MOLECULAR AND CELLULAR BIOLOGY, Apr. 1996, p. 1305–1315
Copyright ? 1996, American Society for Microbiology
Vol. 16, No. 4
Phospholipase C-?1 Interacts with Conserved Phosphotyrosyl Residues
in the Linker Region of Syk and Is a Substrate for Syk
CHE-LEUNG LAW,* KAREN A. CHANDRAN, SVETLANA P. SIDORENKO,†
AND EDWARD A. CLARK
Department of Microbiology, University of Washington,
Seattle, Washington 98195
Received 24 July 1995/Returned for modification 27 September 1995/Accepted 3 January 1996
Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-? (PLC-?)
isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)]
domain of PLC-?1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells
phosphorylated PLC-?1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same
B cells phosphorylated PLC-?1 only on Tyr-771. The ability of Syk to phosphorylate PLC-?1 required antigen
receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a
tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-?1 SH2(C) in
vitro, and induced tyrosyl phosphorylation of endogenous PLC-?1 in vivo. Substitution of Tyr-525 and Tyr-526
at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding
of this variant chimera to PLC-?1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-?1
in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect
the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-?1 SH(C) and
completely eliminated its ability to induce tyrosyl phosphorylation of PLC-?1 in vivo. Thus, an optimal kinase
activity of Syk and an interaction between the linker region of Syk with PLC-?1 are required for the tyrosyl
phosphorylation of PLC-?1.
The B-cell receptor (BCR) complex consists of the surface
immunoglobulin (sIg) noncovalently linked to the Ig?/Ig? het-
erodimer with cytoplasmic tails containing immunoreceptor
tyrosine-based activation motifs responsible for signal trans-
duction (4, 31). Members of the Src family of protein tyrosine
kinases (PTK), including Lyn, Blk, and Fyn, and a member of
the Syk/Zap70 family, Syk, are among the signal-transducing
molecules that interact with the BCR (4, 31).
Stimulation of the BCR complex induces rapid tyrosyl phos-
phorylation of receptor-associated PTKs and activates both
phospholipase C-?1 (PLC-?1) (6) and PLC-?2 (11). The hy-
drolysis of phosphoinositides by activated PLC-? isoforms into
diacylglycerol and inositol phosphates results in activation of
protein kinase C and increases in the concentration of intra-
cellular free Ca2?(2). Ligands of receptor protein tyrosine
kinases (RPTK), e.g., epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF) receptors (5, 9), induce
receptor oligomerization and autophosphorylation, generating
docking sites for Src homology domains (SH2) including the
C-terminal SH2 domain of PLC-?1. This allows RPTK to
phosphorylate and activate PLC-?1. PLC-? isoforms are in
vitro substrates for Src family kinases (23), and SH2 domains
of Src family kinases can bind to tyrosyl-phosphorylated
In leukocytes, the PTKs Syk and Zap-70 are required for the
activation of PLC-? isoforms in cytotoxic T cells (16), a chicken
B-cell line (17, 41), and basophils (32). A lack of Zap-70
expression is the underlying cause of an autosomal recessive
form of severe combined immunodeficiency characterized by
the failure of CD4?T cells to respond to antigen receptor
stimulation and by an absence of peripheral CD8?T cells (1,
8, 10). Recently, we reported that Syk, PLC-?1, and a 120-kDa
phosphoprotein form a complex in human B cells (38). Despite
evidence implying an essential function of the Syk/Zap-70 ki-
nases in the antigen receptor-mediated activation of PLC-?
isoforms in both B and T lymphocytes, relatively little is known
about the nature of the physical connections between PLC-?
isoforms, PTKs, and the antigen receptor in vivo. It is also
unclear which PTK(s) actually phosphorylates PLC-? isoforms
on tyrosines (Tyrs) upon ligation of the antigen receptors.
In this report, we present evidence that (i) PLC-?1 can
directly interact with Syk via its C-terminal SH2 domain; (ii)
Syk isolated from BCR-activated B cells can phosphorylate
PLC-?1 in vitro on a key regulatory Tyr residue that is involved
in the in vivo activation of PLC-?1; and (iii) both the binding
of PLC-?1 SH2(C) to Syk and in vivo tyrosine phosphorylation
of PLC-?1 are dependent on two Tyr residues (Tyr-348 and
Tyr-352), which are located in the linker region joining the
C-terminal SH2 domain and the kinase domain of Syk. We
propose that Syk in lymphocytes is functionally analogous to
receptor PTKs, including the EGF (5) and the PDGF (9)
receptors, in such a way that upon oligomerization, Syk phos-
phorylates itself, is bound by PLC-?1 via its SH2(C) domain,
and thus can phosphorylate and activate PLC-?1.
MATERIALS AND METHODS
Cells, antibodies, and reagents. COS cells and the Burkitt’s lymphoma lines
Daudi and Ramos were maintained as previously described (20). Anti-Syk serum
and anti-phosphotyrosine monoclonal antibody (MAb) PY20 were purchased
from Santa Cruz Biotechnology, La Jolla, Calif.; biotinylated anti-phosphoty-
rosine MAb 4G10 and mouse anti-PLC-?1 were purchased from Upstate Bio-
technology, Lake Placid, N.Y.; and rabbit anti-PLC-?1 serum was a kind gift
from Graham Carpenter, Vanderbilt University. Normal goat IgG F(ab?)2and
* Corresponding author. Mailing address: Department of Microbi-
ology, Box 357242, University of Washington, Seattle, WA 98195.
Phone: (206) 543-7169. Fax: (206) 685-0305. Electronic mail address:
† Permanent address: Kavetsky Institute of Experimental Pathology,
Oncology and Radiology, Ukrainian Academy of Sciences, Kiev,
goat anti-human IgM F(ab?)2were purchased from Jackson ImmunoResearch,
West Grove, Pa. Normal rat IgG was purchased from Pierce, Rockford, Ill. Rat
anti-mouse CD8?, 53-6.72, was obtained from the American Type Culture Col-
lection, Rockville, Md. Glutathione-agarose was purchased from Sigma, St.
Louis, Mo., and protein A- and protein G-Sepharose were purchased from
Pharmacia Biotech, Piscataway, N.J.
Plasmid constructs. For the generation of glutathione S-transferase (GST)
fusion proteins containing the SH2 domains of PLC-?1 and substrates for the in
vitro kinase assay (Fig. 1), cDNAs encoding different regions of PLC-?1 were
generated by using total RNA from Daudi cells as the template and reverse
transcription-PCR with Superscript reverse transcriptase (Life Technologies,
Gaithersburg, Md.). The following primer pairs were used:
5? primer: 5? GCG GGA TCC TCC AAT GAG AAG TGG TTC CAT 3?
3? primer: 5? GCG GAA TTC GGC GTT GGT CTG TGG GAC AGG 3?
5? primer: 5? GCG GGA TCC GAG AGC AAA GAA TGG TAC CAC 3?
3? primer: 5? GCG GAA TTC TGC CTC CTC GTT GAT GGG ATA 3?
5? primer: 5? GCG GGA TCC TCC AAT GAG AAG TGG TTC CAT 3?
3? primer: 5? GCG GAA TTC TGC CTC CTC GTT GAT GGG ATA 3?
5? primer: 5? GCG GGA TCC TCA CCC AAC CAG CTT AAG 3?
3? primer: 5? GCG GAA TTC GGC GTT GGT CTG TGG GAC AGG 3?
5? primer: 5? GCG GGA TCC GAG AGC AAA GAA TGG TAC CAC 3?
3? primer: 5? GCG GAA TTC TCC AGG GCC ACG GGG TTG 3?
Underlined nucleotides indicate BamHI and EcoRI sites in the 5? and 3? primers,
respectively; these sites were used to clone the PCR products into the pGEX-2T
vector (Pharmacia Biotech) for protein production in Escherichia coli
BL21(DE3). For the construction of the mCD8-Syk chimera, a cDNA encoding
the extracellular and transmembrane domains of mouse CD8? (mCD8) was
generated by reverse transcription-PCR with total RNA from a CD8?mouse
cytotoxic T-lymphocyte clone as template, a 5? primer of 5? GCG GCG GCC
GCC CAC ACC ATG GCC TCA CCG TTG ACC 3?, and a 3? primer of 5? GCG
GGA TCC CCT GTG GTA GCA GAT GAG AGT 3?. Underlined nucleotides
are NotI and BamHI sites used for cloning. The coding region of human Syk was
amplified by using the 5? primer 5? GCG GGA TCC GCC ATG GCT GAC AGC
GCC AAC CAC 3?, the 3? primer 5? GCG GAA TTC TTA GTT CAC CAC
GTC ATA GTA GTA ATT 3?, and phSyk-1 (20) as the template. The PCR
product was cut with BamHI and EcoRI (at the underlined nucleotides) and
cloned into the pBluescript II SK?vector (Stratagene, La Jolla, Calif.). The
insert was then excised from the pBluescript plasmid with BamHI and XhoI
and ligated together with the cut mCD8? fragment into NotI-XhoI-cut expres-
sion vector pcDNA3 (Invitrogen, San Diego, Calif.). Site-directed mutagen-
esis was conducted with the Unique site elimination kit (Pharmacia Biotech) and
the appropriate selection and mutagenesis primers. Two variants of the GST-
?1Y771,Y783 with either Tyr-771 or Tyr-783 substituted with phenylalanine
(Phe) to give GST-?1Y771F or GST-?1Y783F, respectively, were generated.
Four variants of mCD8-Syk with either Lys-397 and Lys-402 replaced with Gln
(mCD8-SykATP?), Tyr-296 replaced with Phe (mCD8-SykY296F), Tyr-348 and
Tyr-352 replaced with Phe (mCD8-SykY348F,Y352F), or Tyr-525 and Tyr-526
replaced with Phe (mCD8-SykY525F,Y526F) were also generated. All constructs
were sequenced to confirm sequence fidelity.
Purification of GST fusion proteins. Overnight cultures of E. coli BL21(DE3)
containing the expression plasmids were diluted 10-fold into new medium plus 50
?g of ampicillin per ml and cultured for 1 h at 37?C. Expression of fusion
proteins was induced by the addition of 0.1 mM isopropyl-?-D-thiogalactoside
(IPTG) for 2.5 h at 30?C. Cells were then pelleted and resuspended in phos-
phate-buffered saline (PBS) containing 0.1% Triton X-100, 2 mM phenylmeth-
ylsulfonyl fluoride, 10 ?g of aprotinin per ml, 10 ?g of leupeptin per ml, 1 ?g of
pepstatin per ml, and 100 ?g of soybean trypsin inhibitor per ml. Lysis was done
by sonication for 1 min on ice. Cell debris was removed by centrifugation at
15,000 ? g for 15 min at 4?C. Supernatants were filtered through 0.45-?m-pore-
size filters and then passed over 2 ml of glutathione-agarose preequilibrated in
PBS. The glutathione-agarose beads were washed with 30 volumes of PBS.
Bound GST fusion proteins were eluted with 10 mM of glutathione in 50 mM
Tris (pH 8.0) and dialyzed against PBS. Protein concentrations were determined
by the Bradford assay.
COS cell transfection. The DEAE-dextran method was used to transiently
express cDNAs in COS cells. Briefly, COS cells were seeded at approximately 3
? 106cells per 150-mm-diameter plate 16 h before transfection. The cells were
washed twice with serum-free Dulbecco modified Eagle medium. Transfection
medium containing 400 ?g of DEAE-dextran per ml, 0.1 mM chloroquine, and
3 ?g of cDNA constructs per ml was added. After a 3- to 4-h incubation at 37?C
in a tissue culture incubator, the cells were pulsed with 10% dimethyl sulfoxide
in 1? PBS at room temperature for 2 min and then returned to fully supple-
mented Dulbecco modified Eagle medium. After 24 h, the medium was replaced
with Dulbecco modified Eagle medium containing 5% fetal calf serum. Cells
were harvested for analysis 72 h after transfection.
B-cell stimulation. The Burkitt’s lymphoma cell line Daudi or Ramos was
pelleted and resuspended in fully supplemented RPMI 1640 at 10 ? 106cells per
ml. Cells were allowed to equilibrate at 37?C for 10 to 15 min. Stimulation was
initiated by adding F(ab?)2fragments of goat anti-human IgM to a final concen-
tration of 10 ?g/ml or by adding 2.5 mM H2O2plus 100 ?M sodium orthovana-
date. The control for F(ab?)2fragments of goat anti-human IgM was either
F(ab?)2fragments of normal goat IgG or F(ab?)2fragments of goat anti-mouse
IgM, and the control for H2O2plus orthovanadate was medium. Stimulation was
stopped by dilution of the cell suspensions into ?10 volumes of ice-cold 1?
PBS–0.02% NaN3. The cells were pelleted and washed once more with ice-cold
PBS before lysis in 0.5% Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris
[pH 8.0], 5 mM EDTA) supplemented with protease inhibitors (2 mM phenyl-
methylsulfonyl fluoride, 10 ?g of aprotinin per ml, 10 ?g of leupeptin per ml, 1
?g of pepstatin per ml, and 100 ?g of soybean trypsin inhibitor per ml) and
phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, and 5 mM Na4P2O7).
Immunoprecipitation and Western blotting. Immunoprecipitation, sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the in vitro
kinase assay were performed as described previously (22). Western blotting
(immunoblotting) was performed with an enhanced chemiluminescence (ECL)
kit (Amersham Life Science, Arlington Heights, Ill.). In Western blotting with
GST fusion proteins, the blots were incubated with 20 nM fusion proteins in
Tris-buffered saline (10 mM Tris [pH 8.0], 150 mM NaCl) plus 0.01% Tween 20
and 2.5% bovine serum albumin for 2 h at room temperature. Binding of GST
fusion proteins was detected by an anti-GST MAb (Santa Cruz Biotechnology)
plus horseradish peroxidase-conjugated goat anti-mouse serum (Jackson Immu-
noResearch) and ECL.
FIG. 1. PLC-?1 fusion protein constructs. The locations of Tyr residues in PLC-?1 known to be phosphorylated by activated growth factor receptor PTK are
indicated in the top diagram. Lower diagrams show the different portions of PLC-?1 that were included in each of the GST fusion protein constructs. Replacement
of Y-771 or Y-783 with Phe in GST-?1Y771,Y783 gives GST-?1Y771F or GST-?1Y783F, respectively.
1306 LAW ET AL.MOL. CELL. BIOL.
SH2 domains of PLC-?1 bind to Syk. Since SH2 domains
recognize tyrosyl-phosphorylated ligands (29), we reasoned
that upon stimulation of the sIgM/BCR, the SH2 domains of
PLC-?1 might be responsible for relocalizing PLC-?1 to BCR-
activated PTK(s) to facilitate its own activation. To test this, we
generated GST fusion proteins containing either the N-termi-
nal [GST-?1SH2(N)], C-terminal [GST-?1SH2(C)], or both
[GST-?1SH2(NC)] SH2 domains of PLC-?1 (Fig. 1). B cells
were stimulated by cross-linking IgM receptors or H2O2plus
sodium orthovanadate, which elicit signal transduction events
in lymphocytes resembling antigen receptor-mediated signal-
ing (35–37). Figure 2A shows that GST-?1SH2(NC) recog-
nized similar patterns of tyrosyl-phosphorylated proteins from
B cells activated by either anti-IgM or H2O2plus sodium or-
thovanadate. GST-?1SH2(N) and GST-?1SH2(C) each precipi-
tated a subset of these proteins. Thus, GST-?1SH2(N) precipi-
tated 140- and 70-kDa phosphoproteins whereas GST-?1SH2(C)
precipitated 120-, 80-, 76-, and 70-kDa phosphoproteins (Fig.
2A, upper panel). Western blotting with an anti-Syk serum
revealed that Syk was part of the 70-kDa protein band present
in the GST-?1SH2(NC) precipitates obtained from activated B
cells (Fig. 2A, lower panel). To determine if the interaction
between the SH2 domains of PLC-?1 and Syk was direct, GST
fusion proteins were used in a Western blot of Syk isolated
from resting or anti-IgM-activated B cells. GST-?1SH2(C) and
GST-?1SH2(NC) recognized tyrosyl-phosphorylated Syk isolated
from anti-IgM-activated B cells (Fig. 2B), but GST-?1SH2(N)
and GST alone did not (data not shown). GST-?1SH2(C) also
precipitated a 70-kDa phosphoprotein which was not recog-
nized by the anti-Syk serum (Fig. 2A); however, GST-
?1SH2(C) clearly bound to tyrosyl-phosphorylated Syk on
Western blots (Fig. 2B). It is possible that GST-?1SH2(C)
bound to only a small amount of highly tyrosyl-phosphorylated
Syk in solution, which could not be detected by the anti-Syk
serum used in this study. Alternatively, GST-?1SH2(C) might
precipitate another 70-kDa tyrosyl-phosphorylated protein(s)
in addition to Syk. Taken together, these data demonstrate
that PLC-?1 can directly interact with Syk via its SH2 domains;
the SH2(C) domain probably confers the specificity for inter-
acting with Syk (Fig. 2; also see Fig. 5).
Syk phosphorylates PLC-?1 in vitro. Tyr-472, Tyr-771, Tyr-
783, and Tyr-1254 of PLC-?1 can be phosphorylated in vitro by
activated EGF receptors (15, 26, 44) and in vivo by activated
PDGF receptors (14). Phosphorylation of Tyr-783 and Tyr-
1254 correlates with the activation of PLC-?1 in vivo by PDGF
(14). Since GST-?1SH2(C) bound to tyrosyl-phosphorylated
Syk, we examined if Syk could phosphorylate PLC-?1. GST
fusion proteins containing either Tyr-472 (GST-?1Y472) or
Tyr-771 and Tyr-783 (GST-?1Y771,Y783) of PLC-?1 were
used as substrates in kinase assays (Fig. 1). Syk isolated from
control stimulated or anti-IgM-stimulated B cells had little
activity against GST (Fig. 3A). Syk isolated from control stim-
ulated B cells also did not phosphorylate GST-?1Y472 and had
minimal activity against GST-?1Y771,Y783 (Fig. 3A). In con-
trast, Syk isolated from anti-IgM stimulated B cells not only
showed increased autophosphorylation but also strongly phos-
phorylated GST-?1Y771,Y783 (Fig. 3A). Phosphoamino acid
analysis confirmed that phosphorylation on GST-?1Y771,Y783
was exclusively on Tyr residues (data not shown). Substituting
Tyr-771 or Tyr-783 with Phe greatly reduced the levels of
phosphorylation of these substrates, showing that in vivo-acti-
vated Syk phosphorylated GST-?1Y771,Y783 on both Tyr-771
and Tyr-783 (Fig. 3A). The BCR-associated PTK Lyn also
phosphorylated GST-?1Y771,Y783 (Fig. 3B), but unlike Syk,
this activity was present in unstimulated cells and only mod-
estly increased in response to anti-IgM stimulation. Interest-
ingly, whereas the level of phosphorylation of GST-?1Y771F
by Lyn was greatly reduced, phosphorylation of GST-?1Y783
was similar to that of GST-?1Y771,Y783, suggesting that Lyn,
unlike Syk, phosphorylated GST-?1Y771,Y783 principally on
Tyr-771 (Fig. 3B). Similar results were obtained when another
Src family kinase, Fgr, was tested instead of Lyn (data not
shown). No phosphorylation of GST-?1Y472 by either Syk or
Lyn was detectable. These data show that Syk could phosphor-
ylate PLC-?1 in vitro on a key Tyr (Tyr-783), which is essential
for initiating the enzymatic activity of PLC-?1 (14).
A chimera of mCD8-Syk can be expressed as an active trans-
membrane PTK in COS cells. To study the requirements for
Syk activation, we generated a plasmid construct made up of
the extracellular and transmembrane domains of mCD8?
fused in frame with the complete coding sequence of human
Syk (mCD8-Syk) (Fig. 4A). This construct encodes a mono-
meric PTK of 97 kDa (Fig. 4B, left panel, reducing lanes);
when transiently expressed in COS cells, this plasmid produced
a disulfide-linked oligomeric (?200-kDa) transmembrane fu-
sion protein (Fig. 4B, left panel, nonreducing lanes). Com-
pared with expressing wild-type cytosolic Syk in COS cells,
mCD8-Syk was heavily tyrosyl phosphorylated (Fig. 4B, right
panel) and induced high levels of tyrosyl phosphorylation of
cellular proteins (Fig. 4C). Tyrosyl phosphorylation of both
mCD8-Syk and cellular proteins was strictly dependent on
kinase activity; substitution of the two Lys residues in the
ATP-binding site with Gln (mCD8-SykATP?) reduced the ty-
rosyl phosphorylation of this chimera to background levels
(Fig. 4B). On the other hand, the fusion protein (mCD8-
SykY525F,Y526F) with substitutions of Phe at Tyr-525 and
Tyr-526 (two Tyr residues at positions corresponding to the
autophosphorylation sites in the Src family PTKs [7, 20]) had
reduced kinase activity compared with mCD8-Syk but was still
tyrosyl phosphorylated (Fig. 4B and C). The residual kinase
activity of membrane-associated mCD8-SykY525F,Y526F in
vivo, however, was considerably higher than that of soluble
wild-type Syk (Fig. 4B and C). These results also suggest that
Tyr-525 and/or Tyr-526 are probably not the only Tyr residues
in Syk that can be phosphorylated.
Tyr-525 and Tyr-526 of Syk are required for optimal binding
by the SH2 domains of PLC-?1. We next determined if GST
fusion proteins of PLC-?1 SH2 domains could interact with
either CD8-Syk or mutant CD8-SykY525F,Y526F. GST fusion
proteins of PLC-?1 did not precipitate any detectable tyrosyl-
phosphorylated proteins from mock-transfected COS cells
(Fig. 5A). Both GST-?1SH2(C) and GST-?1SH2(NC) precip-
itated tyrosyl-phosphorylated proteins 68 to 72 kDa and 87 to
97 kDa in size from COS cells expressing either mCD8-Syk or
mCD8-SykY525F,Y526F. The phosphoprotein levels detected
in the CD8-SykY525F,Y526F transfectants with the SH2 fusion
proteins were lower than those in the mCD8-Syk transfectants.
Probing of the precipitates with an anti-Syk serum revealed
that GST-?1SH2(C) and GST-?1SH2(NC) could indeed pre-
cipitate mCD8-Syk (Fig. 5B). In contrast, mCD8-SykY525F,
Y526F was only minimally precipitated by GST-?1SH2(NC)
and was not precipitated by GST-?1SH2(C). These data confirm
the results, obtained with activated B cells, that the C-terminal
SH2 domain of PLC-?1 could mediate binding to tyrosyl-phos-
phorylated Syk (Fig. 2). Furthermore, Tyr-525 and Tyr-526 at the
autophosphorylation site of Syk are apparently required for op-
timal binding of Syk by the SH2 domains of PLC-?1.
Tyr-525 and Tyr-526 of Syk are required for the in vivo
tyrosyl phosphorylation of PLC-?1. The observations that
mCD8-Syk was expressed as an active PTK in COS cells and
VOL. 16, 1996BINDING OF PLC-?1 TO THE LINKER REGION OF Syk 1307
FIG. 2. SH2 domains of PLC-?1 interact with multiple tyrosyl-phosphorylated proteins including Syk. (A) Nonidet P-40 lysates from Daudi B cells stimulated for
5 min with either 10 ?g of control goat IgG (gIgG) per ml, 10 ?g of goat anti-human IgM (g ?-hIgM) per ml, medium (Med.), or hydrogen peroxide (2.5 mM) plus
sodium orthovanadate (100 ?M) (H2O2? VO43?) were precipitated with 5 ?g of GST, GST-?1SH2(N), GST-?1SH2(C), or GST-?1SH2(NC) plus glutathione-agarose.
Bound proteins were resolved by reducing SDS-PAGE and immunoblotted with the anti-phosphotyrosine (anti-PY) MAb 4G10 or anti-Syk serum, respectively. Proteins
of 33 to 35 and 45 to 48 kDa are the GST fusion proteins (dots). Arrowheads indicate precipitated phosphoproteins (see ‘‘SH2 domains of PLC-?1 bind to Syk’’ in
Results). (B) Syk was immunoprecipitated from Daudi cells stimulated with either goat IgG or goat anti-human IgM, resolved by reducing SDS-PAGE, and
immunoblotted with either anti-Syk serum, 4G10 (anti-PY), GST-?1SH2(N), GST-?1SH2(C), or GST-?1SH2(NC). The negative control for the immunoprecipitating
anti-Syk serum was normal rabbit IgG (rIgG). Western blotting with GST alone did not detect any proteins (data not shown).
1308 LAW ET AL.MOL. CELL. BIOL.
can be recognized by the PLC-?1 SH2 domains prompted us
to determine if endogenous PLC-?1 expressed in COS cells
could become tyrosyl phosphorylated upon transfection with
mCD8-Syk. PLC-?1 in both mock-transfected COS cells and
COS cells transfected with soluble wild-type Syk was not ty-
rosyl phosphorylated (Fig. 6). Transfection of mCD8-Syk into
COS cells resulted in tyrosyl phosphorylation of endogenous
PLC-?1. However, transfection of mCD8-SykY525F,Y526F
did not induce tyrosyl phosphorylation of endogenous PLC-?1
in COS cells (Fig. 6), even though the mutated Syk fusion
protein was tyrosyl phosphorylated (Fig. 4B) and augmented
the tyrosyl phosphorylation of other cellular proteins (Fig.
4C). Hence, both the kinase activity and Tyr-525 and Tyr-
526 of Syk were needed for in vivo tyrosyl phosphorylation
The linker region of Syk contains a binding site for PLC-
?1. The mCD8-Syk chimeras that can be tyrosyl phosphor-
ylated in COS cells provided a system to further define the
PLC-?1 binding site in Syk. Although Tyr-525 and Tyr-526
of Syk were needed for optimal binding by SH2 domains of
PLC-?1, these Tyr residues do not conform to the PLC-?1
SH2(C)-binding-site sequence, pTyrXXPro, defined by
Songyang et al. (40); therefore, it seemed possible that they
would not participate directly in PLC-?1 binding. A com-
parison of the amino acid sequences of Syk and Zap-70
revealed 19 conserved Tyr residues; of these, Tyr-348 and
Tyr-352 of Syk and Tyr-315 and Tyr-319 of Zap-70, located
in the linker region between the SH2 domains and the
kinase domains, have the TyrXXPro sequence (Fig. 7). In
addition, Tyr-296 of Syk has the TyrXXPro sequence, but
this motif is not present in Zap-70 (Fig. 7). Additional
variants of mCD8-Syk with Tyr-296 and with Tyr-348 and
Tyr-352 substituted with Phe (mCD8-SykY296F and mCD8-
SykY348F,Y352F) were then generated to determine if
these sites could participate in PLC-?1 binding (Fig. 4A).
Both variants were expressed in COS cells at comparable
levels to that of mCD8-Syk (Fig. 8A, left panel). Phospho-
tyrosine immunoblotting revealed that whereas mCD8-
SykY296F was phosphorylated to the same extent as mCD8-
Syk, the level of phosphorylation of mCD8-SykY348F,Y352F
was slightly lower (Fig. 8A, left panel). This suggested that
Tyr-348 and/or Tyr-352 may be among the autophosphory-
lation sites in Syk. The enzymatic activities of these two
variants were similar to that of mCD8-Syk as indicated by
the levels of tyrosyl phosphorylation of COS cell proteins in
these transfectants (Fig. 8A, right panel). Thus, these Tyr
residues in the linker region probably do not significantly
influence Syk activity. The spectrum of proteins precipitated
by GST-?1SH2(C) and GST-?1SH2(NC) from mCD8-SykY296F
transfectants is similar to that precipitated from mCD8-Syk
transfectants (Fig. 5A and 8B, left panel). The signals obtained
from the mCD8-SykY348F,Y352F transfectant were consider-
ably less intense, in particular the species around 97 kDa (the
size of the different mCD8-Syk fusion proteins) (Fig. 8B, left
panel). Immunoblotting with an anti-Syk serum of the same pre-
cipitates showed that both GST-?1SH2(C) and GST-?1SH2(NC)
precipitated mCD8-Syk and mCD8-SykY296F (Fig. 8B, right
panel). However, even though mCD8-SykY348F,Y352F demon-
strated kinase activity comparable to that of mCD8-Syk or
mCD8-SykY296F and was tyrosyl phosphorylated, GST-
?1SH2(C) did not bind to it and GST-?1SH2(NC) showed
barely detectable binding (Fig. 8B, right panel). Immunoblot-
ting of PLC-?1 precipitates revealed that mCD8-SykY296F,
like mCD8-Syk, was capable of inducing tyrosyl phosphoryla-
tion of PLC-?1 in COS cells whereas mCD8-SykY348F,Y352F
had completely lost this activity (Fig. 8C).
FIG. 3. Phosphorylation of GST PLC-?1 fusion proteins by Syk and Lyn in vitro. Syk (A) and Lyn (B) were immunoprecipitated (Syk IP or Lyn IP) from lysates
of Ramos B cells stimulated with goat anti-mouse IgM (g ?-mIgM) or goat anti-human IgM (g ?-hIgM). Immunoprecipitates were then subjected to in vitro kinase
assays in the presence of the indicated substrates, reducing SDS-PAGE, and autoradiography. A rabbit IgG immunoprecipitate (rIgG IP) plus the GST-?1Y771,Y783
substrate was used as the negative control. Arrowheads show the mobilities of the indicated proteins.
VOL. 16, 1996BINDING OF PLC-?1 TO THE LINKER REGION OF Syk1309
Increasing evidence is now available suggesting a critical role
for Syk in activating PLC-? isoforms in hematopoietic cells
including lymphocytes (1, 8, 16, 17, 41) and basophils (32). In
particular, Syk-negative B cells cannot be activated by BCR
ligation to tyrosyl phosphorylate PLC-?2 (41). Although a
complex of Syk, PLC-?1, and a 120-kDa phosphoprotein in B
cells can be isolated (38), just how Syk actually activates
PLC-?1 is not clear. A recent report showed that SH2 domains
of murine PLC-?1 can bind to tyrosyl-phosphorylated murine
Syk (39). Our results confirm and extend this observation. In
this study, we demonstrate the ability of Syk to tyrosyl phos-
phorylate PLC-?1 on a key regulatory residue in vitro. The
abilities of different mCD8-Syk chimeras to be bound by the
SH2 domains of PLC-?1 in vitro were also correlated to the
abilities of these chimeras to induce tyrosyl phosphorylation of
PLC-?1 in vivo.
Individual SH2 domains of PLC-?1 precipitated distinct but
overlapping sets of tyrosyl-phosphorylated proteins from stim-
ulated B cells (Fig. 2). Phosphoproteins of 140 kDa interacted
preferentially with PLC-?1 SH2(N), whereas phosphoproteins
of about 70 kDa interacted preferentially with PLC-?1
SH2(C). Similar to the SH2 domains of Lyn, Fyn, and Blk (24),
FIG. 4. Expression of mCD8-Syk chimeras in COS cells. (A) Schematics of the different mCD8-Syk constructs used. (B) COS cells were transfected with either the
mCD8-Syk, mCD8-SykATP?, mCD8-SykY525F,Y526F, or phSyk-1 (Syk) constructs. The chimeras and Syk were immunoprecipitated with either anti-mCD8? or
anti-Syk serum, resolved by reducing (R) or non-reducing (NR) SDS-PAGE, and immunoblotted with either anti-Syk serum (left panel) or 4G10 (anti-PY) (right
panel). Arrowheads show the mobilities of the reduced mCD8-Syk chimeras and wild-type Syk. (C) Tyrosyl-phosphorylated proteins from the same transfectants as in
panel B were immunoprecipitated with the anti-phosphotyrosine MAb PY20 (anti-PY), resolved by reducing SDS-PAGE, and immunoblotted with 4G10. Normal
mouse IgG was used as a control (CONT) for PY20.
1310 LAW ET AL.MOL. CELL. BIOL.
the SH2 domains of PLC-?1 also bound to multiple tyrosyl-
phosphorylated proteins (Fig. 2). Immunoblotting identified
Syk as part of the 70-kDa tyrosyl-phosphorylated proteins pre-
cipitated by GST-?1SH2(NC) (Fig. 2A). Since GST-?1SH2(C)
recognized tyrosyl-phosphorylated Syk on Western blots (Fig.
2B), we conclude that the C-terminal SH2 domain of PLC-?1
can directly interact with Syk and that the presence of the
N-terminal SH2 domain increases overall binding to Syk.
Binding of ligands to RPTKs, e.g., the EGF (33, 45), PDGF
(42, 43), and fibroblast growth factor (25) receptors, induces
receptor oligomerization and autophosphorylation, which gen-
erate docking sites for signal transduction molecules including
PLC-?1. Mutation of Tyr-1021 of the PDGF receptor to Phe
blocks both in vivo PLC-?1 binding and ligand-induced in-
crease in intracellular free Ca2?concentration, emphasizing
the importance of the direct physical interaction between
PLC-?1 and activated RPTK (42, 43). Syk is an integral com-
ponent of the BCR in both human (20, 21) and murine (3) B
cells. Within the BCR complex, Syk can associate with the
Ig?-Ig? heterodimer (3, 18) and CD22 (19). Augmentation of
tyrosyl phosphorylation of Syk and its kinase activity are
among the earliest detectable BCR-mediated signal transduc-
tion events preceding the activation of PLC-? isoforms and
intracellular Ca2?mobilization (12, 13, 20). By analogy to the
binding of PLC-?1 to activated RPTKs, we propose that the
interaction of PLC-?1 SH2 domains to tyrosyl-phosphorylated
Syk directs PLC-?1 to the sIgM-BCR during receptor activa-
tion. Since the PLC-?1 SH2 fusion proteins could precipitate
additional phosphoproteins from activated B-cell lysates (Fig.
2), it is also possible that PLC-?1 interacts with other regula-
tory proteins during B-cell activation. In fact, the 140-kDa
protein precipitated by the N-terminal SH2 fusion protein was
recently identified to be the B-cell-associated surface antigen
CD22, and both Syk and PLC-?1 can be found associated with
CD22 in activated B cells (19).
Activated EGF receptor phosphorylates PLC-?1 at Tyr-472,
Tyr-771, Tyr-783, and Tyr-1254 in vitro (15, 44). Substitution of
Tyr-783 and Tyr-1254 of PLC-?1 profoundly inhibits the
PDGF-induced hydrolysis of inositol phospholipids in vivo
(14). In T lymphocytes, antigen receptor ligation also induces
phosphorylation of these Tyr residues in PLC-?1 (27). How-
ever, the PTK(s) responsible for the tyrosyl phosphorylation of
PLC-? isoforms in lymphocytes remains to be defined. Mu-
tagenesis studies on the GST-?1Y771,Y783 substrate for Syk
revealed that Tyr-771 and Tyr-783 are two major residues
phosphorylated by Syk (Fig. 3), suggesting that Syk can poten-
tially activate PLC-?1 by phosphorylating Tyr-783 of PLC-?1.
The in vitro tyrosyl phosphorylation of GST-?1Y771,Y783 by
Syk is specific, since (i) cross-linking of BCR, a potent stimulus
for the enzymatic activity of Syk, dramatically augments its
activity toward GST-?1Y771,Y783 (Fig. 3); (ii) Tyr residues in
other parts of PLC-?1, e.g., Tyr-472, were not phosphorylated
by Syk (Fig. 3); and (iii) Syk does not phosphorylate other
proteins in vitro, including CD22 and enolase, a substrate for
variety of PTKs including the Src family kinases (data not
shown). Although the Src family kinases can phosphorylate
PLC-?1 and PLC-?2 (23) and their N-terminal unique regions
bind to PLC-?2 (30) in vitro, their roles in the activation of
PLC-? isoforms during antigen receptor-mediated lymphocyte
activation are still unclear. In this study, the Src family kinases
Lyn and Fgr preferentially phosphorylated GST-?1Y771,Y783
on Tyr-771 but not Tyr-783, the key residues involved in
FIG. 5. Tyr-525 and Tyr-526 of Syk are required for binding by GST-?1SH2(C). COS cells were transfected with either the vector alone (Mock), mCD8-Syk, or
mCD8-SykY525F,Y526F constructs. Cell lysates were precipitated with 5 ?g of GST, GST-?1SH2(N), GST-?1SH2(C), or GST-?1SH2(NC) plus glutathione-agarose.
Precipitates were resolved by reducing SDS-PAGE and immunoblotted with either 4G10 (anti-PY) (A) or anti-Syk serum (B). Part of the lysate from the mCD8-Syk
and mCD8-SykY525F,Y526F transfectants was also immunoprecipitated with anti-mCD8? and immunoblotted with anti-Syk to demonstrate comparable levels of
transgene expression. Arrowheads show the mobilities of the mCD8-Syk chimera and an additional 72-kDa phosphoprotein precipitated by GST-?1SH2(C) and
VOL. 16, 1996 BINDING OF PLC-?1 TO THE LINKER REGION OF Syk1311
PLC-?1 activation in vitro (Fig. 3; data not shown). We also
could not detect Lyn among the phosphoproteins that were
precipitated by the SH2 domains of PLC-? shown in Fig. 2
(data not shown). Moreover, in the DT40 chicken B-cell line,
anti-IgM induces the activation of Syk and PLC-?2 and aug-
mentation of intracellular free Ca2?concentration even in the
absence any detectable Src family kinases (41), suggesting that
an Src family kinase is not obligatory for the activation of
PLC-?2. With the present observation that Tyr-783 of GST-
?1Y771,Y783 could be phosphorylated by Syk in vitro (Fig. 3),
we propose that Syk may be the PTK that phosphorylates
PLC-?1 in vivo upon antigen receptor stimulation of B cells.
Expression of a mCD8-Syk construct in COS cells alone
resulted in the generation of an active transmembrane PTK
(Fig. 4). Soluble wild-type Syk, however, showed undetectable
tyrosyl phosphorylation and much lower overall PTK activity
(Fig. 4B and C). Clustering of Syk activates its activity (16), and
mCD8-Syk is oligomeric when expressed in COS cells (Fig.
4A); therefore, it is possible that oligomerization of mCD8-Syk
in COS cells alone results in its autophosphorylation and en-
zymatic activation. Active mCD8-Syk might be able to activate
the other PTK(s) present in COS cells. Hence, the overall
tyrosyl phosphorylation of the various mCD8-Syk chimera
might represent a summation of autophosphorylation and
phosphorylation by the other activated PTK(s) in COS cells.
Similar to Syk isolated from stimulated B cells (Fig. 3), the
mCD8-Syk chimera could phosphorylate GST-?1Y771,Y783 in
vitro on Tyr (data not shown) and could be recognized by
GST-?1SH2(C) and GST-?1SH2(NC) (Fig. 4 and 7). This not
only further supports the notion that the specificity of interac-
tion between Syk and PLC-?1 is conferred by the C-terminal
SH2 domain of PLC-?1 but also provides us with a convenient
tool to define the PLC-?1-binding site(s) in Syk.
Substitution of Tyr-525 and Tyr-526 resulted in reduced
autophosphorylation activity of the chimera and tyrosyl phos-
phorylation of cellular proteins in COS cells (Fig. 4). Interest-
ingly, this substitution almost completely eliminated the ability
of mCD8-SykY525F,Y526F to be bound by the PLC-?1 SH2
fusion proteins, even though this Syk mutant retained consid-
erable kinase activity (Fig. 3 and 4). Possible explanations for
these results are as follows: (i) PLC-?1 SH2 domains bind
directly to the autophosphorylation sites at Tyr-525 and Tyr-
526, and (ii) substitution of Tyr-525 and Tyr-526 reduces the
enzymatic activity in Syk; consequently, mCD8-SykY525F,
Y526F is not able to phosphorylate the appropriate Tyr(s) in
Syk which constitutes the actual binding site for the SH2 do-
mains of PLC-?1. Using a phosphopeptide library, Songyang et
al. showed that both SH2 domains of PLC-?1 bind phos-
phopeptides of pTyr-hydrophobic-X-hydrophobic with differ-
ent specificities for the amino acids 3? to the pTyr residue (40).
Since the Tyr-525–Tyr–Lys–Ala–Gln sequence at the auto-
phosphorylation site does not conform to this preferred target
sequence for PLC-?1 SH2(C), the second possibility seems
PLC-?1 binds to the pTyr-Ile-Ile-Pro-Leu-Pro motif in the
cytoplasmic tail of the PDGF receptor (28, 40). This sequence
agrees with the pTyrXXPro motif which the C-terminal SH2
FIG. 6. Tyr-525 and Tyr-526 of Syk are required for CD8-Syk-induced tyrosyl
phosphorylation of PLC-?1 in vivo. COS cells were transfected with either the
vector (Mock), mCD8-Syk, mCD8-SykY525F,Y526F, or phSyk-1 (Syk) con-
structs. PLC-?1 was immunoprecipitated with a rabbit anti-PLC-?1 serum, re-
solved by SDS-PAGE, and immunoblotted with either 4G10 (anti-PY) or a
mouse anti-PLC-?1 MAb.
FIG. 7. Amino acid sequence comparison between human Syk (hSyk) and human Zap-70 (hZap70). Linker regions (A) and parts of the kinase domains (B) of hSyk
and hZap70 were compared. Dashes indicate conserved residues, and dots indicate gaps. Conserved Tyr residues are marked by asterisks. TyrXXPro motifs are
underlined, and Tyr residues at the autophosphorylation sites are in italics.
1312 LAW ET AL.MOL. CELL. BIOL.
domain of PLC-?1 has been predicted to bind by using a
phosphopeptide library (40). Since the C-terminal SH2 domain
of PLC-?1 apparently conferred the specificity for binding to
tyrosyl-phosphorylated Syk (Fig. 2, 5, and 7), it is possible that
a pTyrXXPro motif in Syk is the target sequence for PLC-?1.
Thirty-one conserved Tyr residues are present in Syk from
different mammalian species (20, 34). Three of them, all lo-
cated in the linker region joining the SH2 domains of Syk to its
kinase domain, show sequence identity with the TyrXXPro
motif (Fig. 7). Both Syk and Zap-70 have been implicated in
the activation of PLC-? isoforms, and Zap-70 can substitute
for Syk in restoring the anti-IgM-induced PLC-?2 in the Syk?
FIG. 8. PLC-?1 binds to the linker region of Syk. (A) COS cells were transiently transfected with the mCD8-Syk, mCD8-SykY296F, or mCD8-SykY348F,Y352F
constructs. Chimeras were immunoprecipitated by anti-mCD8, resolved by reducing SDS-PAGE, and immunoblotted with either an anti-Syk serum or 4G10 (anti-PY)
(left panel). Cellular tyrosyl-phosphorylated proteins were immunoprecipitated by PY20, resolved by reducing SDS-PAGE, and immunoblotted with 4G10 (right panel).
(B) Cell lysates from the transfectants in panel A were precipitated with GST, GST-?1SH2(N), GST-?1SH2(C), or GST-?1SH2(NC). Proteins were resolved by
reducing SDS-PAGE and immunoblotted with either 4G10 (anti-PY) (left panel) or an anti-Syk serum (right panel). Dots on the right side of the anti-PY blot indicate
mobilities of the GST fusion proteins and their partially degraded products. (C) PLC-?1 was precipitated from cell lysates of the transfectant in panel A, resolved by
reducing SDS-PAGE, and immunoblotted with either 4G10 (anti-PY) or an anti-PLC-?1 serum.
VOL. 16, 1996 BINDING OF PLC-?1 TO THE LINKER REGION OF Syk1313
DT40 chicken B cell (17); therefore, corresponding TyrXXPro
motifs may be present in Zap-70 as well. A comparison
between human Syk and Zap-70 indeed revealed the presence
of two such motifs at Tyr-315 and Tyr-319 of Zap-70 (Fig.
7). Site-directed mutagenesis on the mCD8-Syk chimera
showed that substitution of either Tyr-296 or Tyr-348 and
Tyr-352 in Syk with Phe had little effect on the enzymatic
activities of these variant chimeras (Fig. 8A). However, mCD8-
SykY348F,Y352F was phosphorylated slightly less than mCD8-
Syk or mCD8-SykY296F (Fig. 8A), suggesting that Tyr-348
and/or Tyr-352 is among the phosphorylation sites in Syk. At
the same time, the interaction between the SH2 domains of
PLC-?1 and mCD8-SykY348F,Y352F became almost unde-
tectable (Fig. 8B). In contrast, substitution of Tyr-296 had
virtually no effect. These results demonstrate that Tyr-348
and/or Tyr-352 in the linker region of Syk constitutes a func-
tional domain in Syk that is responsible for the binding of
PLC-?1. It is still unclear if both Tyr residues are needed to
mediate interaction with PLC-?1. However, Tyr-352–Ala–
Asp–Pro appears to give a better fit to the pTyr-hydrophobic-
X-hydrophobic sequence than Tyr-348–Glu–Ser–Pro does.
The biological significance of the in vitro interaction be-
tween mCD8-Syk and PLC-?1 SH2(C) is supported by the
ability of mCD8-Syk to induce tyrosyl phosphorylation of en-
dogenous PLC-?1 in COS cells (Fig. 6). Substitution of Tyr-
525 and Tyr-526 in Syk, as illustrated by mCD8-SykY525F,
Y526F, produced a chimera with reduced kinase activity,
greatly diminished its recognition by GST-?1SH2(C) and GST-
?1SH2(NC) in vitro (Fig. 5), and eliminated tyrosyl phosphor-
ylation of PLC-?1 in vivo (Fig. 6). Hence, an optimal kinase
activity of Syk is necessary for the induction of tyrosyl phos-
phorylation of PLC-?1 in vivo. Moreover, the results obtained
from the mCD8-SykY348F,Y352F chimera demonstrate that
as well as the kinase activity, a functional binding site on
activated Syk for PLC-?1 is needed for the in vivo phosphor-
ylation of PLC-?1 (Fig. 8B and C).
In conclusion, our data are consistent with a model in which
BCR engagement results in clustering of the receptor associ-
ated Syk. Expression of the mCD8-Syk chimera in COS cells
would mimic the BCR-induced clustering. According to this
model (Fig. 9), auto- or transphosphorylation of Syk molecules
occurs upon clustering, creating docking sites for PLC-?1.
PLC-?1 would then bind to Syk through its C-terminal SH2
domain and become phosphorylated by Syk on key regulatory
Tyr residues (Fig. 9). It remains to be determined if phosphor-
ylation of PLC-?1 by Syk is sufficient to fully activate the
phospholipase activity of PLC-?1. It is most likely that addi-
tional regulatory factors are involved, because both Syk and
PLC-?1 can bind to other cellular proteins in B lymphocytes
including CD22 (19). Data obtained in this study also do not
rule out the possibility that another PTK(s) is involved. Hence,
the docking of PLC-?1 to Syk may localize PLC-?1 to the
proximity of an undefined PTK(s) required for full PLC-?1
activation (Fig. 9).
We thank Stephen J. Klaus and Friederike Siebelt for their critical
review of the manuscript.
This work was supported by NIH grants GM42508 and RR00166.
Che-Leung Law is a Special Fellow of the Leukemia Society of Amer-
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VOL. 16, 1996 BINDING OF PLC-?1 TO THE LINKER REGION OF Syk1315