(Received for publication, October 26, 1995)
From the
We demonstrate that the CD19 receptor associates with the 1
family integrin receptors on human B-cell precursors as well as mature
B-lymphocytes, and engagement of the
1 family integrin receptors
with monoclonal antibody homoconjugates leads to rapid activation of
the CD19-associated protein-tyrosine kinases (PTK) and results in
hyperphosphorylation of CD19 on tyrosine residues. Our findings prompt
the hypothesis that homoconjugate-induced integrin clustering may
effect the approximation and, by intermolecular cross-phosphorylation,
activation of the CD19-associated PTK and subsequent tyrosine
phosphorylation of the CD19 receptor. The ability of the
1 family
integrin receptors to transmit a biochemical signal triggering the
CD19-linked multifunctional PTK pathway provides a possible explanation
for the pleiotropic biologic responses generated through adhesive
VLA-4- and VLA-5-mediated contacts.
Integrins are heterodimeric integral plasma membrane proteins
that mediate cell-cell as well as cell-extracellular matrix
adhesion(1, 2, 3, 4) . All members
of the integrin superfamily exist as -heterodimers. The
1
integrin common chain CD29 can associate with distinct
chains to
form the very late antigens (VLA), (
)which serve as
receptors for extracellular matrix
proteins(1, 2, 3, 4) . Members of
the VLA
1 integrin subfamily of adhesion molecules are thought to
play a pivotal regulatory role in human B-cell ontogeny by mediating
cell-cell and cell-stroma interactions of B-cell precursors
(BCP)(1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
Two members of the
1 or VLA integrin family expressed on normal
and leukemic BCP are VLA-4 (heterodimer of
1CD29 subunit with
/CD49d subunit) and VLA-5 (heterodimer of
1CD29
subunit with
/CD49e
subunit)(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
VLA-4 binds to vascular adhesion molecule-1 (VCAM-1) on stromal cells,
and it recognizes three distinct cell adhesion sites in the
COOH-terminal heparin binding domain and in adjacent alternatively
spliced IIICS domain of fibronectin, which is the major component of
the extracellular matrix in lymphohematopoietic
microenvironments(16, 17, 18, 19, 20, 21) .
VLA-5 binds to the RGD motif in the central cell binding domain located
in the 10th type-III repeat in A and B subunits of
fibronectin(3, 13) . In addition to mediating cell
adhesion, the engagement of
1 family integrin molecules influence
several other important cellular events (1, 2, 3, 4) . The molecular
mechanisms by which the engagement of VLA-4 and/or VLA-5 generates
pleiotropic biologic responses has not been deciphered. However, recent
studies demonstrated that signaling through these adhesion receptors
leads to enhanced tyrosine phosphorylation of multiple protein
substrates, suggesting the involvement of protein-tyrosine kinases
(PTK) in the generation of physiologically significant biochemical
events after the ligation of the
1 family integrin
receptors(4, 22, 23, 24, 25, 26, 27, 28) .
Recent studies indicated that the CD19 receptor may be a common
response element involved with PTK signaling stimulated through diverse
B-cell surface molecules, such as the B-cell antigen receptor, CD40
receptor, and CD72 receptor(29) . Here, we present experimental
evidence that 1) the CD19 receptor is physically associated with the
1 family integrin receptors on human BCP as well as mature
B-lymphocytes, and 2) engagement of the
1 family integrin
receptors leads to rapid activation of the CD19-associated PTK and
results in hyperphosphorylation of CD19 on tyrosine residues. The
physical association between CD19 and
1 family integrins suggests
that homoconjugate-induced integrin clustering may effect the
approximation, activation of the CD19-associated PTK by intermolecular
cross-phosphorylation, and subsequent tyrosine phosphorylation of the
CD19 receptor. The ability of the
1 family integrin receptors to
transmit a biochemical signal leading to activation of the CD19-linked
PTK pathway, which is coupled to other functionally important receptors
as well as several distal second messengers(29, 30) ,
provides an explanation for the pleiotropic responses generated through
adhesive contacts mediated by VLA-4 and VLA-5.
To study tyrosine phosphorylation of CD19 receptor, CD19
receptor was immunoprecipitated using the anti-CD19 mAb B43 from whole
cell lysates of B-lineage lymphoid cells following the engagement of
various B-cell surface receptors. In brief, following various
stimulation regimens, cells (5 10
cells/sample)
were solubilized in 0.5 ml of 1% Nonidet P-40 lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, plus 1
mM EDTA) containing 0.1 mM sodium orthovanadate and
0.1 mM sodium molybdate as phosphatase inhibitors, 10
µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride as protease inhibitors on ice for 30
min. Lysates were spun twice at 12,000
g for 15 min at
4 °C prior to immunoprecipitation. 100 µg of cell lysates were
immunoprecipitated with 3 µg of B43 antibody for 90 min at 4
°C. The immune complexes were collected with 50 µl of a 1:1
(v/v) slurry of protein A-Sepharose (Sigma) in Nonidet P-40 buffer. The
immunoprecipitates were washed four times with Nonidet P-40 buffer (150
mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% (v/v) Nonidet
P-40, 1 mM Na
VO
, 1 mM sodium
molybdate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 10 µg/ml aprotinin), resuspended in 50 µl of 2
SDS reducing sample buffer, and boiled. Samples were run on
10.5% SDS-polyacrylamide gel electrophoresis gels and subsequently
immunoblotted with either APT (5 µg/ml) or polyclonal anti-CD19 (5
µg/ml) antibodies, using a previously detailed immunoblotting
procedure(29) . To study tyrosine phosphorylation of Lyn kinase
and Lyn kinase-associated protein substrates, we immunoprecipitated Lyn
from Nonidet P-40 lysates of NALM-6 cells (200 µg of cell
lysates/sample) with a rabbit anti-Lyn antibody (2 µl/200 µg of
lysate) and subjected the Lyn immune complexes to APT Western blot
analysis, as described(29) .
I-Labeled protein A
(1 µCi/ml; specific activity = 30 µCi/µg; ICN
Biomedicals) was used to detect APT reactive or anti-CD19 reactive
proteins in the CD19 or Lyn immune complexes. After a 30-min incubation
with
I-labeled protein A, blots were washed three times
in rinsing buffer, dried, and autoradiographed using a XAR-5 film
(Kodak).
Figure 1: Expression of VLA-4 and VLA-5 on human B-cell precursors correlates with CD19 receptor expression. Fluorescence-activated cell sorter-correlated two-parameter displays of NALM-6 cells two-color-stained with B43/anti-CD19-PE and 15A8/anti-VLA-4-FITC or SAM-1/anti-VLA-5-FITC. Cells were incubated with the B43-PE/15A8-FITC pair or B43-PE/SAM-1-FITC pair for 30 min on ice, washed twice to remove unbound antibody, and analyzed on a FACStar Plus flow cytometer for CD19/VLA-4 and CD19/VLA-5 co-expression. Antibodies were used at 1 µg/ml final concentration, and all dilutions were in PBS. Controls included unstained cells as well as cells stained with control antibodies MsIgG-PE and MsIgG-FITC, which are not reactive with human cells.
Figure 2:
Engagement of the VLA-4 or VLA-5 adhesion
receptors induces tyrosine phosphorylation of multiple
electrophoretically distinct substrates in human pre-B and mature
B-cells. NALM-6 pre-B (shown in A) and AK mature B (shown in B) cells (5 10
cells/sample at a density
of 5
10
cells/ml) were stimulated with
homoconjugates (1 µg/ml) of anti-VLA-4, anti-VLA-5, or anti-CD19
monoclonal antibodies for the indicated periods to engage and dimerize
the respective target receptors. Controls included unstimulated cells (C, CON), cells treated with PBS, as well as cells stimulated
with Fab`
goat anti-human IgM (1 µg/ml). After the
indicated incubation periods, cells were lysed with SDS lysis buffer
and boiled. Equivalent amounts of protein were fractionated on 10.5%
polyacrylamide gels, transferred to Immobilon polyvinylidene difluoride
membranes, and immunoblotted with APT antibodies followed by incubation
with
I-protein A (1 µCi/ml) and autoradiography for
detection of phosphotyrosyl protein substrates. Molecular masses (in
kilodaltons) of the protein substrates were calculated from prestained
molecular size markers run as standards.
The CD19 receptor is
physically and functionally associated with Src family PTK, of which
Lyn kinase is the predominant member in human pre-B-cells and mature
B-lymphocytes(29) . The engagement of the CD19 receptor with a
high affinity anti-CD19 monoclonal antibody or its homoconjugate
rapidly activates the associated PTK and results in tyrosine
phosphorylation of CD19(29) . In whole cell lysates of
pre-B-cells as well as mature B-cells, a 95-kDa phosphoprotein
substrate showed rapid and strong tyrosine phosphorylation after
engagement of the 1 integrin family adhesion receptors VLA-4 or
VLA-5 (Fig. 2). Based on its molecular mass, we hypothesized
that the 95-kDa tyrosine phosphorylated protein could be the CD19
receptor, which has been shown to become tyrosine-phosphorylated
following engagement of various B-cell surface
receptors(29, 30) . To test this hypothesis, we
subjected CD19 immune complexes from the lysates of NALM-6 pre-B-cells,
which were stimulated with an anti-VLA-5
VLA-5 homoconjugate,
to APT Western blot analysis. As shown in Fig. 3A, left
panel, CD19 showed increased tyrosine phosphorylation within 1 min
after VLA-5 cross-linking. The abundance of the CD19 receptor protein,
as estimated by immunoblotting, did not change during the course of the
experiment (Fig. 3A, right panel), suggesting increased
enzymatic activity of CD19-associated PTK. Cross-linking of the VLA-4
receptor on NALM-6 cells with an anti-VLA-4xVLA-4 homoconjugate also
resulted in rapid tyrosine phosphorylation of the CD19 receptor (data
not shown). Similarly, CD19 receptor of AK mature B-cells showed
increased tyrosine phosphorylation within 5 min after VLA-5
cross-linking (Fig. 3B, left panel) or VLA-4
cross-linking (data not shown).
Figure 3:
Tyrosine phosphorylation of CD19 upon
cross-linking of VLA-4 or VLA-5 receptors. NALM-6 pre-B (shown in A) and AK mature B (shown in B) cells (10
10
/sample) were stimulated with an anti-VLA-5xVLA-5 mAb
homoconjugate (1 µg/ml) for the indicated periods to engage and
dimerize the respective target receptors. Subsequently, CD19 receptor
was immunoprecipitated from Nonidet P-40 lysates, and equal amounts
were subjected either to APT Western blot analysis (left panel of A and B) or to anti-CD19 Western blot
analysis (right panel of A), as described under
``Experimental Procedures.'' In parallel, LYN
immunoprecipitates from Nonidet P-40 lysates of AK mature B-cells
stimulated with anti-VLA-5xVLA-5 mAb homoconjugate (1 µg/mL) were
also examined by APT Western blot analysis for tyrosine phosphorylation
of LYN and LYN-associated kinase substrates (B, right panel). Arrowheads indicate the position of
CD19.
Lyn is the predominant PTK
associated with the CD19 receptor in B-lineage lymphoid
cells(29, 30) . To obtain preliminary information
about the possibility that the increased tyrosine phosphorylation of
CD19 after VLA-5 cross-linking could be due to increased PTK activity
of the CD19-associated Lyn kinase, Lyn immune complexes from Nonidet
P-40 lysates of AK cells were subjected to APT Western blot analysis.
As shown in Fig. 3B, right panel, engagement of the
VLA-5 receptor resulted in increased tyrosine phosphorylation of Lyn
protein within 5 min. Concomitant with enhanced tyrosine
phosphorylation of Lyn between 5 and 20 min after VLA-5 cross-linking,
CD19 became detectable in the Lyn immune complexes as a tyrosine
phosphorylated protein substrate (Fig. 3B, right
panel). These results indicated that activation of CD19-associated
PTK, such as Lyn, may be an integral component of the signal
transduction cascade triggered by 1 integrin family adhesion
receptors.
Figure 4:
Physical and functional associations
between 1 family surface adhesion receptors and CD19. A,
VLA-4 and VLA-5 receptors were immunoprecipitated from Nonidet P-40
lysates of unstimulated NALM-6 pre-B-cells (10
10
cells/sample), and the immune complexes were subjected to
anti-CD19 Western blot analysis, as described under ``Experimental
Procedures.'' No 1
Ab indicates mock-immunoprecipitated lysates to which no primary
antibody was added. B, AK mature B-cells (10
10
cells/sample) were stimulated with homoconjugates (1 µg/ml)
of anti-VLA-4 or anti-VLA-5 monoclonal antibodies for the indicated
periods and then lysed in Nonidet P-40 buffer. Equal amounts of lysate
(200 µg of protein/reaction mixture) were immunoprecipitated with
B43(anti-CD19) mAb (6 µg/200 µg of lysate protein), and immune
complex protein kinase assays were performed, as described under
``Experimental Procedures.'' Molecular masses (in
kilodaltons) of the phosphoprotein substrates were calculated from
prestained molecular size markers run as standards. Arrowheads indicate the position of CD19.
CD19 is associated with
Src family PTK, including Lyn, in resting pre-B and mature B-cells. To
explore the possibility that homoconjugate-induced integrin clustering
might activate the CD19-associated PTK, we first examined whether the
PTK activity in CD19 immunoprecipitates is increased after
cross-linking VLA-4 or VLA-5. For these experiments, mature B-cells
were stimulated with anti-VLA-4 VLA-4 or anti-VLA-5
VLA-5 homoconjugates, cell lysates were immunoprecipitated with
anti-CD19 mAb B43, and immune complex kinase assays were performed. As
shown in Fig. 4B, the kinase activity of the CD19
immune complexes was enhanced in a time-dependent manner following
stimulation with anti-VLA-4
VLA-4 or anti-VLA-5
VLA-5.
The immunoprecipitated CD19 molecule was also phosphorylated during the
kinase reactions (Fig. 4B). As reported previously, the
major phosphoprotein species associated with CD19 migrated in the
50-60-kDa region. Notably, a closely spaced 53/56-kDa doublet,
most likely corresponding to the autophosphorylated products of the two
alternatively spliced LYN transcripts, was induced by engagement of the
VLA-5 receptor but not the VLA-4 receptor.
After engagement of the
CD19 receptor Src family PTK, of which Lyn is the predominant member in
pre-B as well as mature B-cells, are activated and phosphorylated on
tyrosine residues(29) . Syk kinase is also activated after CD19
ligation and associates with the tyrosine-phosphorylated CD19 receptor. ()Previous studies have implicated Src family PTK in
integrin-linked signaling events(4) . Activated Src kinase has
been shown to associate with integrin-dependent cytoskeletal complexes
in platelets and fibroblasts(4) . Syk kinase has also been
shown to associate with integrin-dependent cytoskeletal structures and
to become activated in platelets after engagement of
integrins(4) . To examine the activation of the Lyn and Syk
kinases, both of which are intimately linked to the CD19-linked signal
transduction pathway, by signaling through
1 family integrin
receptors, VLA-4 and VLA-5, were cross-linked on the surface of NALM-6
pre-B-cells and AK mature B-cells by addition of the appropriate
homoconjugates, and the PTK activities of Lyn and Syk were estimated by
immune complex kinase assays. As shown in Fig. 5,
homoconjugate-induced dimerization of the VLA-5 receptor caused rapid
activation of Lyn and Syk kinases in both NALM-6 and AK cells. By
comparison, VLA-4 cross-linking did not significantly stimulate the Lyn
kinase in either cell line (Fig. 6). Unlike Lyn, Syk kinase was
activated following engagement of not only the VLA-5 receptor, but the
VLA-4 receptor as well (Fig. 6).
Figure 5:
Engagement of the VLA-5 adhesion receptor
stimulates Lyn and Syk kinases in human pre-B and mature B-cells. AK
mature B (depicted in A and B) and NALM-6 pre-B
(depicted in C and D) cells were stimulated with an
anti-VLA-5 VLA-5 mAb homoconjugate (1 µg/ml) for the times
indicated, pelleted, and lysed in Nonidet P-40 buffer. Lyn (depicted in A and C) or Syk (depicted in B and D) tyrosine kinases were immunoprecipitated from these
lysates, and immune complex protein kinase assays were performed, as
described under ``Experimental Procedures.'' In A-D, No 1
Ab indicates mock-immunoprecipitated lysates to which no primary
antibody was added.
Figure 6:
Engagement of the VLA-4 adhesion receptor
stimulates Syk kinase in human pre-B and mature B-cells. AK mature B
(depicted in A and B) and NALM-6 pre-B (depicted in C and D) cells were stimulated with an anti-VLA-4
VLA-4 mAb homoconjugate (1 µg/ml) for the times indicated,
pelleted, and lysed in Nonidet P-40 buffer. Lyn (depicted in A and C) or Syk (depicted in B and D)
tyrosine kinases were immunoprecipitated from these lysates, and immune
complex protein kinase assays were performed, as described under
``Experimental Procedures.'' In A-D, No
1
Ab indicates
mock-immunoprecipitated lysates to which no primary antibody was added. Arrowheads indicate the position of CD19 which is detected in
Syk immune complexes from lyates of anti-VLA-4
VLA-4 stimulated
cells.
CD19 is a physiologically important multifunctional surface
receptor, which is expressed throughout the B-cell ontogeny. It is
physically and functionally associated with Src family PTK to form
transmembrane receptor tyrosine kinases with ancillary
signal-transducing functions(29, 30) . Src family PTK
in these CD19-PTK complexes act as signal transducers and couple CD19
to downstream cytoplasmic signaling pathways(29, 30) .
CD19 has been shown to play an important coreceptor role in optimal
signal transduction through the B-cell antigen
receptor(29, 30) . More recent studies demonstrated
that the CD19 signals can complement and modulate the signals through
several other B-cell receptors as well, indicating that the co-receptor
role of CD19 is not restricted to the B-cell antigen
receptor(29, 30) . CD19 may serve as an ancillary
signal transduction unit that facilitates an effective communication
between different B-cell receptors for the generation of an optimal
B-cell immune response(29, 30) . Recent studies in
CD19-deficient mice demonstrated that CD19 is very important for
initial B-cell activation by T-cell-dependent antigens and development
of a memory cell compartment(39) . Furthermore, CD19 appears to
be required for antigen-driven expansion and maintenance of B-1 subset
in the B-cell compartment(39) . Other studies have indicated
that the membrane-associated CD19 receptor-Lyn enzyme complex serves as
a regulator of apoptosis in human B-cell precursors (37) as
well as mature B-cells(31) . The results presented herein
regarding the physical and functional association of the CD19 receptor
with 1 integrin family adhesion receptors extend these earlier
studies and offer the first insights into the role of the CD19
coreceptor and CD19-associated PTK in signal transduction through the
VLA-4 and VLA-5 receptors.
VLA-4 mediates cell-cell interactions in
the contexts of homing(3) , homotypic
aggregation(40, 41) , and cognate interactions between
B and T cells(42) . For example, VLA-4 functions as a homing
receptor on both mouse and human lymphocytes, facilitating their
attachment to Peyer's Patch high endothelial venule
cells(3, 41) . Ryan et al.(7, 8) reported that VLA-4 interaction with its
ligand VCAM-1 mediates the adhesion of human BCP to cultured bone
marrow stroma cells. Similarly, Dittel et al.(9) found that IL-7 responsive human BCP adhere to bone
marrow stroma via the VLA-4/VCAM-1 interactions, reminiscent of the
observations by Miyake et al.(43) that VLA-4 on mouse
pre-B-cells mediates their adhesion to bone marrow stroma. Leukemic BCP
have been shown to bind to purified fibronectin via VLA-4 and
VLA-5(12) . According to the findings of Bradstock et al.(11) the adhesion of leukemic BCP to bone marrow
fibroblasts occurs predominantly through interaction of VLA-4 with
VCAM-1. Furthermore, the ability of leukemic BCP to migrate into stroma
depends on 1 integrin expression(10, 13) .
Previous studies demonstrated that treatment of leukemic BCP with
antibodies to
1 common subunit or a mixture of anti-VLA-4 and
anti-VLA-5 antibodies abrogates their ability to migrate into bone
marrow stroma(13, 44) . Notably, infusion of a
blocking antibody to the
4 subunit of the VLA-4 receptor in
primates has been shown to cause a 200-fold increase in circulating
hematopoietic progenitor cells, implicating the VLA-4 integrin in the
regulation of the in vivo migration and trafficking of
hematopoietic progenitor cells(45) .
Here, we presented
experimental evidence that CD19 receptor is physically associated with
the 1 family integrin receptors on human BCP as well as mature
B-lymphocytes and engagement of the
1 family integrin receptors
leads to rapid activation of the CD19-associated PTK and results in
hyperphosphorylation of CD19 on tyrosine residues. The physical
association between CD19 and
1 family integrins prompts the
hypothesis that homoconjugate-induced integrin clustering may effect
the approximation and, by intermolecular cross-phosphorylation,
activation of the CD19-associated PTK and subsequent tyrosine
phosphorylation of the CD19 receptor. The ability of the
1 family
integrin receptors to transmit a biochemical signal leading to
activation of the CD19-linked PTK pathway, which is coupled to other
functionally important receptors as well as several distal second
messengers(29, 30) , provides an explanation for the
pleiotropic responses generated through adhesive contacts mediated by
VLA-4 and VLA-5.