From the
Phospholipase C
The B cell antigen receptor
(BCR)
B
cells express two isozymes of PLC,
PLC
The fact that p72
Three observations
further support our model of the BCR-initiated signaling cascade.
First, the peak of p72
We cannot, however, state unequivocally that this
interaction is direct rather than mediated via an as yet unidentified
intermediary protein. If involved, this protein is unlikely to be one
of the other receptor-associated PTKs, as the lack of tyrosine
phosphoproteins with molecular weights consistent with src family PTKs
in PLC
The
relative contributions of PLC
The precise mechanism of p72
We thank Dr. Tony Pawson for the generous gift of the
PLC
-catalyzed inositol phospholipid hydrolysis,
a critical step in B cell antigen receptor signaling leading to second
messenger generation and proliferation, depends upon tyrosine kinase
activation. The B cell antigen receptor-associated tyrosine kinases
p53/56
, p59
,
p55
, and p72
are assumed
to participate in receptor-initiated signaling. It is unknown, however,
which of these kinases is involved in the tyrosine phosphorylation and
resulting activation of phospholipase C
in response to antigen
receptor cross-linking. We have used a fusion protein containing the
tandem src homology-2 (SH2) domains of phospholipase C
1
(PLC
1) to identify B cell kinases which associate with PLC
1.
Using an in vitro kinase assay, we demonstrate SH2-dependent
association of tyrosine kinase activity from anti-µ-stimulated B
cells. The PLC
1 SH2 domains associate with a prominent
70-72-kDa tyrosine phosphoprotein from anti-µ-stimulated, but
not resting, B cells. Immunoblotting and secondary immunoprecipitation
studies definitively identify this protein as
p72
. These results imply a physical interaction
between PLC
1 and p72
in antigen
receptor-stimulated B cells. This conclusion is confirmed by our
ability to co-immunoprecipitate p72
and
PLC
1 from lysates of anti-µ-stimulated B cells. These results
implicate p72
in the activation of phospholipase
C
1 during B cell antigen receptor signaling.
(
)
-associated src family protein tyrosine
kinases (PTKs) p53/56
,
p59
, and p55
, and the
non-src family PTK p72
, become activated in B
cells within seconds following BCR
cross-linking
(1, 2, 3) . It is assumed, although
not formally proven, that each of these PTKs plays an important role in
transducing BCR-initiated signals for B cell activation. Studies using
PTK inhibitors have shown that tyrosine kinase activity is a
requirement for phosphatidylinositol 4,5-bisphosphate hydrolysis in B
cells following BCR cross-linking (4-6). This critical step in
the pathway generates the second messengers diacylglycerol and inositol
1,4,5-trisphosphate, which are responsible for protein kinase C
activation and a component of the increase in levels of intracellular
calcium, respectively
(7, 8, 9) . Hydrolysis of
phosphatidylinositol 4,5-bisphosphate is catalyzed by
phosphatidylinositol-specific phospholipase C (PLC)
(10) .
1 and
2
(11) . Both
isozymes are phosphorylated on tyrosine following BCR
stimulation
(4, 11, 12, 13) . Tyrosine
phosphorylation increases the activity of PLC
1, and may be the
principal means of its activation in
vivo(14, 15) . In B cells, it has been assumed,
although not demonstrated, that this phosphorylation is mediated by one
or more of the receptor-associated PTKs which are activated immediately
following BCR cross-linking. Recent studies in a
p72
-deficient avian B cell line suggest that
p72
is required for BCR-coupled tyrosine
phosphorylation of PLC
2 and inositol 1,4,5-trisphosphate
production (16). However, these cells may not accurately model normal B
cells as they also lacked expression of the src family PTKs
p55
and p59
. Studies to
determine PLC
-PTK associations at the molecular level are
necessary to establish the direct involvement of particular PTK(s) in
this important BCR signaling pathway.
1 activation by the
platelet-derived growth factor and epidermal growth factor receptor
PTKs is known to depend upon an interaction between the SH2 domains of
PLC
1 and phosphorylated tyrosine residues of the activated
receptor
PTK
(17, 18, 19, 20, 21) .
Extending this model to the BCR, which is noncovalently coupled to
several cytoplasmic PTKs, PLC
1 interaction with the PTK(s)
involved in its regulation is predicted to be dependent upon a physical
interaction between one or both of the src homology-2 (SH2) domains of
PLC
1 and phosphorylated tyrosine residue(s) on the BCR-activated
kinase. Exploiting this model of PLC
-PTK interaction, we have used
a glutathione S-transferase (GST) fusion protein containing
the two tandem SH2 domains of PLC
1 to identify PTK(s) which
associate with PLC
1 following B cell activation through the BCR.
Mice
Balb/c mice were purchased from The Jackson
Laboratory (Bar Harbor, ME) and maintained in our animal colony.
Preparation of Splenic B Cells
Mice were killed by
cervical dislocation and spleens were removed aseptically. Cell
suspensions were made by grinding spleens between the frosted ends of
two glass microscope slides. The suspension was depleted of T cells by
anti-Thy-1.2 antibody (HO-13-4) and lysis in rabbit complement
(Pel-Freez Biologicals, Rogers, AR); erythrocytes were removed by
osmotic shock. Finally, B cells were purified by centrifuging over a
step gradient of 50/75% Percoll (Pharmacia, Piscataway, NJ). This
preparation is routinely 85-95% B cells (IgM B220
).
Purification of Bacterial GST Fusion Proteins
500
ml of LB bacterial growth medium containing ampicillin was inoculated
1:10 with an overnight culture of Escherichia coli DH5F`
harboring either the pGEXKG vector
(22) (a derivative of the
pGEX2T vector, Pharmacia, Piscataway, NJ) encoding only GST, or the
pGEXKG-PLC plasmid encoding GST and amino acids 549-755 of bovine
PLC
1, which comprise the tandem SH2 domains of PLC
1 (a gift
of Dr. Tony Pawson, Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto). Following 1 h of shaking at 37 °C
(A
= 0.4), fusion protein production was
induced by addition of
isopropyl-
-D-thio-galactopyranoside (Stratagene Cloning
Systems, La Jolla, CA) to 0.5 mM. Cells were grown an
additional 3 h at 37 °C with shaking, and then harvested by
centrifugation. Cell pellets were lysed in 10 ml of phosphate-buffered
saline, 1% Triton X-100 with 0.2 mg/ml lysozyme, 2 mM
phenylmethylsulfonyl fluoride (PMSF), and 50 mM EDTA.
Following sonication to reduce viscosity, cell debris was pelleted by
centrifugation in a Sorvall SS34 rotor at 15,000 rpm for 20 min.
Glycerol was added to the supernatant to a concentration of 20%; the
supernatant was aliquoted and stored at -80 °C. Fusion
proteins were purified from supernatants by affinity chromatography on
glutathione-Sepharose 4B (Pharmacia, Piscataway, NJ). The amounts of
GST and PLC
1 SH2-GST bacterial lysate used to coat the Sepharose
beads were adjusted so that similar amounts of the two proteins would
be present in precipitation experiments. Fusion protein-coated beads
were washed four times in phosphate-buffered saline, 2% Triton X-100
with 2 mM PMSF and 50 mM EDTA, and then twice in 0.5%
Nonidet P-40 (Calbiochem) kinase assay lysis buffer (0.5% Nonidet P-40,
150 mM NaCl, 10 mM Tris, pH 7.3, 0.4 mM
EDTA, 10.8 µg/ml aprotinin, 1.5 µg/ml each of leupeptin,
pepstatin A, chymostatin, and antipain, 2 mM PMSF, 2
mM sodium orthovanadate, 10 mM NaF). Washed, fusion
protein-coated glutathione-Sepharose beads were used to precipitate B
cell lysates as described below.
Preparation of B Cell Lysates
Following
purification, B cells were resuspended in Hank's balanced salt
solution without fetal calf serum at a density of 2 10
cells/ml. Following a 15-min equilibration in a 37 °C water
bath, cells were stimulated for the time points indicated in the
figures using 30 µg/ml goat anti-mouse µ heavy chain antibodies
(Jackson Immunoresearch, West Grove, PA) or 20 µg/ml F(ab`)
fragments of goat anti-mouse µ heavy chain antibodies
(Jackson Immunoresearch, West Grove, PA) as a polyclonal activator.
Cells were pelleted and lysed with 0.5% Nonidet P-40 kinase assay lysis
buffer on ice for 10 min. Lysates were cleared by microcentrifugation
at 14,000 rpm for 10 min at 4 °C. Cleared lysates were precipitated
with fusion protein-coated glutathione-Sepharose beads (30-40
µl of a 50% slurry per sample) on a rotator overnight at 4 °C.
Lysate-adsorbed, fusion protein-coated beads were washed four times
with ice-cold 0.5% Nonidet P-40 kinase assay lysis buffer, boiled in 2
reducing sample buffer (125 mM Tris pH 6.8, 20% (w/v)
glycerol, 10% (v/v) 2-mercaptoethanol, 4.6% SDS), and then eluted
proteins were fractionated using SDS-7.5% polyacrylamide gel
electrophoresis (PAGE). Fractionated proteins were electroblotted onto
Hybond-ECL nitrocellulose filters (Amersham Corp.) for use in
immunoblot analysis. Alternatively, lysate-adsorbed fusion
protein-coated beads were washed as stated above and then subjected to
the in vitro kinase assay or the thrombin cleavage procedure.
In Vitro Kinase Assay
The in vitro kinase
reaction was performed essentially as in Ref. 23. Washed
lysate-adsorbed fusion protein-coated beads (40 µl of a 50% slurry
per sample) were washed once with ice-cold kinase assay wash buffer
(150 mM NaCl, 10 mM Tris-HCl, pH 7.3, 2 mM
PMSF, 2 mM sodium orthovanadate), once with kinase assay
buffer (10 mM MgCl, 10 mM HEPES, pH 7.0,
2 mM PMSF, 2 mM sodium orthovanadate), and then
resuspended in 40 µl of kinase assay buffer containing 10 µCi
of [
-
P]ATP (6,000 Ci/mmol, 10 mCi/ml;
DuPont NEN). Beads were incubated for 10 min at 30 °C, and then
washed three times with ice-cold 0.5% Nonidet P-40 kinase assay lysis
buffer. Proteins were eluted from the beads by boiling in 2
reducing sample buffer, and then fractionated using SDS-10% PAGE. Gels
were fixed for 2 h in 20% methanol, 10% acetic acid, dried, and then
subjected to autoradiography. In one experiment, the gel was fixed,
treated with 1 N KOH for 2 h at 55 °C (to selectively
hydrolyze phosphoserine and phosphothreonine), neutralized in fixative,
dried, and then subjected to autoradiography
(24) .
Immunoblot Analysis
Membranes were incubated
overnight in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris,
pH 8, 137 mM NaCl, 0.05% Tween 20) containing 2% bovine serum
albumin to block nonspecific binding. To detect tyrosine-phosphorylated
substrates, blocked membranes were incubated for 90 min with the
monoclonal antibody 4G10 (Upstate Biotechnology Inc., Lake Placid, NY),
followed by horseradish peroxidase-conjugated sheep anti-mouse Ig
secondary antibody (Amersham) for 90 min. To detect
p72, membranes were incubated with rabbit IgG
directed against a bacterial fusion protein containing the
COOH-terminal SH2 domain of p72
(a gift of Drs.
Ellen Pure and Mark Forman, The Wistar Institute, Philadelphia, PA) for
90 min, washed, and then incubated with horseradish
peroxidase-conjugated F(ab`)
fragments of donkey
anti-rabbit Ig antibodies (Jackson Immunoresearch). PLC
1 was
detected using a mixture of monoclonal anti-PLC
1 antibodies
(Upstate Biotechnology Inc.) followed by the horseradish
peroxidase-conjugated sheep anti-mouse Ig secondary antibody listed
above. p59
, p55
, and
p53/56
were detected in a similar manner using
antibodies given to us by Dr. Joseph Bolen (Bristol-Myers Squibb,
Princeton, NJ). Following three 15-min washes in TBST, membranes were
developed using the enhanced chemiluminescence (ECL) system (Amersham)
followed by autoradiography. Membranes were stripped of bound
antibodies by incubating in stripping buffer (62.5 mM
Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS) for 30 min
in a 50 °C shaking water bath, followed by three 15-min washes in
TBST.
Thrombin Cleavage and Immunoprecipitation of
p72
The thrombin cleavage procedure was
modified from Ref. 22. Washed lysate-adsorbed fusion protein-coated
beads (40 µl of a 50% slurry per sample) were washed once with
thrombin cleavage buffer (150 mM NaCl, 50 mM Tris, pH
8, 2.5 mM CaCl, 0.1% 2-mercaptoethanol, 2
mM sodium orthovanadate, 100 ng/ml okadaic acid), and then
incubated in 1 ml/sample of thrombin cleavage buffer containing 12
µg of human thrombin (Sigma) for 20 min at ambient temperature.
Samples were placed on ice, and PMSF, aprotinin, leupeptin, pepstatin
A, chymostatin, and antipain were added to the final concentrations
listed previously. Samples were centrifuged to pellet the
glutathione-Sepharose beads. The supernatant was removed and incubated
with 5 µl/sample of anti-p72
rabbit
antiserum (Upstate Biotechnology Inc.) overnight at 4 °C, followed
by 50 µl of a 50% slurry of protein A-Sepharose (Sigma) for 3 h at
4 °C. Immunoprecipitations were washed 4 times in 0.5% Nonidet P-40
immunoprecipitate wash buffer (10 mM Tris, pH 8, 0.5% Nonidet
P-40, 0.5% SDS) with protease and phosphatase inhibitors added to the
final concentrations listed previously. Immunoprecipitated proteins
were eluted by boiling in 2
reducing sample buffer. SDS-PAGE,
electroblotting, and anti-phosphotyrosine immunoblot analysis were
performed as stated above.
Immunoprecipitation of PLC
2 1
10
B cells were stimulated as before and lysed in 1%
Nonidet P-40 lysis buffer (1% Nonidet P-40, 10 mM Tris, pH 8)
with protease and phosphatase inhibitors added. Lysates were precleared
by rotating for 1 h at 4 °C with 30 µl of a 50% slurry of
protein A-Sepharose (Sigma). Following centrifugation to pellet the
protein A-Sepharose beads, lysates were incubated with 6 µg of a
mixture of monoclonal anti-PLC
1 antibodies (Upstate Biotechnology
Inc.) on ice for 30 min. 30 µl of a 50% slurry of protein
A-Sepharose was added and lysates were rotated at 4 °C overnight.
Immunoprecipitates were washed 4 times with 0.5% Nonidet P-40
immunoprecipitate wash buffer and then eluted by boiling in 2
reducing sample buffer. SDS-PAGE, electroblotting, and
anti-p72
and anti-PLC
1 immunoblot analysis
were performed as stated above.
RESULTS
The PLC
To determine
whether the PLC1 SH2 Domain Fusion Protein Precipitates
Tyrosine Kinase Activity from B Cell Lysates
1 SH2 domains associate with PTKs from
BCR-stimulated B cells, we used the PLC
1 SH2-GST fusion protein to
precipitate proteins from lysates of anti-receptor (anti-µ heavy
chain) antibody-stimulated splenic B cells. Precipitated proteins were
then assayed for kinase activity in an in vitro kinase assay
in the presence of [
-
P]ATP. Evidence for
SH2-associated tyrosine kinase activity was determined by the presence
of phosphoprotein bands in the SDS-PAGE gel after treatment with
potassium hydroxide to eliminate phosphoserine and phosphothreonine
(not shown). As shown in Fig. 1, demonstrable kinase activity was
observed in lanes containing PLC
1 SH2-GST fusion
protein-precipitated material from B cells stimulated for 30 and 60 s
with anti-µ antibody. Several substrates are present in both lanes,
presumably representing autophosphorylated kinase(s) and/or B cell
proteins co-precipitated with these kinases as well as proteins
contained within the bacterial lysates from which the fusion protein
was isolated. Importantly, no kinase activity was precipitated by the
GST protein alone (Fig. 1), or in the absence of B cell lysate
(not shown), indicating that the kinase activity present in these
precipitates is derived from the B cell lysates, and that its
association with the fusion protein is dependent upon the presence of
the PLC
1 SH2 domains. The PLC
1 SH2 Domain Fusion Protein Precipitates a 70-72-kDa
Tyrosine-phosphorylated Protein from Lysates of Anti-µ-stimulated B
Cells-To identify the PTK(s) that associate with the PLC
1
SH2-GST fusion protein, we took advantage of the fact that the
BCR-associated PTKs are themselves activated by tyrosine
phosphorylation. Exploiting this characteristic, we determined whether
the PLC
1 SH2 domains bind to tyrosine-phosphorylated proteins
following BCR signaling in vivo. We performed immunoblot
analysis with the monoclonal anti-phosphotyrosine antibody 4G10 on
proteins precipitated from B cell lysates with the PLC
1 SH2 fusion
protein (Fig. 2). A prominent tyrosine-phosphorylated protein
with an approximate molecular mass of 70-72 kDa was observed in
lysates of anti-µ-stimulated, but not resting, B cells. The
intensity of this protein band is at its highest level in precipitates
from B cells that have been stimulated with anti-µ antibodies for 1
min. By 2 min post-stimulation, the intensity of this band has
decreased. Other, higher molecular weight tyrosine-phosphorylated
proteins are also precipitated from anti-µ-stimulated B cell
lysates by the PLC
1 SH2-GST fusion protein. The intensity of these
higher molecular mass bands, relative to the 70-72 kDa band,
suggest that they represent tyrosine-phosphorylated proteins which have
a lower affinity for the PLC
1 SH2-GST fusion protein.
Alternatively, they may represent tyrosine-phosphorylated proteins
which co-associate with the 70-72-kDa phosphoprotein, rather than
bind directly to the SH2 domains of PLC
1. The GST-only fusion
protein did not precipitate any tyrosine-phosphorylated proteins from
the B cell lysates, and PLC
1 SH2-GST fusion protein in the absence
of B cell lysate did not contain detectable tyrosine-phosphorylated
proteins. Whole cell lysates from unstimulated and
anti-µ-stimulated B cells were included on the blot as a positive
control, to show that protein tyrosine phosphorylation was induced upon
BCR stimulation. The 70-72-kDa Tyrosine-phosphorylated Protein Precipitated by the
PLC
1 SH2 Domain Fusion Protein from Lysates of
Anti-µ-stimulated B Cells Reacts with
Anti-p72
Antibodies on
Immunoblots-Our studies to this point indicated association
of the PLC
1 SH2 domains with both tyrosine kinase activity and an
inducible tyrosine phosphoprotein of 70-72 kDa. Because one
possible mechanism of the physical interaction of PLC
1 with its
activating tyrosine kinase is between the SH2 domains of PLC
1 and
a phosphotyrosine residue on the activated kinase, we considered that
the 70-72-kDa tyrosine phosphoprotein represented the PTK which
activates PLC
1. In this regard, among the PTKs known to be
activated as a consequence of anti-µ-induced BCR cross-linking,
p72
was an obvious candidate for the PLC
1
SH2-associated kinase. To test this possibility, we used the PLC
1
SH2-GST fusion protein to precipitate proteins from B cell lysates as
before, and then performed anti-p72
immunoblot
analysis on these proteins (Fig. 3A). The
anti-p72
antibody detected a 72-kDa protein in
the PLC
1 SH2-GST-precipitated lysates from B cells stimulated with
anti-µ for 2 min. A number of other proteins were detected, but
they are also present in the fusion protein-only (no B cell lysate)
precipitate. Importantly, the 72-kDa band is not present in the fusion
protein-only (no B cell lysate) lane, indicating that it is of B cell,
and not bacterial, origin. Stripping and reprobing this blot with
anti-phosphotyrosine antibodies allowed us to determine that the
p72
band overlays exactly with the 72-kDa
tyrosine phosphoprotein (not shown). Finally, immunoblot analysis using
antibodies to p55
,
p53/56
, and p59
failed to
detect any PLC
1 SH2-specific binding of these PTKs, indicating
specificity of the PLC
1 SH2 domains for p72
(not shown).
Figure 1:
In vitro kinase assay of proteins precipitated from B cell lysates by the
SH2 domains of PLC1. Splenic B cells (2
10
/lane) were stimulated with 30 µg/ml goat anti-mouse
µ antibodies for 30 or 60 s, and then lysed in 0.5% Nonidet P-40
kinase assay lysis buffer. Lysates were precipitated with fusion
protein-coated Sepharose beads: either a fusion protein comprised of
GST alone (GST lanes) or of GST and the tandem SH2 domains of
PLC
1 (SH2-GST lanes). Following precipitation, the beads
were washed, and an in vitro kinase assay was performed in the
presence of [
-
P]ATP. Proteins were eluted
from the beads with 2
reducing sample buffer, fractionated by
SDS-10% PAGE, and then detected by autoradiography. The apparent sizes
(in kilodaltons) and positions of prestained molecular weight standards
are indicated on the right; time points (in seconds) of
anti-µ stimulation are indicated at the top. Evidence for
SH2-associated tyrosine kinase activity was determined by the presence
of phosphoprotein bands in the SDS-PAGE gel after treatment with
potassium hydroxide to eliminate phosphoserine and phosphothreonine
(not shown).
Figure 2:
Detection of a 70-72-kDa
tyrosine-phosphorylated protein in PLC1 SH2 domain precipitates
from lysates of anti-µ-stimulated B cells. Murine splenic B cells
were stimulated, lysed, and precipitated with GST fusion proteins as in
Fig. 1 (GST-SH2-SH2 lanes contain precipitations done with the
PLC
1 SH2 domain-containing fusion protein.) Precipitated proteins
were eluted from the beads with 2
reducing sample buffer,
fractionated by SDS-7.5% PAGE, and electroblotted onto nitrocellulose
filters. Filters were probed with the anti-phosphotyrosine monoclonal
antibody 4G10 followed by horseradish peroxidase-conjugated sheep
anti-mouse Ig antibodies. Detection was by ECL followed by
autoradiography. Whole cell lysate lanes contain lysates from 5
10
unstimulated or anti-µ-stimulated B cells. Time
points (in minutes) of anti-µ stimulation are indicated at the
top of the figure; n.l., fusion protein-coated beads
only (no lysate). The apparent sizes (in kilodaltons) and positions of
prestained molecular weight standards are indicated on the
right.
Figure 3:
Detection of p72 in PLC1 SH2 domain
precipitates from lysates of anti-µ-stimulated B cells. A,
PLC
1 GST-SH2 fusion protein precipitations from
anti-µ-stimulated B cell lysates followed by SDS-7.5% PAGE and
electroblotting were done as described in the legend to Fig. 2. Blots
were probed with rabbit anti-p72 antibodies followed by horseradish
peroxidase-conjugated F(ab`)
fragments of donkey
anti-rabbit Ig antibodies. Detection was by ECL followed by
autoradiography. p72 is indicated by an arrow. B,
murine splenic B cells were stimulated, lysed, and precipitated with
the PLC
1 GST-SH2 fusion protein-coated Sepharose beads as
described in the legend to Fig. 1. Following several washes, the beads
were treated with thrombin, which cleaves the fusion protein between
the GST domain and the SH2 domains. The supernatant from this
treatment, which contained the PLC
1 SH2 domains and any proteins
bound to them, was then immunoprecipitated with anti-p72 antibodies and
protein A-Sepharose beads. Precipitated proteins were eluted from the
beads with 2
reducing sample buffer, fractionated by SDS-7.5%
PAGE, and electroblotted onto a nitrocellulose filter. The filter was
probed with the anti-phosphotyrosine monoclonal antibody 4G10 followed
by horseradish peroxidase-conjugated sheep anti-mouse Ig antibodies.
Detection was by ECL followed by autoradiography. Time points (in
minutes) of anti-µ stimulation are indicated at the top of
the figure; n.l., fusion protein-coated beads only (no
lysate). The apparent sizes (in kilodaltons) and positions of
prestained molecular weight standards are indicated on the right of both panels; p72 is indicated by an
arrow.
p72
We also evaluated whether we could
immunoprecipitate p72Can be Immunoprecipitated
from B Cell Proteins Which Bind to the PLC
1 SH2
Domains
from the B cell proteins
which bind to the PLC
1 SH2-GST fusion protein. We took advantage
of a proteolytic cleavage site in the PLC
1 SH2-GST fusion protein.
Thrombin cleaves between the GST domain and the amino-terminal SH2
domain of the PLC
1 SH2-GST fusion protein, releasing the SH2
domains into the supernatant, along with any bound proteins. The GST
domains remain bound to the glutathione-Sepharose beads, and can be
removed. We immunoprecipitated p72
from this
supernatant, and subjected the immunoprecipitated proteins to
anti-phosphotyrosine immunoblot analysis with 4G10
(Fig. 3B). We observed a tyrosine-phosphorylated protein
of approximately 72 kDa that is immunoprecipitated by
anti-p72
antiserum from PLC
1 SH2 domain
bound proteins. This tyrosine-phosphorylated protein is
immunoprecipitated by anti-p72
antibodies from
lysates of B cells stimulated with anti-µ antibodies for 1 min,
indicating that the major protein that associates with the SH2 domains
of PLC
1 in this system is the PTK p72
.
p72
These data indicate that
p72Can Be
Co-immunoprecipitated with PLC
1 from Lysates of
Anti-µ-stimulated B Cells
from anti-µ-stimulated B cells is able
to bind to the PLC
1 SH2-GST fusion protein. In order to determine
whether the interaction between p72
from
anti-µ-stimulated B cells and the PLC
1 SH2-GST fusion protein
predicts what occurs in vivo during B cell antigen receptor
signal transduction, we wished to establish whether
p72
is associated with PLC
1 in lysates of
antigen receptor-stimulated B cells. PLC
1 was immunoprecipitated
from lysates of anti-µ-stimulated B cells, and
anti-p72
immunoblot analysis was performed on
the immunoprecipitated proteins (Fig. 4).
Anti-p72
immunoblot analysis of anti-PLC
1
immunoprecipitates shows that p72
is associated
with PLC
1 in lysates of anti-µ-stimulated B cells
(Fig. 4, right panel, lane 3). p72
is not present in lanes containing proteins pre-cleared by
protein A-Sepharose beads (Fig. 4, right panel, lane 2).
This blot was stripped and re-probed with anti-PLC
1 antibodies to
show the efficiency of the anti-PLC
1 immunoprecipitation in this
experiment (Fig. 4, left panel). In summary, the ability
to co-immunoprecipitate p72
and PLC
1 from
lysates of BCR-stimulated B cells supports the physiological relevance
of the p72
-PLC
1 association defined by
these studies.
Figure 4:
Detection of p72 in anti-PLC1
immunoprecipitates from lysates of anti-µ-stimulated B cells.
Lysates from 2
10
resting or anti-µ-stimulated
B cells were precleared by incubation with protein A-Sepharose
(pre-clear lanes) and then incubated with 6 µg of
anti-PLC
1 monoclonal antibodies and protein A-Sepharose
(anti-PLC
1 immunoppt. lanes). Precipitated proteins were
eluted by boiling in 2
reducing sample buffer; SDS-10% PAGE and
electroblotting were done as described in the legend to Fig. 3. Filters
were probed with anti-p72 antibodies (right panel) as
described in the legend to Fig. 3, stripped, and re-probed with
anti-PLC
1 monoclonal antibodies followed by horseradish
peroxidase-conjugated F(ab`)
fragments of donkey anti-mouse
Ig antibodies (left panel). Detection was by ECL followed by
autoradiography. Whole cell lysates from 5
10
anti-µ-stimulated B cells is shown (whole lysate
lanes) as a control. The apparent sizes (in kilodaltons) and
positions of prestained molecular weight standards are indicated on the
right; p72 is indicated by an
arrow.
DISCUSSION
We have shown that a fusion protein containing the SH2
domains of PLC1 can precipitate tyrosine kinase activity from
lysates of BCR-stimulated murine splenic B cells. We have also shown
that the PLC
1 SH2 domains precipitate a 72-kDa tyrosine
phosphoprotein which is identified as p72
based
upon reactivity with anti-p72
antibodies both on
immunoblots and in secondary immunoprecipitation experiments. These
results imply that p72
is the tyrosine kinase
responsible for PLC
1 phosphorylation following BCR stimulation
in vivo. Consistent with this possibility is our demonstration
of p72
-PLC
1 association in vivo.
Specifically, we have demonstrated co-immunoprecipitation of
p72
and PLC
1 from lysates of
anti-µ-stimulated B cells. Reciprocal experiments in which
immunoprecipitates of p72
were tested for
PLC
1 co-immunoprecipitation were negative. We believe that this
inconsistency with the anti-PLC
1 immunoprecipitations shown here
is due to the amount of p72
immunoprecipitated
under these conditions. In our hands, the anti-p72
antibody immunoprecipitates a relatively small proportion of the
total p72
protein present in B cells. Because
PLC
1 may be only one of the substrates of p72
in B cells, only a fraction of the p72
protein would be found associated with PLC
1. Therefore, the
relative inefficiency of the anti-p72
antibody
in immunoprecipitation experiments makes this approach to the
experiment less efficient than testing anti-PLC
1
immunoprecipitates for the presence of p72
.
can interact with the SH2
domains of PLC
1 in this in vitro system and is found
complexed with PLC
1 in lysates of anti-µ-stimulated B cells
suggests that p72
interacts with PLC
1
in vivo during BCR-initiated signal transduction. This leads
us to postulate that p72
, and not one of the src
family PTKs, is the PTK which directly activates PLC
1 by
phosphorylating it in B cells following BCR cross-linking. Thus, we
conclude that PLC
1 interacts with activated p72
during BCR-initiated signal transduction, and that this
interaction involves PLC
1 SH2 domains and activation-associated
phosphotyrosine residues of p72
. This
interaction then leads to the phosphorylation and activation of
PLC
1, and subsequent signaling events.
association with the
PLC
1 SH2 domains (at 1 min post-BCR stimulation, Fig. 2)
occurs before the peak of PLC
1 tyrosine phosphorylation (2-5
min post-BCR stimulation).
(
)
Second, a recent
study demonstrated that a p72
-deficient avian B
cell line could not hydrolyze inositol phospholipids in response to
anti-IgM stimulation. These cells were also unable to phosphorylate
PLC
2 in response to anti-IgM stimulation. Transfection of a
kinase-deficient form of p72
was unable to
restore anti-IgM-stimulated inositol phospholipid hydrolysis,
indicating that this signaling event depended directly upon the kinase
activity of p72
(16) . Third, another
study showed that aggregation of a CD16/syk, but not a CD16/fyn,
chimeric cell surface molecule could induce tyrosine phosphorylation of
PLC
1 in TCR
Jurkat T cells
(25) . Our
results extend these studies by showing that this interaction can be
mediated via the PLC
1 SH2 domains and occurs in the more
physiological context of non-transformed B cells in which the levels of
BCR-associated PTKs are under normal cellular control. Taken together,
these observations substantiate our conclusion that
p72
is directly responsible for the
phosphorylation of PLC
and the initiation of phosphatidylinositol
4,5-bisphosphate hydrolysis in response to stimulation through BCR
in vivo.
1 SH2-GST precipitations shows (Fig. 2). Also, the
inability of our immunoblot studies to show p59
,
p53/56
, or p55
association with the PLC
1 SH2 domains argues against this
possibility (not shown). The exact nature of the PLC
1 SH2 domain
interaction with p72
with regards to the
possibility of intermediary proteins remains to be evaluated.
1 and PLC
2 to signaling through
BCR is not known. Other groups have reported that PLC
2 is the
predominant isoform expressed in B
cells
(11, 13, 26) . However, we observe equal or
higher expression of PLC
1 in primary murine B cells by immunoblot
analysis.
(
)
Because both isoforms are
phosphorylated on tyrosine following BCR cross-linking, both are
presumed to participate in the signaling
pathway
(4, 11, 12, 13) . However, the
SH2 domains of PLC
1 and PLC
2 are not identical. The
NH
- and COOH-terminal SH2 domains are 70 and 76%
homologous, respectively, between the two isoforms
(27) . This
leaves open the possibility that PLC
2 may associate with
p72
but at a different affinity than PLC
1
does, or that it may associate with a different kinase altogether. This
could be another example of redundancy in the BCR signaling pathway, or
it could be a means of regulating the response to antigen receptor
stimulation, depending on the levels of the different PTKs and PLC
isoforms expressed.
activation, or the activation of any of the BCR-associated PTKs,
is currently unknown, although there is some evidence that the src
family PTKs (p53/56
in particular) may be able
to phosphorylate and activate p72
(28, 29).
Whether all of the BCR-associated PTKs are activated simultaneously by
clustering of BCR during cross-linking, or whether they are activated
in some sort of BCR-initiated sequence is an area under study.
1 SH2 domain fusion protein construct, Dr. Bill Dougall for his
advice on fusion protein expression, and Drs. Ellen Pure and Mark
Forman for providing us with the anti-p72
antiserum which we used for the immunoblot analysis. Our
gratitude also to Drs. Marian Birkeland and Mark Forman for their
critical reading of this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.