5% n-dodecyl-ß-D-maltoside (Ultrol Grade; Calbiochem, La Jolla, CA) (21, 22). For GST pulldown assays, Ramos cells were unstimulated or stimulated with goat anti-human IgM F(ab')2 Ab fragments (1:100; BioSource International, Camarillo, CA), lysed, and analyzed for binding to the GST-Abl SH2 domain as previously described (21, 22).
Kinase assays
Ramos or Raji cells (2 x 107
cells/ml) were stimulated with anti-IgM fragments as above or with
LPS (100 µg/ml) and immediately washed twice with ice cold hypotonic
buffer (Buffer C) (24) containing 1 mM sodium vanadate.
Cells were resuspended in three volumes of Buffer C containing a
cocktail of protease and phosphatase inhibitors (10 µg/ml aprotinin,
10 µg/ml leupeptin, 1 mM PMSF, 1 mM sodium vanadate, 2 mM sodium
pyrophosphate, and 25 mM ß-glycerophosphate, pH 7.5) and lysed by
passage through a 25-gauge needle. Nuclei and insoluble components were
removed by centrifugation (24). The remaining soluble,
cytoplasmic fraction (50 µg) was diluted into Triton lysis buffer
(4) containing protease and phosphatase inhibitors. c-Abl
was immunoprecipitated with the monoclonal anti-Abl Ab Ab-3
(Oncogene Research Products, Cambridge, MA). Samples were washed, and
c-Abl kinase activity was measured as previously described
(4) except that kinase assays were incubated for 20 min at
room temperature. For CD19 in vitro phosphorylation assays, c-Abl was
immunoprecipitated with Ab-3 or K12 (Santa Cruz Biotechnology, Santa
Cruz, CA) from 293T cells transfected with the pSR-c-Abl
or pSR-c-AblK290R plasmids. c-Abl kinase assays were
performed as above.
For mass spectrometric analysis, in vitro phosphorylation of CD19 was
performed as above except that cells were lysed in kinase lysis buffer
(4) lacking all detergents, but containing 0.5%
n-dodecyl-ß-D-maltoside, and
immunoprecipitates were washed four times with lysis buffer and twice
with kinase buffer. Kinase assays were performed as above except the 1
mM cold ATP was used in the absence of
[
-32P]ATP and reactions were at 37°C for
30 min.
Immunoprecipitation and Western blotting
Transfected Bosc23 cells were lysed in a buffer of 50 mM Tris-HCl, pH 8.0; 0.5% Nonidet P-40; 150 mM NaCl; and 2 mM EDTA or RIPA buffer containing the inhibitors described above. Ramos cells were stimulated as above and lysed with a 1% Triton X-100 buffer containing 6 mM HEPES, pH 7.5; 4 mM Tris-HCl, pH 7.5; 130 mM NaCl; 15 mM KCl; 3 mM MgCl2; 1 µM CaCl2; 1 mM DTT; and protease and phosphatase inhibitors as above. Lysates were immunoprecipitated with the indicated Abs or controls, and immunocomplexes were washed three to six times with lysis buffer. Western blotting for c-Abl was performed with the anti-Abl Ab 8E9 (PharMingen, San Diego, CA). CD19 protein was detected with a mixture of the anti-CD19 antisera 5381 and 5382. Phosphotyrosine was detected using a mixture of the anti-phosphotyrosine Abs PY99 (Santa Cruz Biotechnology) and 4G10 (Upstate Biotechnology, Lake Placid, NY).
CD19 phosphorylation mapping by mass spectrometry
The phosphorylation of CD19 by c-Abl was determined by analyzing aliquots of the kinase reaction by liquid chromatography/mass spectrometry. The system consisted of an HP1090 liquid chromatograph (Hewlett-Packard, Palo Alto, CA), a 250 µm id x 15 cm Poros R2 (PerSeptive Biosystems, Cambridge, MA) reversed-phase capillary column, and an API-III triple quadrupole mass spectrometer (PE Sciex, Thornhill, Ontario, Canada) equipped with electrospray ionization. For phosphorylation site identification, phosphopeptides were isolated from solution tryptic or Asp-N digests of c-Abl-phosphorylated CD19 using immobilized metal-ion affinity chromatography (25, 26, 27). Aliquots of digests were loaded onto a 25 µl GaCl3-charged Sepharose-NTA (Qiagen, Chatsworth, CA) immobilized metal-ion affinity chromatography column equilibrated with 50 mM MES buffer, pH 5.5, containing 1 M NaCl. After extensive washing with MES buffer, retained phosphopeptides were eluted and collected with 500 mM ammonium bicarbonate, pH 8.5. Isolated phosphopeptides were desalted using small reversed phase pipet tip microcolumns and loaded into nanoelectrospray needles (Protana A/S, Odense, Denmark). Positive ion nanoelectrospray mass spectra were acquired using a quadrupole time-of-flight mass spectrometer (Micromass, Manchester, U.K.) fitted with the Z-spray ion source. Enriched phosphopeptides were sequenced by tandem mass spectrometry, allowing specific phosphorylation site identification.
Isolation of lipid membrane rafts
Lipid rafts were isolated as previously described (28). Briefly, Ramos cells were stimulated as above and lysed in 1 ml 0.05% Triton X-100 in TNE (25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 5 mM EDTA; 30 mM sodium pyrophosphate; 10 mM ß-glycerophosphate; 1 mM sodium orthovanadate; and protease inhibitors as above). Lysates were diluted with an equal volume of 80% sucrose in TNE and transferred into an ultracentrifuge tube. The lysates were overlaid with 7 ml 30% sucrose in TNE and then with 4 ml 5% sucrose in TNE. The samples were centrifuged 17 h at 200,000 x g at 4°C. The rafts were isolated from the interface between the 5 and 30% sucrose layers and solubilized by the addition of n-octyl ß-D-glucopyranoside to 50 mM. The soluble fraction at the bottom of the tube was also collected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether complete deletion of c-Abl alters BCR-induced
cell proliferation we assayed the IgM-induced proliferative response of
splenic B cells from wild-type and abl2
knockout (complete c-Abl-null) mice. Purified splenic B cells were
incubated with increasing amounts of anti-IgM
F(ab')2 Ab fragments, and proliferation was
determined by measuring [3H]thymidine
incorporation (Fig. 1
A). The
proliferative capacity of c-Abl-null splenic B cells was reduced (43 to
73%) compared with that of cells from wild-type littermates. This
difference was observed over an Ab range of 10–160 µg/ml. The
reduced proliferative capacity of the c-Abl-null cells was specific for
BCR activation, as LPS-induced thymidine incorporation was similar in
wild-type and c-Abl-null B cells in liquid culture assays (Fig. 1B). Similar results were observed in B cells from
abl2 mice crossed with the outbred strain
CD-1 (data not shown). These data are in agreement with results
obtained with the ablm1 mutant mice
(7). Taken together, these data demonstrate that the
proliferative response of splenic B cells to anti-IgM requires the
full-length c-Abl tyrosine kinase.
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Because c-Abl is required for maximal proliferation of B cells
following activation of the BCR, c-Abl may be regulated downstream of
the BCR. To determine whether c-Abl is regulated following activation
of the BCR, Ramos cells were stimulated with anti-human IgM
F(ab')2 Ab fragments and then fractionated into
soluble cytosol, insoluble membrane/cytoskeleton, and nuclear
fractions. As depicted in Fig. 2
(top), c-Abl kinase activity was elevated in the
soluble fraction following stimulation of the BCR (1.7 ± 0.2-fold
after 5 min, n = 3). This increase in kinase activity
was paralleled by an increase in c-Abl protein levels in the cytosol
(Fig. 2, middle). No alteration of c-Abl protein levels or
kinase activity was apparent in either the Triton-insoluble
membrane/cytoskeletal or nuclear fractions (data not shown). The purity
of the fractionation was verified by Western blotting for proteins
specific to each fraction (data not shown). Similar results were
obtained in another cell line, Raji (data not shown). LPS treatment of
B cells in liquid culture did not result in an alteration of c-Abl
protein levels or kinase activity (data not shown). These data
demonstrate that c-Abl is regulated following engagement of the
BCR.
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Because c-Abl is regulated by activation of the BCR, it was of
interest to identify signaling molecules that associate with or are
regulated by c-Abl. The BCR coreceptor CD19 is highly tyrosine
phosphorylated and interacts with SH2-containing proteins following B
cell activation (12, 13). Two phosphotyrosines in CD19
have been identified as potential binding sites for the Abl SH2 domain
(18). To determine whether the c-Abl SH2 domain interacts
with CD19, lysates from unstimulated or stimulated Ramos cells were
incubated with a GST-Abl SH2 domain fusion protein. As shown in Fig. 3A, the c-Abl SH2 domain
interacted with a tyrosine-phosphorylated protein of 95 kDa only
after activation of the BCR. No interaction with the phosphoprotein was
observed with GST alone. This protein was not detected when lysates
were precleared with an anti-CD19 Ab, but remained when lysates
were not precleared or were precleared with an irrelevant Ab. These
results suggest that the tyrosine-phosphorylated protein is CD19 or is
in a complex with CD19. Western blotting with antisera against CD19
demonstrated that CD19 was present in the GST-Abl SH2 pulldown only
after stimulation (Fig. 3B). Direct interaction of the c-Abl
SH2 domain with CD19 was observed in Far Western blots (data not
shown). These results demonstrate that tyrosine-phosphorylated CD19 can
associate with the c-Abl SH2 domain following B cell activation.
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A) and, conversely, CD19 was
present in c-Abl immunoprecipitates (data not shown) only when the
proteins were coexpressed. Western blotting of whole cell lysates (WCL)
and of the immunoprecipitates with Abs to c-Abl and CD19 verified the
expression of each protein (Fig. 4A). Similar results were
obtained with different Abs to c-Abl and CD19, raised against distinct
epitopes in the two proteins (data not shown). Neither protein was
observed when control Abs were used.
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B). Surprisingly, the complex
was not quantitatively altered following BCR stimulation (Fig. 4B). Together, these results demonstrate that c-Abl and CD19
form a constitutive molecular complex in vivo in unstimulated Ramos
cells, and following BCR stimulation the interaction is mediated in
part by the c-Abl SH2 domain with tyrosine-phosphorylated
CD19. CD19 is a substrate of the c-Abl tyrosine kinase
CD19 can interact with the SH2 domains of multiple proteins
(12), including that of c-Abl (Fig. 3). However, little is
known regarding the kinases that directly phosphorylate CD19. To
determine whether CD19 is a substrate of the c-Abl kinase, we first
examined whether CD19 was phosphorylated in the presence of c-Abl in
vivo. As shown in Fig. 5, immunoprecipitated CD19 was tyrosine phosphorylated only when CD19 was
coexpressed with wild-type c-Abl. No phosphorylation was observed in
cells lacking c-Abl or CD19. Furthermore, this phosphorylation was
dependent upon the presence of c-Abl kinase activity, as coexpression
of a kinase inactive c-Abl (K290R) did not result in phosphorylation
of CD19.
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A and 5).
Overexpressed CD19 in the absence of c-Abl migrated as a doublet of
80–95 kDa. When coexpressed with wild-type c-Abl, the relative
amounts of the CD19 doublet were altered such that the lower band was
more prominent (Figs. 4A and 5). The levels of the 95-kDa
form of CD19, which is heavily tyrosine phosphorylated, were reduced in
the presence of kinase-active c-Abl (compare Fig. 5 upper
panel to middle panel). Furthermore, in several
experiments expression of c-Abl wild type induced the accumulation of
an even faster migrating form of CD19 of 75 kDa (Fig. 4A). This species may represent a cleavage product of CD19,
or CD19 molecules that are differentially posttranslationally modified,
as the predicted molecular mass of unmodified CD19 is 65 kDa. These
results may also suggest that the hyperphosphorylated form of CD19 is
targeted for internalization and/or degradation in the presence of
kinase-active c-Abl.
We next examined whether c-Abl could directly phosphorylate CD19 in
vitro. As shown in Fig. 6A,
c-Abl produced in 293T cells phosphorylated the GST-CD19 tail
encompassing amino acids 362–540, as well as the known c-Abl substrate
GST-Crk. No phosphorylation of GST alone was observed (data not shown).
Furthermore, only wild-type c-Abl and not a kinase-inactive mutant
(K290R) could phosphorylate CD19, ruling out the presence of a
coprecipitating kinase (Fig. 6A). These results demonstrate
that CD19 is a substrate of c-Abl in vitro.
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B).
Unphosphorylated GST-CD19 tail (top) had a molecular
mass of 45,540 Da, whereas that of GST-CD19 phosphorylated by c-Abl
was 45,629 Da. This shift in molecular mass reflects the additional
mass of one phosphate group. The minor peaks to the left of the major
peaks represent CD19 minus one methionine residue in the
unphosphorylated and phosphorylated states. These results suggest that
only one site on CD19 is phosphorylated by c-Abl. However, the data can
be interpreted to mean that c-Abl phosphorylates CD19 at one specific
site, or that c-Abl phosphorylates one site on any single CD19
molecule, but that multiple individual tyrosines may be targeted. To
determine the identity of the tyrosine(s) phosphorylated by c-Abl,
phosphopeptide sequence analysis was performed. The results revealed
that only tyrosine (Y)490 of CD19 was phosphorylated by c-Abl. This
site of phosphorylation of CD19 by c-Abl is the same as that predicted
by phage display analysis (19). c-Abl and CD19 colocalize in detergent-insoluble lipid rafts
To further examine whether endogenous c-Abl and CD19 colocalize in
B cells, we investigated whether both proteins are present in lipid
rafts isolated from Ramos cells. Lipid rafts are
sphingolipid/cholesterol-enriched membrane microdomains that have been
proposed to function as platforms for signal transduction events and
are rich in signaling molecules including the subunits of the BCR, the
Lyn tyrosine kinase, CD20, CD40, and phospholipase C (PLC)-2 in B
cells (28, 29, 30, 31). The data in Fig. 7 (top and
middle) demonstrate that c-Abl and CD19 are present in the
detergent-insoluble lipid rafts in Ramos cells. Both proteins are
constitutively associated with the membrane rafts, and their presence
in lipid rafts is not altered following BCR stimulation. We show that
the Lyn tyrosine kinase is also present in lipid rafts (Fig. 7, bottom), as previously reported (28, 29). The
concentration of the c-Abl tyrosine kinase appeared to be equivalent
between the soluble fraction and the lipid rafts under the conditions
used here. In contrast, there was less CD19 present in the lipid rafts
than in the soluble fraction. Interestingly, the relative level of
tyrosine-phosphorylated CD19 in the lipid rafts was elevated relative
to that found in the soluble fraction (Fig. 8). Thus, a higher proportion of CD19
molecules, or available tyrosines on CD19, are phosphorylated in this
membrane microdomain following BCR stimulation. Overall, these results
demonstrate that c-Abl and CD19 colocalize in the detergent-insoluble
lipid raft microsignaling domain. This colocalization may have a
functional significance given the high fraction of
tyrosine-phosphorylated CD19 present in this fraction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or that express a truncated c-Abl lacking the carboxyl
terminus (7). It is noteworthy that mice with a targeted
deletion of CD19 exhibit a similar reduction of BCR-induced
proliferation (32). This suggests that c-Abl and CD19 may
act in a similar pathway leading to BCR-mediated growth regulation of B
cells. The reduced proliferation of B cells lacking c-Abl may be the
result of increased apoptosis. In this regard, embryos from mice that
are doubly deficient for c-abl and the
c-abl-related kinase arg, and B cell lines
derived from the mutant ablm1 mice have an
increased susceptibility to apoptosis (8, 33), whereas
cells expressing the activated Abl oncoproteins are resistant to
apoptosis induced by a variety of stimuli (reviewed in Ref.
34). Alternatively, the reduced BCR-mediated proliferation
in c-Abl- and CD19-deficient B cells may be due to a signaling defect
in a proliferative pathway(s) modulated by these two molecules. In this
regard, CD19 may modulate signaling thresholds and function to enhance
the proliferative response to external signals (12).
Similar roles may be ascribed to c-Abl. Thus, in the absence of c-Abl,
reduced phosphorylation of CD19 and other c-Abl substrates may impair
recruitment of signaling components to the BCR complex and reduce the
proliferative or survival potential of B cells.
The link between c-Abl and CD19 is further strengthened by our
observation that the subcellular distribution of c-Abl is regulated
following engagement of the BCR (Fig. 2). The c-Abl protein levels and
kinase activity increase in the cytosol following stimulation of the
BCR in two independent B cell lines. One possible explanation for these
findings is described below. We have shown that c-Abl and CD19 form a
constitutive complex in B cells that does not appear to be increased
following stimulation of the BCR (Fig. 4B). This observation
is similar to that reported for the association of CD19 and the Lyn
tyrosine kinase. Lyn and CD19 form a constitutive complex in primary B
cells, and BCR stimulation increases Lyn association with CD19 by only
2-fold (17). Tyrosine phosphorylation and activation of
the Lyn tyrosine kinase have been shown to produce a significant
decrease in the binding capacity of the Lyn SH2 domain for
phosphotyrosine-containing targets (35). Thus, the reduced
affinity of the activated Lyn kinase for tyrosine-phosphorylated CD19
may result in the release of Lyn from the CD19 complex into the
cytosol. Similarly, following BCR engagement, a pool of c-Abl in the
vicinity of the BCR may be autophosphorylated or phosphorylated by Src
family kinases (4), and the tyrosine-phosphorylated c-Abl
may then be released into the cytosol as a consequence of the decreased
binding affinity between activated c-Abl and CD19. This possibility is
consistent with our observation of increased c-Abl kinase in the
cytosol following BCR stimulation (Fig. 2).
We have also shown here for the first time that the BCR coreceptor CD19 is a substrate for the c-Abl kinase in vitro and in vivo. Although tyrosine phosphorylation of CD19 downstream of numerous signals has been reported (12, 13, 14), the kinases that mediate the phosphorylation of CD19 downstream of these signals have not been identified. Recently, the Lyn tyrosine kinase was shown to phosphorylate CD19 in vitro and in vivo (11, 17). Significantly, CD19 was not tyrosine phosphorylated in resting or activated B cells isolated from Lyn-/- mice following BCR ligation. In contrast, CD19 was tyrosine phosphorylated in B cells from wild-type mice, and this phosphorylation was elevated following BCR stimulation (17). Here we show that c-Abl phosphorylates CD19 at a specific tyrosine, Y490. Specific tyrosine-phosphorylated sites on CD19 have been shown to interact with various SH2-containing proteins (12). However, Y490 has not been reported or predicted to interact with known SH2-domain containing proteins (17), although it is in the context of a putative SH2-binding motif (YXXP, where X is any amino acid; Ref. 18). Interestingly, Y490 lies between two binding sites for the SH2 domains of the p85 subunit of phosphatidylinositol 3-kinase and the Src family tyrosine kinases (Y482 and Y513) (12, 17). Thus, it is possible that phosphorylation of the c-Abl site may modulate binding of other proteins to these adjacent phosphotyrosines. In this case, phosphorylation of Y490 would serve to modulate CD19 complex interactions rather than to directly recruit complex components. Alternatively, phosphorylation of Y490 by c-Abl may recruit an SH2-containing protein that participates in CD19 signaling. Recruitment of such a protein may affect the binding of phosphatidylinositol 3-kinase or Src kinases to the adjacent sites or may regulate other BCR-mediated signaling pathways.
In addition to its ability to phosphorylate CD19, we demonstrate that
c-Abl can associate with this BCR coreceptor. The SH2 domain of c-Abl
associates with CD19 only after stimulation of B cells, suggesting that
tyrosine phosphorylation of CD19 is required for this interaction.
However, coimmunoprecipitation of endogenous CD19/c-Abl complexes from
Ramos B cells revealed that these proteins are constitutively
associated (Fig. 4B). Furthermore, the coimmunoprecipitated
c-Abl/CD19 complex did not appear to be increased after BCR
stimulation. In agreement with these data, we have also observed that
c-Abl kinase activity is not required for this interaction (data not
shown). These data suggest that these proteins associate in the absence
of detectable CD19 tyrosine phosphorylation (Figs. 4A and 5,
and data not shown) or B cell activation (Fig. 4B).
Similarly, as described above, the Lyn tyrosine kinase forms a
constitutive complex with CD19 that is only slightly elevated following
BCR stimulation (17). Activation of c-Abl and/or
hyperphosphorylation of CD19 may result in a qualitative change in the
complex that causes an unstable SH2-mediated interaction between c-Abl
and CD19 such that an increase in stable complexes containing CD19 and
c-Abl are not observed following BCR stimulation. Furthermore, it has
been documented that substrate/kinase interactions are difficult to
isolate, such as that described for the interaction of the Src tyrosine
kinase and its substrate, the lipid raft-associated phosphoprotein
associated with glycosphingolipid-enriched microdomains/CSK binding
protein (36, 37).
Results from phage display analysis predict that the c-Abl SH2 domain
may bind to phosphorylated Y391 and Y421 of CD19 (18).
These phosphorylated tyrosines of CD19 are the sites of interaction
with the SH2 domain of the Vav protein, and tyrosine to phenylalanine
(F) mutation (Y391F, Y421F) of these sites reduces this interaction and
alters signal transduction downstream of the BCR and CD19
(38, 39, 40). However, not all the effects of the tyrosine to
phenylalanine mutations can be accounted for by the loss of Vav binding
to CD19. The CD19 Y391F and Y421F mutants exhibit reduced
Ca2+ flux and activation of Erk following
stimulation of the BCR alone or costimulation of both the BCR and CD19
(38, 39, 40). Significantly, the observed decrease in Erk
activation cannot be due to loss of Vav binding, as Erk activation is
unaltered in BCR-stimulated B cells from Vav-deficient mice (38, 41). This suggests that SH2-containing proteins other than Vav
may bind to these sites to regulate distinct signaling pathways.
Recently, PLC-2 has also been shown to interact with these residues
and may be important for signaling downstream of CD19
(40). Our results suggest that c-Abl may be yet a third
protein that interacts with these sites on CD19. Thus, the c-Abl kinase
may alter protein associations with CD19, as well as recruit and
phosphorylate substrates to transduce signals downstream of
CD19.
In addition to complex formation between c-Abl and CD19, we have
demonstrated for the first time that c-Abl and CD19 colocalize as
components of the detergent-insoluble lipid raft microsignaling domains
(Fig. 7). These microsignaling domains are dynamic sites of signal
transduction, and accumulating evidence suggests that lipid rafts play
a crucial role in immune receptor signal initiation and propagation
(42). We and others have found that c-Abl, CD19, and the
Src tyrosine kinase family member Lyn are constitutively associated
with lipid rafts in B cells (Fig. 7) (28, 29).
Furthermore, proteins including the BCR Ig and Ig subunits and
PLC-2 have been shown to translocate into lipid rafts following BCR
stimulation (28, 29). Both CD19 (Fig. 8) and Lyn
(29) are hyperphosphorylated in lipid rafts following B
cell stimulation, suggesting that these proteins are important
mediators of signaling within lipid rafts. Interestingly, in resting T
cells the linker for activation of T cells (LAT) adaptor protein and
the Src family member Lck have been identified as constitutive
components of lipid rafts (43). The localization of both
the adaptor protein LAT and the tyrosine kinase Lck to membrane rafts
is required for efficient signal transduction downstream of the TCR.
Lck and LAT are believed to function as integral mediators of early
signal transduction by respectively phosphorylating and recruiting
numerous signaling proteins to these microsignaling domains (reviewed
in Ref. 43). A similar scenario may be envisioned in B
cells where the constitutive association of Lyn, c-Abl, and CD19 in
lipid rafts may play a role in the transduction of early signaling
events downstream of the BCR.
In summary, the data presented here suggest a model for signaling
events downstream of the BCR. Crosslinking of the BCR results in its
translocation to rafts where Lyn, c-Abl, and CD19 are preassembled. Lyn
becomes activated and phosphorylates the BCR CD79 subunit, the CD19
coreceptor, and other proteins. Phosphorylated CD19 then acts as a
scaffolding protein to bind SH2 domains, such as that of the c-Abl
tyrosine kinase. Once recruited to CD19, c-Abl may be activated via the
CD19/Src amplification loop (11, 17), as we have
previously shown that Src kinases phosphorylate and activate c-Abl
(4). Tyrosine phosphorylation of CD19 leads to the
recruitment of additional signaling molecules (12, 17)
that may serve as substrates for the c-Abl tyrosine kinase. These
signaling molecules may together contribute to the regulation of
survival and/or proliferative pathways downstream of the BCR. The
absence of c-Abl in B cells may affect the formation of a fully
functional CD19 signaling complex, leading to the reduced proliferation
observed in c-Abl-null B cells following activation of the
BCR.
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Acknowledgments |
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Footnotes |
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2 P.A.Z. and M.G. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ann Marie Pendergast, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710.
4 Abbreviations used in this paper: SH, Src homology; BCR, B cell Ag receptor; WCL, whole cell lysate; Y, tyrosine; F, phenylalanine; TNE, 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 30 mM sodium pyrophosphate, 10 mM ß-glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors; PLC, phospholipase C; LAT, Tinker for activation of T cells.
Received for publication December 7, 1999. Accepted for publication September 25, 2000.
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