MOLECULAR AND CELLULAR BIOLOGY, Nov. 2004, p. 9986–9999
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 22
Mechanism of B-Cell Receptor-Induced Phosphorylation and
Activation of Phospholipase C-?2
Yeun Ju Kim,1,2Fujio Sekiya,1Benoit Poulin,1† Yun Soo Bae,2and Sue Goo Rhee1*
Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland,1and Center for Cell Signaling Research and Division of Molecular
Life Sciences, Ewha Womans University, Seoul, South Korea2
Received 24 February 2004/Returned for modification 2 May 2004/Accepted 17 August 2004
Phospholipase C-?2 (PLC-?2) plays an important role in B-cell signaling. Phosphorylation of various
tyrosine residues of PLC-?2 has been implicated in regulation of its lipase activity. With the use of antibodies
specific for each of the putative phosphorylation sites, we have now shown that PLC-?2 is phosphorylated on
Y753, Y759, and Y1217 in response to engagement of the B-cell receptor in Ramos cells, as well as in murine
splenic B cells. In cells stimulated maximally via this receptor, the extent of phosphorylation of Y1217 was
three times that of Y753 or of Y759. Stimulation of Jurkat T cells or platelets via their immunoreceptors also
elicited phosphorylation of Y753 and Y759 but not that of Y1217. A basal level of phosphorylation of Y753 was
apparent in unstimulated lymphocytes. The extent of phosphorylation of Y753 and Y759, but not that of Y1217,
correlated with the lipase activity of PLC-?2. Examination of the effects of various pharmacological inhibitors
and of RNA interference in Ramos cells suggested that Btk is largely, but not completely, responsible for
phosphorylation of Y753 and Y759, whereas phosphorylation of Y1217 is independent of Btk. Finally, phos-
phorylation of Y1217 and that of Y753 and Y759 occurred on different PLC-?2 molecules.
The phospholipase C-? (PLC-?) isozymes PLC-?1 and
PLC-?2 are 50% identical in amino acid sequence and share
the same domain organization, including the arrangement of
an NH2-terminal pleckstrin homology (PH) domain, catalytic
X and Y domains, two Src homology 2 (SH2) domains, and
one SH3 domain (Fig. 1A). PLC-?1 is expressed in all tissues
examined, whereas the expression of PLC-?2 is largely re-
stricted to a subset of cells of the hematopoietic lineage (4, 34,
50). Genetic evidence suggests that the functions of the two
isozymes may not overlap. Targeted disruption of the PLC-?1
gene thus results in embryonic death in mice (22), whereas
deficiency of PLC-?2 in mice is not lethal but manifests devel-
opmental abnormalities in B cells with consequent severe im-
munodeficiency as well as dysfunction of platelets and mast
Both PLC-?1 and PLC-?2 are activated by tyrosine phos-
phorylation. An essential step in the activation of PLC-?1 is
phosphorylation of tyrosine 783 (Y783), which is induced by
stimulation of receptors (such as those for platelet-derived
growth factor [PDGF] or epidermal growth factor [EGF]) with
intrinsic protein tyrosine kinase (PTK) activity or of receptors
(such as B- or T-cell antigen receptors) that are linked to the
activation of nonreceptor PTKs (4, 34, 50). PLC-?1 may be
additionally phosphorylated on Y771 and Y1253. The function
of phosphorylation on Y771 or on Y1253, which is not required
for induction of the lipase activity of PLC-?1, is thus far un-
known. PLC-?1 phosphorylation on Y1253 occurs substantially
in growth factor-stimulated cells but is undetectable in leuko-
cytes stimulated via immunoreceptors. Phosphorylation on
Y771 also occurs in growth factor-stimulated cells but to an
extent much smaller than that apparent for Y783 and Y1253
Given that PLC-?2 is much more abundant than is PLC-?1
in B cells and that it is an essential component in signaling of
the B-cell antigen receptor (BCR), the mechanism of PLC-?2
activation has been studied most extensively in these cells. The
binding of antigen to the BCR induces the activation of Src
family PTKs Lyn, Fyn, and Blk (27), which results in phosphor-
ylation of the cytoplasmic tails of the BCR components. These
phosphorylated tails provide docking sites for the SH2 do-
mains of the PTK Syk, and BCR-associated Syk then phos-
phorylates various adapter proteins. The adapter BLNK (B-
cell linker protein; also known as SLP-65) appears particularly
important for PLC-?2 activation (23, 28). Phosphorylated
BLNK provides a scaffold for the assembly of a macromolec-
ular complex that includes PLC-?2 and Btk, a member of the
Tec family of PTKs. Activated Syk also contributes to the
production of phosphatidylinositol 3,4,5-trisphosphate (PIP3)
by activating phosphatidylinositol (PI) 3-kinase (11, 32, 38). An
important function of PIP3is to bind the PH domain of Btk,
thereby facilitating recruitment of the kinase to the cell mem-
brane, probably at lipid rafts. Tyrosine-phosphorylated BLNK
and PIP3thus promote nucleation of a signaling complex
known as a signalosome (12); formation of the signalosome
concentrates PLC-?2 in lipid rafts, where it is phosphorylated
by colocalized nonreceptor PTKs and gains access to its sub-
Engagement of the BCR results in the recruitment into the
signalosome and consequent activation of members of three
distinct families (Src, Syk, and Tec) of nonreceptor PTKs. All
three types of PTKs are able to phosphorylate PLC-?2 in vitro
(29, 35, 49). However, it is not clear which kinase (or kinases)
* Corresponding author. Mailing address: Building 50, Room 3523,
50 South Dr., MSC 8015, Bethesda, MD 20892. Phone: (301) 496-9646.
Fax: (301) 480-0357. E-mail: email@example.com.
† Present address: Interactions Cellulaires Neuroendocriniennes,
UMR 6544-CNRS-Universite ´ de la Me ´diterrane ´e, 13916 Marseille,
is responsible for PLC-?2 phosphorylation in B cells. Gene
disruption of Lyn or Syk resulted in inhibition of BCR-induced
phosphorylation of PLC-?2 (45). However, given that tyrosine
phosphorylation of BLNK, production of PIP3, and activation
of Btk all occur downstream of Lyn/Syk activation, whether or
not PLC-?2 is a direct substrate of Lyn or Syk in B cells is
difficult to prove. PLC-?2 has been proposed to be a substrate
for Btk on the basis of the observations that the extent of
BCR-induced tyrosine phosphorylation of PLC-?2 was in-
creased in a murine B-cell line that overexpresses Btk (10, 37),
and was substantially (but not completely) decreased in a
chicken B-cell line (DT-40) deficient in Btk (44). BCR stimu-
lation also failed to induce the formation of inositol 1,4,5-
trisphosphate (IP3) or an increase in the cytosolic free Ca2?
concentration in the Btk-deficient DT-40 cells. Results ob-
tained with human B cells that express mutant forms of Btk,
however, appeared inconsistent with a major role for this ki-
nase in the phosphorylation of PLC-?2. Mutations in the Btk
gene are responsible for X-linked agammaglobulinemia
(XLA) in humans, and, as in the Btk-deficient DT-40 cells, the
IP3and Ca2?responses of B cells derived from individuals with
XLA were shown to be markedly attenuated. In contrast to the
chicken cells, however, the XLA cells exhibited a near-normal
increase in PLC-?2 tyrosine phosphorylation in response to
BCR stimulation (10).
We previously identified Y753 and Y759 as the sites of
PLC-?2 phosphorylation (31). Subsequently, Rodriguez et al.
(35) showed that phosphorylation of Y753 and Y759 is essen-
tial for the activation of PLC-?2 by comparing the abilities of
wild-type and Tyr3Phe double-mutant (Y753F/Y759F) forms
of human PLC-?2 to restore the Ca2?response to BCR stim-
ulation in a derivative of DT-40 cells made deficient in PLC-
?2. Immunoblot analysis with antibodies to tyrosine phosphate
failed to detect phosphotyrosine residues in the Y753F/Y759F
mutant immunoprecipitated from the BCR-stimulated cells,
suggesting that Y753 and Y759 are the major sites of phos-
phorylation in stimulated B cells. In contrast, Watanabe et al.
(49) proposed Y1197 and Y1217, in addition to Y753 and
Y759, as sites of phosphorylation by Btk on the basis both of in
vitro phosphorylation assays and of the observation that mu-
tation of all four Tyr residues was necessary to eliminate com-
pletely the BCR-induced phosphorylation of human PLC-?2
expressed in DT-40 cells.
We have now generated antibodies specific for PLC-?2
phosphorylated on either Y753, Y759, Y1197, or Y1217 and
have used these antibodies to study the BCR-induced phos-
phorylation of PLC-?2.
MATERIALS AND METHODS
Materials and cells. Convulxin was obtained from Sigma. PDGF-BB (rat,
recombinant) was from R&D Systems. LY294002, ?-cyano-?-hydroxy-?-methyl-
N-(2,5-dibromophenyl)propenamide (LFM-A13), phorbol myristate acetate
(PMA), bisindolylmaleimide X (BIM), and PP1 were from Calbiochem.
Ramos, an Epstein-Barr virus-negative Burkitt’s lymphoma cell line (26), and
J.gamma1, a PLC-?1-deficient derivative of the Jurkat E6 cell line (19) kindly
provided by R. T. Abraham, were maintained in RPMI 1640 medium supple-
mented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and strepto-
mycin (100 ?g/ml). Null TV-1 cells, which were derived from PLC-?1?/?mouse
embryonic fibroblasts (22) and were kindly provided by G. Carpenter, were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS, penicillin (100 U/ml), and streptomycin (100 ?g/ml). Platelets
were isolated as described previously (9), without aspirin treatment, from the
blood of healthy volunteers with acid-citrate-dextrose as an anticoagulant.
Mouse spleens (5 to ?10 spleens combined for each preparation) were taken
from anesthetized animals and dispersed through a nylon mesh to generate a
single-cell suspension. After hemolysis by osmotic shock, splenocytes were
washed and incubated with anti-CD43 and anti-Mac-1 antibody-conjugated mag-
netic microbeads (Miltenyi Biotec, Auburn, Calif.). Resting B cells were recov-
ered in the flow-through fraction after separation with an AutoMacs magnetic
cell sorter (Miltenyi Biotec) as suggested by the manufacturer.
Antibodies. Antibodies to PLC-?2 phosphorylated on Y753, Y759, Y1197, or
Y1217 (anti-pY753, anti-pY759, anti-pY1197, and anti-pY1217, respectively)
were prepared by injection of rabbits with the phosphorylated peptides INSL
(pY)DVSR (corresponding to residues 749 to 757 of human PLC-?2, with pY
representing phosphorylated Tyr), VSRM(pY)VDPS (residues 755 to 763),
FIG. 1. Characterization of antibodies specific for PLC-?2 phos-
phorylated at each of four Tyr residues. (A) Domain organization and
tyrosine phosphorylation sites of PLC-?1 and PLC-?2. The two
isozymes share a domain organization that includes an NH2-terminal
PH domain, an EF-hand domain, catalytic X and Y domains, a split
PH domain (indicated by P and H), two SH2 domains, an SH3 domain,
and a C2 domain. (B) Alignment of the amino acid sequences of
human PLC-?1 and PLC-?2 between the SH2 and SH3 domains.
Identical or similar residues are indicated by | and ?, respectively.
Tyrosine residues in bold are sites of phosphorylation. (C) Specificity
of antibodies to phosphorylated forms of PLC-?2. Null TV-1 cells were
infected with recombinant vaccinia viruses encoding either wild-type
(WT) PLC-?2 or Tyr3Phe mutants (Y753F, Y759F, and Y1217F)
thereof. They were then deprived of serum overnight and either left
unstimulated or stimulated for 10 min with PDGF (0.1 ?g/ml) in the
presence of 2 mM H2O2and 1 mM sodium vanadate. Cell lysates were
subjected to immunoblot analysis with antibodies to PLC-?2 or with
antibodies specific for PLC-?2 phosphorylated on Y753, Y759, Y1197,
or Y1217, as indicated (?).
VOL. 24, 2004PHOSPHORYLATION AND ACTIVATION OF PLC-?2 9987
EEEL(pY)SSSR (residues 1193 to 1201, with Cys1200replaced with Ser to avoid
conjugation with hemocyanin through this residue), and QLFL(pY)DTHQ (res-
idues 1213 to 1221), respectively. All peptides were acetylated at their NH2
termini, had a Gly-Gly-Gly linker sequence followed by a Cys-amide added to
their COOH termini, and were conjugated with keyhole limpet hemocyanin via
the SH group of the terminal Cys. Peptides were manufactured by BioSynthesis.
Antisera were depleted of cross-reactive antibodies by incubation with the
phosphorylated peptides not used as the antigen (for example, pY759, pY1197,
and pY1217 peptides for anti-pY753) in order to prevent possible recognition of
phosphorylated sites other than the target. Although specific antibodies are
usually purified with the use of the immobilized peptide antigen (with the non-
phosphorylated peptide for negative selection and then with the phosphorylated
peptide for positive selection) (42), we found that this approach resulted in the
loss of high-affinity antibodies for the pY753 and pY759 peptides as a result of
difficulty in eluting them from the affinity gel.
Rabbit polyclonal anti-PLC-?2 antibody (Q20) was obtained from Santa Cruz
Biotechnology, mouse monoclonal anti-Btk was obtained from BD Transduction
Laboratories, mouse monoclonal anti-human CD19 was obtained from BD Bio-
sciences Pharmingen, rabbit polyclonal anti-Syk and mouse monoclonal an-
tiphosphotyrosine (4G10) were obtained from Upstate Biotechnology, rabbit
polyclonal anti-phosphoAkt (pThr308) was obtained from Cell Signaling, and
F(ab?)2fragments of goat anti-human immunoglobulin M (IgM) (Fc5? specific)
and F(ab?)2fragments of goat anti-mouse IgM (?-chain specific) were obtained
from Jackson ImmunoResearch. Mouse monoclonal anti-CD3 (OKT-3) was
kindly provided by L. Samelson. A mixture of anti-PLC-?1 monoclonal antibod-
ies was described previously (41). Anti-pY783 PLC-?1 antibody was from Bio-
Expression of PLC-?2 mutants. Point mutations that result in the substitution
of phosphorylation site Tyr residues with Phe were introduced into human
PLC-?2 cDNA with the use of the pMT2 vector and a QuikChange site-directed
mutagenesis kit (Stratagene). The coding sequences of the mutant and wild-type
PLC-?2 cDNAs were then transferred to the pSC11 vector for the production of
recombinant vaccinia viruses (6). Null TV-1 cells were exposed for 3 h to the
resulting viruses and then cultured for 24 h in DMEM containing 1% FBS.
Cell stimulation, cell lysis, immunoprecipitation, and immunoblot analysis.
Stimulation of vaccinia virus-infected Null TV-1 cells and preparation of cell
lysates were described elsewhere (39). Ramos and Jurkat cells were harvested by
centrifugation, washed twice, and resuspended in ice-cold RPMI 1640 supple-
mented with 25 mM HEPES-NaOH (pH 7.4) at a density of 5 ? 107cells/ml.
Platelets were suspended in Tyrode’s solution. The cells were equilibrated at
37°C for 10 min and then stimulated, after which 0.25 volume of ice-cold 5? lysis
buffer [100 mM HEPES-NaOH (pH 7.4), 5% Triton X-100, 1% sodium dodecyl
sulate (SDS), 5 mM EGTA, 5 mM EDTA, 100 mM NaF, 5 mM sodium vanadate,
25-?g/ml leupeptin, 25-?g/ml aprotinin, 0.5 mM 4-(2-aminoethyl)benzenesulfo-
nylfluoride] was added and the resulting mixture was incubated for 10 min on ice
before centrifugation at 40,000 ? g for 10 min at 4°C. The resulting supernatants
(cell lysates) were subjected to SDS-polyacrylamide gel electrophoresis. The
separated proteins were transferred electrophoretically to a piece of nitrocellu-
lose membrane, which was then probed with appropriate antibodies. Bound
antibodies were detected with alkaline phosphatase-conjugated secondary anti-
bodies (KPL) and the chemiluminescent reagent CDP-Star (Tropix). Images
were captured and analyzed with a Kodak Image Station 440 equipped with a
cooled charge-coupled device camera.
For immunoprecipitation, cell lysates were incubated with rocking for 1 h at
4°C with an excess amount of antibodies and then for an additional 2 h with
protein G-Sepharose (Amersham Pharmacia Biotech). The beads were recov-
ered by centrifugation, washed twice with lysis buffer and once with water,
suspended to the original lysate volume in SDS sample buffer, and boiled. The
eluted proteins were subjected to immunoblot analysis as described above.
RNAi. For RNA interference (RNAi), a small interfering RNA (siRNA) that
targets human Btk mRNA was based on nucleotides 895 to 913 relative to the
translation start site (16). Custom SMARTpool Plus siRNAs that target human
Syk as well as a control RNA (Scramble) were supplied by Dharmacon. Cells
were washed once with ice-cold phosphate-buffered saline, suspended in elec-
troporation buffer (Amaxa) at a density of 108cells/ml, and subjected to elec-
troporation with siRNA (1 ?M) with an Amaxa Nucleofector apparatus and a
2.0-mm electroporation cuvette.
Measurement of inositol phosphates. Ramos cells were labeled for 36 h with
myo-[2-3H]inositol (2.5 ?Ci/ml) (Amersham) in inositol-free DMEM containing
2% FBS. After washing, the cells were suspended in DMEM containing 25 mM
HEPES-NaOH (pH 7.4) at a density of 1 ? 106to 2 ? 106cells/ml and LiCl (20
mM) and test agents were added. The cells (100 ?l) were equilibrated at 37°C for
10 min, stimulated, and then mixed with 9 volumes of 5% perchloric acid. The
perchloric acid extracts of cells were centrifuged to remove debris, and the
resulting supernatants were neutralized with 2 M KOH before analysis of [3H]
inositol phosphates by high-performance liquid chromatography (HPLC) on a
Partisil SAX-10 ion-exchange column and with a Flo-One on-line radiometric
detector (Packard), as described previously (39).
HPLC analysis of PLC-?2. Ramos cell lysates (prepared from 4 ? 108cells)
were mixed with 3 ml of packed heparin-Sepharose CL-6B (Amersham Phar-
macia Biotech) that had been equilibrated with a solution containing 20 mM
Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM EGTA. After incubation for 1 h at
4°C with rocking, the mixture was poured into a column and washed with
equilibration buffer supplemented with 20 mM NaCl. The bound proteins were
eluted with 5 ml of equilibration buffer supplemented with 0.5 M NaCl. The
eluted proteins were diluted 10-fold with equilibration buffer and then subjected
to fractionation by HPLC on a heparin-5PW column (7.5-mm inside diameter by
7.5 cm; TosoHaas) that had been equilibrated with the equilibration buffer.
Bound proteins were eluted with a linear gradient of NaCl (0 to 1 M) in the
equilibration buffer over 50 min at a flow rate of 1 ml/min. Fractions were
collected at 1-min intervals, and those containing PLC-?2 were identified by
Characterization of antibodies specific for phosphorylated
PLC-?2. The four residues Y753, Y759, Y1197, and Y1217 of
PLC-?2 have been implicated as the sites of phosphorylation
on the basis of evidence from various indirect experimental
approaches (31, 35, 49). Like Y771 and Y783 of PLC-?1, Y753
and Y759 of PLC-?2 are located in the linker region between
the catalytic X and Y domains (Fig. 1A). Alignment of the
sequences of PLC-?1 and PLC-?2 reveals that Y759 of PLC-?2
corresponds to Y783 of PLC-?1 (Fig. 1B). However, Y753 of
PLC-?2 aligns poorly with Y771 of PLC-?1; it corresponds
instead to Y775 of PLC-?1, but there is no evidence that this
site is phosphorylated. PLC-?2 does not contain a Tyr residue
corresponding to Y771 of PLC-?1. The COOH-terminal re-
gions of PLC-?1 and PLC-?2 share little sequence similarity;
PLC-?2 does not contain a Tyr residue corresponding to
Y1253 of PLC-?1, and, conversely, PLC-?1 does not possess
Tyr residues corresponding to Y1197 and Y1217 of PLC-?2.
Attempts to map the phosphorylation sites of PLC-?2 mole-
cules isolated from stimulated B cells have not been successful
(35), probably because the extent of phosphorylation is low in
BCR-stimulated cells (see below).
To confirm the identity of the tyrosine phosphorylation sites
of PLC-?2 and to provide a means with which to monitor
phosphorylation at each site quantitatively, we prepared rabbit
polyclonal antibodies specific for PLC-?2 phosphorylated on
either Y753, Y759, Y1197, or Y1217. The specificity of these
various antibodies was tested with PLC-?1-deficient fibroblasts
(Null TV-1 cells) that had been infected with recombinant
vaccinia viruses encoding either wild-type PLC-?2 or Tyr3Phe
phosphorylation site mutants (Y753F, Y759F, and Y1217F).
The cells were stimulated with a combination of PDGF and
pervanadate (an irreversible inhibitor of protein tyrosine phos-
phatases [PTPs] that induces the activation of a variety of
receptor and nonreceptor PTKs), and cell lysates were then
subjected to immunoblot analysis (Fig. 1C). None of the four
antibody preparations recognized wild-type PLC-?2 before cell
stimulation, but they all yielded a single immunoreactive band
at a position corresponding to the molecular size of PLC-?2
after cell stimulation, indicating that they are specific for phos-
phorylated forms of the enzyme. The sequence specificity of
anti-pY753, anti-pY759, and anti-pY1217 was demonstrated
by the observation that mutation of the target Tyr residue
9988 KIM ET AL.MOL. CELL. BIOL.
prevented the recognition of PLC-?2 by the corresponding
antibodies but not that by the other three antibody prepara-
tions. The corresponding specificity test could not be per-
formed with anti-pY1197 because, for an unknown reason, we
failed to establish the viral vector for the Y1197F mutant.
Phosphorylation of PLC-?2 in various cell types. We next
compared the patterns of PLC-?2 phosphorylation among
Ramos (human B lymphoma) cells stimulated via the BCR,
human platelets stimulated via the collagen receptor, a PLC-
?1-deficient derivative of Jurkat (19) (human T lymphoma)
cells stimulated via the T-cell antigen receptor (TCR), and
PDGF-stimulated Null TV-1 fibroblasts expressing human
PLC-?2. (Given that PLC-?1 is much more abundant than is
PLC-?2 in parental Jurkat cells, we used a PLC-?1-deficient
version of this cell line to avoid possible interference.) Each
cell type was stimulated with a saturating concentration of
ligand, and the duration of stimulation was chosen to maximize
the extent of PLC-?2 phosphorylation. Substantial phosphor-
ylation of Y759 was apparent in each cell type in response to
receptor stimulation (Fig. 2A).
In Fig. 2A, the amounts of lysate protein applied to the gel
were adjusted to yield similar immunoblot intensities with anti-
pY759 for the different cell types. Taking this fact into account,
the extent of receptor-induced Y759 phosphorylation de-
creased in the rank order of Null TV-1 cells ? Jurkat cells ?
platelets ? Ramos cells. Phosphorylation of Y1197 was not
detected in any of the four cell types. Given that marked
phosphorylation of Y1197 was detected in Null TV-1 cells
stimulated with PDGF plus pervanadate (Fig. 1C) as well as in
B cells stimulated with pervanadate (Fig. 3A), the lack of
phosphorylation of this residue in cells stimulated via receptors
was not likely due to the unavailability of Y1197 for phosphor-
ylation. Ligand-induced phosphorylation of the other COOH-
terminal Tyr residue, Y1217, was apparent in Ramos cells but
not in platelets, Jurkat cells, or Null TV-1 cells; the faint bands
apparent with a slightly lower (platelets and Null TV-1 cells) or
higher (Jurkat cells) mobility relative to that of PLC-?2 were
attributable to nonspecific interactions of anti-pY1217 or the
secondary antibodies. Ligand-induced phosphorylation of
PLC-?2 on Y753 was observed in Ramos cells, Jurkat cells, and
platelets; the band intensity for pY753 was very low compared
with that for pY759 in Null TV-1 fibroblasts, however. Anti-
pY753 yielded a single band in unstimulated Ramos and Jurkat
cells but a doublet in the stimulated cells. The band detected in
the unstimulated cells appeared not to be attributable to rec-
ognition of nonphosphorylated PLC-?2, because the antibod-
ies did not yield this band with unstimulated platelets or Null
TV-1 cells. Even when the amount of unstimulated platelet
lysate loaded onto the gel was increased so that the amount of
PLC-?2 was similar to that for the Ramos cell sample, no
positive band was observed (Fig. 2B). Furthermore, PLC-?2
immunoprecipitated from unstimulated Ramos cells with anti-
PLC-?2 was equally reactive with anti-pY753 as was PLC-?2 in
the lysate, suggesting that the band was not due to an unknown
lysate protein (Fig. 2B). Serum deprivation did not reduce the
level of Y753 phosphorylation in Ramos cells (data not
shown). These results suggest that some PLC-?2 molecules are
constitutively phosphorylated on Y753 in lymphocytes and that
receptor stimulation further increases the extent of Y753 phos-
phorylation as well as induces phosphorylation at other sites,
resulting in the production of at least two different populations
of pY753-containing PLC-?2 molecules with different electro-
BCR-induced PLC-?2 phosphorylation was also examined in
mouse splenic B cells. Although the extents of phosphorylation
were lower than those in Ramos cells, PLC-?2 was phosphor-
ylated on Y753, Y759, and Y1217 but not on Y1197, as in
Ramos cells (Fig. 2C). Constitutive phosphorylation of Y753
FIG. 2. Phosphorylation of PLC-?2 in various cell types. (A) Ramos
cells, platelets, Jurkat cells, and human PLC-?2-expressing Null TV-1
cells were stimulated (or not) with anti-IgM (25 ?g/ml) for 1 min, with
convulxin (100 ?g/ml) for 2 min, with OKT-3 (0.3 ?g/ml) for 1 min, or
with PDGF (0.1 ?g/ml) for 10 min, respectively. Cell lysates were then
subjected to immunoblot analysis with the indicated antibodies (?-).
The amounts of protein applied to the gel were adjusted so that the
lysates from the different cells yielded similar intensities on the blot
with anti-pY759. (B) Lysates of unstimulated platelets and Ramos cells
(WL) and proteins immunoprecipitated (IP) with anti-PLC-?2 from
the lysate of Ramos cells (IP ?2) were subjected to immunoblot anal-
ysis with anti-PLC-?2 and anti-pY753. The amounts of protein loaded
onto the gel were adjusted to give similar blot intensities with anti-
PLC-?2. (C) Ramos cells and mouse splenic B cells were stimulated
with anti-IgM (25 ?g/ml) for 1 min. Cell lysates were then subjected to
immunoblot analysis with indicated antibodies. The amounts of pro-
tein applied to the gel were adjusted as in panel A.
VOL. 24, 2004 PHOSPHORYLATION AND ACTIVATION OF PLC-?2 9989
was also apparent in unstimulated splenic B cells. Faint bands
seen in the blots with anti-pY759 and anti-pY1217 are likely
due to a small fraction of cells activated during isolation from
spleen (Fig. 2C).
BCR-induced PLC-?2 phosphorylation. The stoichiometry
of phosphorylation at each site of PLC-?2 was studied in BCR-
stimulated Ramos cells. To obtain fully phosphorylated PLC-
?2, we stimulated the cells with pervanadate. PLC-?2 appeared
maximally phosphorylated after this treatment at all sites; no
further increase in the reactivity of each phospho-specific an-
tibody was observed with higher concentrations of pervanadate
or with longer incubation periods. In all five immunoblots with
anti-PLC-?2, anti-pY753, anti-pY759, anti-pY1197, or anti-
pY1217, the PLC-?2 bands obtained with the pervanadate-
treated cells were sharper and migrated much more slowly
than those obtained with untreated cells or those stimulated
via the BCR (Fig. 3A). These results strongly suggested that
most PLC-?2 molecules in the pervanadate-treated cells were
phosphorylated at all possible sites, probably including serine
phosphorylation sites: PLC-?2 can be heavily phosphorylated
on unidentified serine residue(s) in B cells upon stimulation
(H. W. Rho, C.-W. Lee, D. J. Park, P.-G. Sue, and S. G. Rhee,
unpublished observation). We therefore defined 100% phos-
phorylation at a given position as the corresponding immuno-
blot intensity obtained with PLC-?2 from cells stimulated with
pervanadate. The amount of lysate of pervanadate-treated
cells applied to the gel shown in Fig. 3A was only 2% of the
amounts of the lysates applied to the other lanes.
Stimulation with a saturating concentration of anti-IgM for
1 min (time needed for maximal phosphorylation) resulted in
the phosphorylation of 0.45, 0.50, and 1.5% of PLC-?2 mole-
cules on Y753, Y759, and Y1217, respectively. Incubation of
cells with vanadate, a reversible inhibitor of PTPs, before stim-
ulation with anti-IgM increased the level of phosphorylation
on Y753 and Y759 by a factor of 2 to 3, whereas that of Y1217
phosphorylation was little affected. Consistent with the low
level of PLC-?2 phosphorylation in BCR-stimulated cells even
in the presence of vanadate, no noticeable broadening or mo-
bility shift of the PLC-?2 band was apparent in the blot with
anti-PLC-?2. However, the PLC-?2 band detected by anti-
pY753 in stimulated cells appeared broad (Fig. 3A) or as a
doublet (Fig. 2A), suggesting that pY753 is distributed among
PLC-?2 molecules with different states of overall phosphory-
lation. The PLC-?2 band detected by anti-pY759 in stimulated
cells was also broad, albeit less so than that detected by anti-
pY753. Phosphorylation on Y1197 was not detected in BCR-
stimulated cells even in the presence of vanadate; this failure
to detect phosphorylation on Y1197 was not due to insensitiv-
ity of anti-pY1197, given that the intensity of the PLC-?2 band
detected with anti-pY1197 was similar to those of the bands
detected by the other phospho-specific antibodies when cells
were treated with pervanadate.
In contrast to those detected by anti-pY753 or anti-pY759,
the PLC-?2 band detected by anti-pY1217 in stimulated cells
was sharp. In response to BCR stimulation in the absence of
vanadate, 1.5% of PLC-2 molecules were phosphorylated on
Y1217 and 0.45 to 0.5% were probably phosphorylated on both
Y753 and Y759 (phosphorylation of Y753 and Y759 appears to
occur on the same molecules, as shown below). If molecules
phosphorylated on Y753 and Y759 were also phosphorylated
on Y1217, the immunoreactivity detected with anti-pY1217
would be expected to be resolved into two bands (one for the
?1% of PLC-?2 monophosphorylated on Y1217 and the other
for the 0.45 to 0.5% of the enzyme that is trisphosphorylated)
FIG. 3. PLC-?2 phosphorylation in Ramos cells stimulated by BCR
engagement. (A) Ramos cells were left unstimulated (control) or were
stimulated either for 1 min with anti-IgM (25 ?g/ml) in the absence
(?-IgM) or presence (?-IgM ? V) of 5 mM sodium vanadate or for 10
min with pervanadate (PV; derived from 5 mM sodium vanadate and
2 mM H2O2). Cell lysates were then subjected to immunoblot analysis
with the indicated antibodies (?-). The amount of lysate protein ap-
plied to the gel for the pervanadate-treated cells was 2% of that
applied in the other lanes. Numbers under each panel represent the
percentage of PLC-?2 molecules phosphorylated on the corresponding
residue calculated on the basis of the assumption that PLC-?2 mole-
cules were fully phosphorylated in the pervanadate-treated cells; they
are means of values from three independent experiments. (B) Ramos
cells were either left unstimulated (Control) or stimulated for 1 min
with anti-IgM (25 ?g/ml) in the presence of 5 mM sodium vanadate
(?-IgM ? V) (a). In a separate experiment, they were either left
unstimulated (Control) or stimulated with pervanadate (PV; 5 mM
sodium vanadate and 2 mM H2O2) for 10 min (b). Lysate proteins were
subjected to fractionation by HPLC on a heparin-5PW column with a
linear NaCl gradient. The resulting fractions were subjected to immu-
noblot analysis with the indicated antibodies.
9990 KIM ET AL.MOL. CELL. BIOL.
or at least to appear as a broad band. The sharpness of this
band thus argues against this possibility.
We attempted to investigate whether phosphorylation on
Y753, Y759, and Y1217 is present in the same molecules by
precipitating PLC-?2 with one phospho-specific antibody and
probing the precipitates with the other antibody. However,
none of the three antibody preparations proved suitable for
immunoprecipitation. We therefore took another approach.
Ramos cells were left unstimulated or stimulated either with
anti-IgM or with pervanadate, and the resultant cell lysates
were then fractionated by HPLC on a heparin column with an
NaCl gradient (Fig. 3B). PLC-?2 molecules derived from the
resting cells peaked in fraction 14. This preparation consisted
of mostly unphosphorylated protein but contained small
amounts (?0.3%) of PLC-?2 phosphorylated on Y753 (Fig.
3A). Distribution of this Y753 monophosphorylated protein
was indistinguishable from the bulk of unphosphorylated
PLC-?2 detected by anti-PLC-?2 (Fig. 3Ba, control), indicating
that phosphorylation of Y753 alone had no effect on the be-
havior on the heparin column. When stimulated with anti-IgM
in the presence of vanadate, 1 to ?1.5% of PLC-?2 molecules
were phosphorylated on each of three sites (Fig. 3A). The
HPLC analysis of the anti-IgM-stimulated sample revealed
that phosphorylated PLC-?2 molecules detected by anti-pY753
or by anti-pY759 peaked in fraction 18, whereas those detected
by anti-pY1217 overlapped with PLC-?2 molecules detected
with anti-PLC-?2 (Fig. 3Ba, ?-IgM ? V). This result indicates
that the pool of PLC-?2 molecules phosphorylated on Y1217 is
different from that phosphorylated on Y753 and Y759. Phos-
phorylation on Y753 alone was insufficient to cause the shift
from fraction 14 to fraction 18 (as seen with the resting cell
samples), but the elution profiles of pY753- and pY759-con-
taining molecules were similar, with both peaking in fraction
18. This finding, together with the finding that activation of
PLC-?2 requires phosphorylation on both Y753 and Y759,
suggests that Y753 and Y759 are phosphorylated within the
While the stimulation through the BCR could elicit tyrosine
phosphorylation of only small fractions of PLC-?2 molecules,
the treatment with a high dose of pervanadate was able to
induce phosphorylation of most, if not all, of them (Fig. 3A).
When the lysate of pervanadate-treated cells was subjected to
the HPLC analysis, PLC-?2 protein was indeed recovered in
the fraction that peaked at 18, and almost no PLC-?2 was
detected in fraction 14 (Fig. 3Bb). This peak of protein de-
tected by anti-PLC-?2 overlapped with the profile detected
with anti-pY759 (Fig. 3Bb) and with anti-pY753 or anti-
pY1217 (not shown).
The time courses of phosphorylation of PLC-?2 at each site
were examined in Ramos cells stimulated with a saturating
concentration of anti-IgM. The extent of phosphorylation was
maximal within 1 min for Y753, Y759, and Y1217 (Fig. 4A).
The time courses for Y753 phosphorylation and Y759 phos-
phorylation were similar, whereas the extent of phosphoryla-
tion of Y1217 decreased at a slower rate. This result, together
with the observation that vanadate enhanced BCR-induced
phosphorylation at Y753 and Y759 but not that at Y1217,
suggests that the linker region residues might be dephosphor-
ylated by a PTP different from that responsible for dephos-
phorylation of the COOH-terminal residue. Stimulation of
murine splenic B cells with anti-IgM yielded a pattern of
PLC-?2 phosphorylation similar to that obtained with Ramos
cells (Fig. 4B).
Identification of PTKs that phosphorylate PLC-?2 in re-
sponse to BCR stimulation. PTKs that belong to the Src fam-
ily, most notably Lyn, initiate BCR signaling by phosphorylat-
ing several components in the BCR. Syk is then recruited by
the phosphorylated receptor chains and activated to phosphor-
ylate many cellular substrates such as BLNK (28). We tested
the effect of PP1, an inhibitor of Src family kinases, on the
phosphorylation of PLC-?2 in Ramos cells. Phosphorylation of
Y753, Y759, and Y1217 induced by anti-IgM was markedly
inhibited by PP1 (Fig. 5A). The higher-mobility band observed
with anti-pY753, which corresponds to constitutively phos-
phorylated PLC-?2 molecules, was resistant to PP1 under the
experimental condition, however. As expected, BCR-induced
tyrosine phosphorylation of cellular proteins in general was
FIG. 4. Time courses of BCR-induced PLC-?2 phosphorylation.
(A) Ramos cells were stimulated with anti-IgM (25 ?g/ml) for the
indicated times, after which cell lysates were subjected to immunoblot
analysis with the indicated antibodies (?-) (upper panel). The intensi-
ties of the PLC-?2 bands were quantified and plotted against time
(lower panel); data are means of four independent determinations and
are expressed as a percentage of the maximal value for each site after
subtraction of the corresponding basal level of phosphorylation.
(B) Mouse splenic B cells were stimulated with anti-IgM (25 ?g/ml) for
the indicated times, after which cell lysates were subjected to immu-
noblot analysis with the indicated antibodies.
VOL. 24, 2004PHOSPHORYLATION AND ACTIVATION OF PLC-?2 9991
inhibited by PP1, as revealed by immunoblot analysis with
antiphosphotyrosine (Fig. 5A).
Given the lack of specific pharmacological inhibitors of Syk
family kinases, we used RNAi to deplete Ramos cells of Syk. It
was necessary to transfect the cells with the Syk siRNA twice
with an interval of 2 days to achieve a substantial reduction
(?70%) in the extent of Syk expression 4 days after the initial
transfection (Fig. 5B). Depletion of Syk resulted in marked
inhibition of BCR-induced phosphorylation of PLC-?2 at all
three sites (Fig. 5B). Again, the lower-mobility (more highly
phosphorylated) band detected with anti-pY753 was more re-
sponsive to Syk depletion than was the higher-mobility band.
The Tec family kinase Btk has been implicated in the phos-
phorylation and activation of PLC-?2 in chicken DT-40 cells
(44). Experiments with XLA cells also revealed that activation
of PLC-?2 required functional Btk, although the overall level
of phosphorylation of PLC-?2 measured with antiphosphoty-
rosine did not appear to be affected in these cells (10). We
evaluated the role of Btk in the phosphorylation of PLC-?2 at
each site by using PI 3-kinase inhibitors. Given that PIP3bind-
ing to Btk’s PH domain is required to recruit Btk to plasma
membranes where Btk phosphorylates its substrates, inhibition
of PIP3synthesis is expected to block Btk function. At satu-
rating concentrations of LY294002, an inhibitor of PI 3-kinase,
BCR-induced phosphorylation of PLC-?2 on Y753 and Y759
in Ramos cells was inhibited by ?60 to 70%, whereas that of
Y1217 was minimally affected (Fig. 6A). We also employed
wortmannin, another potent PI 3-kinase inhibitor, and ob-
served a similar result (Fig. 6B).
As a measure of PI 3-kinase inhibition, phosphorylation of
Akt on Thr308was monitored in parallel. Phosphorylation of
Akt appeared much more sensitive to the PI 3-kinase inhibitors
than was PLC-?2 phosphorylation (Fig. 6B). The serine/thre-
onine kinase Akt possesses a PH domain, which recognizes
PIP3as well as PI(3,4)P2, and translocates to membranes upon
cellular synthesis of these phosphoinositides. Akt is then phos-
phorylated on Thr308by PDK1 (3-phosphoinositide-dependent
protein kinase 1) and on Ser473by another kinase, and both
reactions also depend on PI(3,4)P2and/or PIP3(1). Thus, a
relatively small change in PI 3-kinase activity can have a more
profound effect on Akt activation than on Btk activation. Al-
ternatively, because intracellular concentration of PIP3is the
net result of phosphorylation by PI 3-kinase and dephosphor-
ylation by PTEN and because PIP3is not evenly distributed
throughout the plasma membranes (14, 15, 18, 51), Akt and
Btk might be activated by different pools of PIP3, and the PIP3
pool that promoted nucleation of the BCR signalsome of Btk,
BLNK, PLC-?2, etc., might be less sensitive to PI 3-kinase
inhibition than the pool responsible for Akt activation. We also
tested the effect of PI 3-kinase inhibitors in mouse splenic B
cells. As observed in Ramos cells, LY 294002 (Fig. 6D) or
wortmannin (not shown) caused a large, but incomplete, re-
duction in BCR-induced phosphorylation on Y753 and Y759
but not on Y1217.
Involvement of Btk was studied by treating Ramos cells with
LFM-A13, a Btk inhibitor rationally designed on the basis of
the structure of the kinase (30). Similar to the PI 3-kinase
inhibitors, LFM-A13 caused ?70% inhibition of PLC-?2 phos-
phorylation on Y753 and Y759 without affecting Y1217 phos-
phorylation (Fig. 6C). LFM-A13 did not alter Akt phosphor-
ylation on Thr308, however. The actions of LY 294002 and
LFM-A13 appeared to be selective even at the high concen-
trations used, given that the overall pattern of BCR-induced
tyrosine phosphorylation was not affected (Fig. 6E). These
results suggest that phosphorylation of Y753 and Y759 resi-
dues largely depends on Btk, whereas phosphorylation of
Y1217 is independent of Btk. Given that the phosphorylation
of the linker region residues was not completely blocked by
saturating doses of LY294002, wortmannin, or LFM-A13,
however, PTKs other than Tec family members likely partici-
pate directly in the phosphorylation of these two sites.
The role of Btk was also studied by RNAi. Three days after
FIG. 5. Effects of inhibition of Src family kinases or of Syk deple-
tion on BCR-induced PLC-?2 phosphorylation in Ramos cells. (A) Ef-
fects of Src family inhibition. Ramos cells were treated for 10 min with
the indicated concentrations of PP1 or vehicle before stimulation for 1
min with anti-IgM (25 ?g/ml). Cell lysates were then subjected to
immunoblot analysis with the indicated antibodies (?-) to PLC-?2
(upper 4 panels) or with antiphosphotyrosine (lower panel). The po-
sitions of molecular size standards are shown in kilodaltons in the
bottom panel. (B) Effects of Syk depletion. Ramos cells were trans-
fected (or not) twice with an interval of 2 days with Syk siRNA. Four
days after the initial transfection, they were subjected to immunoblot
analysis with anti-Syk or anti-PLC-?2 (upper panel); alternatively, they
were stimulated (or not) for 1 min with anti-IgM (25 ?g/ml) before
immunoblot analysis with the indicated antibodies (lower panel).
9992 KIM ET AL.MOL. CELL. BIOL.
transfection of Ramos cells with a Btk-specific siRNA, the
expression of Btk was reduced to 31% ? 4% (mean ? stan-
dard error; n ? 4) of that apparent in control cells (Fig. 6F).
The BCR-induced phosphorylation of PLC-?2 on Y753 or
Y759 was reduced by 55% ? 9% and 63% ? 2%, respectively,
in these Btk-depleted cells, whereas the level of Y1217 phos-
phorylation was 99% ? 5% of that in control cells (Fig. 6F).
These data thus provide further support for the notion that
BCR-induced phosphorylation of PLC-?2 is achieved by both
Btk-dependent and Btk-independent mechanisms.
Effects of changes in PKC activity on the phosphorylation
and activation of PLC-?2. Protein kinase C (PKC) down-reg-
ulates Btk activity by directly phosphorylating Btk on Ser180
(25). We tested the effects of the PKC inhibitor BIM and the
PKC activator phorbol myristate acetate (PMA) on PLC-?2
phosphorylation in Ramos cells (Fig. 7A). BCR-induced phos-
phorylation of PLC-?2 on Y753 and Y759 was markedly po-
tentiated by BIM and inhibited by PMA. In contrast, phos-
phorylation of Y1217 was not affected by either reagent,
consistent with the operation of Btk-dependent and Btk-inde-
pendent mechanisms of PLC-?2 phosphorylation. We also in-
vestigated the consequences of the BIM effect of PLC-?2 phos-
phorylation. Exposure of Ramos cells that had been labeled
with myo-[3H]inositol to anti-IgM in the presence of LiCl re-
sulted in the accumulation of [3H]inositol phosphates (Fig.
7B). The sum of the amounts of inositol monophosphate (IP),
FIG. 6. Effects of inhibition or depletion of Btk on BCR-induced PLC-?2 phosphorylation in Ramos cells. (A) Effect of LY294002 (LY) on
PLC-?2 phosphorylation. Ramos cells were treated for 10 min with vehicle or the indicated concentrations of LY294002 before stimulation (or not)
for 1 min with anti-IgM (25 ?g/ml). Cell lysates were then subjected to immunoblot analysis with the indicated antibodies (?-) (left panels). The
immunoblot intensities were corrected for the basal level of phosphorylation, normalized by the corrected value for stimulated cells pretreated with
vehicle, and plotted against inhibitor concentration (right panels); data are means ? standard error of values from four independent experiments.
(B) Effects of wortmannin (Wort.) and LY294002 on PLC-?2 phosphorylation and Akt phosphorylation on Thr308. Ramos cells were treated for
10 min with the indicated concentrations of inhibitors before stimulation (or not) for 1 min with anti-IgM (25 ?g/ml). Cell lysates were then
subjected to immunoblot analysis with the indicated antibodies. (C) Effect of LFM-A13 on PLC-?2 and Akt phosphorylation. Ramos cells were
treated for 10 min with vehicle or the indicated concentrations of LFM-A13 before stimulation (or not) for 1 min with anti-IgM (25 ?g/ml). Cell
lysates were then subjected to immunoblot analysis with the indicated antibodies. (D) Effect of LY 294002 on PLC-?2 phosphorylation in splenic
B cells. Murine B cells were treated with indicated concentrations of the inhibitor for 10 min before stimulation for 1 min with anti-IgM (25 ?g/ml).
Cell lysates were then subjected to immunoblot analysis with the indicated antibodies. (E) Effects of LY294002 and LFM-A13 on overall tyrosine
phosphorylation of cellular proteins. Lysates of Ramos cells similar to those in panels A and C were subjected to immunoblot analysis with
antiphosphotyrosine. The positions of molecular size standards are shown in kilodaltons. (F) Effects of Btk depletion on PLC-?2 phosphorylation.
Ramos cells were transfected with a control RNA or Btk siRNA. Three days after transfection, the cells were left unstimulated or stimulated for
1 min with anti-IgM (25 ?g/ml). Cell lysates were subjected to immunoblot analysis with the indicated antibodies.
VOL. 24, 2004PHOSPHORYLATION AND ACTIVATION OF PLC-?2 9993
inositol bisphosphate (IP2), and inositol trisphosphate (IP3)
increased linearly with time for up to 30 min (Fig. 7C). BIM
promoted the BCR-induced production of inositol phosphates
(Fig. 7C), indicating that the activation of PLC-?2 is depen-
dent on the phosphorylation of Y753 and Y759 but is not
affected by Y1217 phosphorylation. Neither PMA nor BIM, by
themselves, had any effect on basal PLC-?2 phosphorylation or
[3H]inositol phosphate generation.
We next examined the role of Btk in the effect of BIM on
PLC-?2 phosphorylation and activation. Pretreatment with
LY294002 completely inhibited the potentiating effects of BIM
both on the phosphorylation of Y759 (Fig. 7D) and Y753 (data
FIG. 7. Effects of changes in PKC activity on the phosphorylation and activation of PLC-?2 in Ramos cells. (A) Effects of BIM and PMA on
PLC-?2 phosphorylation. Ramos cells were incubated for 10 min in the absence or presence of 2.5 ?M BIM or 1 ?M PMA before stimulation (or
not) for 1 min with anti-IgM (25 ?g/ml). Cell lysates were then subjected to immunoblot analysis with the indicated antibodies (?-) (right panel).
The relative blot intensities for stimulated cells (after correction for basal values) were determined as means ? standard error from three
independent experiments (left panel). (B) Detection of [3H]inositol phosphates produced in unstimulated and BCR-stimulated cells. Ramos cells
that had been metabolically labeled with myo-[3H]inositol were left unstimulated or were stimulated for 30 min with anti-IgM (25 ?g/ml) in the
presence of 20 mM LiCl. [3H]inositol phosphates in cell extracts were analyzed with an HPLC system equipped with an on-line radioactivity
detector. (C) Time course of phosphoinositide hydrolysis. Cells labeled with myo-[3H]inositol were treated for 10 min with LiCl in the absence
(closed circles) or presence (open circles) of 2.5 ?M BIM and then stimulated for the indicated times with anti-IgM (25 ?/ml). [3H]inositol
phosphates (IP ? IP2? IP3) in cell extracts were then measured as in panel B. (D and E) LY294002 sensitivity of the effect of BIM on
BCR-induced PLC-?2 phosphorylation (D) and on PLC activity (E). Ramos cells were treated for 10 min in the absence or presence of 2.5 ?M
BIM, 100 ?M LY294002, or 2.5 ?M BIM plus 100 ?M LY294002 before stimulation for 1 min with anti-IgM (25 ?/ml). Cell lysates were subjected
to immunoblot analysis with anti-pY759 (inset). The relative blot intensities were determined as means from three independent experiments. In
panel E, cells labeled with myo-[3H]inositol were treated with BIM or LY294002 and then stimulated with anti-IgM as in panel D in the presence
of 20 mM LiCl for 30 min. The amounts of [3H]inositol phosphates in cell extracts were then measured. Data are expressed as relative PLC activity
after subtraction of basal values.
9994KIM ET AL.MOL. CELL. BIOL.
not shown) as well as on in vivo PLC activity (Fig. 7E) in
BCR-stimulated cells. Similar effects were observed with LFM-
A13 as with LY294002 (data not shown). These results pro-
vided further support for a mediator role of Btk in BCR-
induced PLC-?2 phosphorylation and activation.
Role of PI 3-kinase in BCR-induced PLC-?2 activation. Ac-
tivation of PI 3-kinase has been considered a requirement for
BCR-induced PLC-?2 activation, and this requirement has
been attributed to the role of PIP3in the membrane recruit-
ment of Btk (12). Our results now indicate that the recruited
Btk activates PLC-?2 by phosphorylating it on Y753/Y759
(Fig. 6 and 7). We have shown that PIP3does not affect growth
factor-induced PLC-?1 phosphorylation on the critical residue
Y783 but serves as an activating factor for the Y783-phosphor-
ylated enzyme (39). We therefore investigated whether ty-
rosine-phosphorylated PLC-?2 is also activated by PIP3.
LY294002 inhibited the BCR-induced PLC activation in
Ramos cells in a dose-dependent manner (Fig. 8A). At 30 or
100 ?M, LY294002 inhibited PLC activity by 70 and 80%,
respectively, while the same concentrations of this drug re-
duced the extent of BCR-induced PLC-?2 phosphorylation on
Y753/Y759 by 40 and 60%, respectively (Fig. 5A). PIP3thus
appears to act both at the level of Btk to up-regulate PLC-?2
phosphorylation and as an activator of the lipase activity of the
To provide further support for this conclusion, we examined
the effects of Btk RNAi followed by treatment with LY294002
(Fig. 8B). Transfection of Ramos cells with Btk siRNA alone
inhibited the BCR-induced activation of PLC by 40% and the
BCR-induced phosphorylation of PLC-?2 on Y759 by 45%.
Exposure of the Btk-depleted cells to a saturating concentra-
tion (100 ?M) of LY294002 resulted in only a slight further
reduction in the extent of BCR-induced Y759 phosphoryla-
tion, whereas the extent of PLC activation was reduced by half
compared with that apparent in the cells subjected to RNAi
alone. These results are thus consistent with a dual role for
PIP3in BCR-induced PLC-?2 activation.
Given that Ramos cells express PLC-?1 and that BCR en-
gagement induces phosphorylation of this isozyme on the crit-
ical residue Y783 (39), it was important to assess the possible
contribution of PLC-?1 to BCR-induced PLC activity. The
amount of PLC-?1 in Ramos cells is only one-fourth of that of
PLC-?2; the BCR-induced phosphorylation of PLC-?1 is sim-
ilar to that of PLC-?2 in terms of kinetics and stoichiometry;
and the two isozymes exhibit similar specific activities in vitro
(data not shown). Furthermore, LY294002 and Btk RNAi each
inhibited PLC-?1 phosphorylation on Y783 (Fig. 8B). On the
basis of these observations, the presence of PLC-?1 in Ramos
cells does not appear to undermine the conclusion that PIP3
plays a dual role in BCR-induced PLC-?2 activation.
CD19 is a BCR-associated coreceptor that functions as an
enhancer of BCR signaling. This transmembrane protein con-
tains two Tyr residues that are phosphorylated in response to
cross-linking of CD19 molecules and then serve as binding sites
for the SH2 domains of the p85 regulatory subunit of PI 3-ki-
nase (3, 47). This interaction with CD19 results in the activa-
tion of PI 3-kinase. We tested whether additional stimulation
of PI 3-kinase via CD19 would enhance BCR-induced PLC
activation. Costimulation of Ramos cells with anti-IgM and
anti-CD19 resulted in a greater accumulation of inositol phos-
FIG. 8. Role of PI 3-kinase in BCR-induced PLC-?2 activation in
Ramos cells. (A) Concentration dependence of PLC inhibition by
LY294002. Cells labeled with myo-[3H]inositol were treated for 10 min
with the indicated concentrations of LY294002 in the presence of 20
mM LiCl and then stimulated for 30 min with anti-IgM (25 ?g/ml).
[3H]inositol phosphates in cell extracts were then measured. Data are
means ? standard error of three independent determinations. (B) Ef-
fects of Btk RNAi and PI 3-kinase inhibition on BCR-induced PLC
activity and phosphorylation of PLC-?2 and PLC-?1. Ramos cells were
transfected with a control RNA (closed symbols) or Btk siRNA (open
symbols). After 3 days, the cells were labeled with myo-[3H]inositol,
treated for 10 min with 100 ?M LY294002 (triangles) or vehicle (cir-
cles) in the presence of LiCl, and then stimulated for the indicated
times with anti-IgM (25 ?g/ml). [3H]inositol phosphates in cell extracts
were then measured (left panel); data are representative of one of
three independent determinations. Alternatively, the siRNA-trans-
fected cells were treated with 100 ?M LY294002 for 10 min before
stimulation with anti-IgM (25 ?g/ml) for 1 min; cell lysates were then
subjected to immunoblot analysis with the indicated antibodies (?-)
(right panel). (C) Effect of CD19 costimulation on BCR-induced PLC
activity and PLC-?2 phosphorylation. Cells labeled with myo-[3H]
inositol were stimulated in the presence of LiCl for the indicated times
with anti-IgM (25 ?/ml) (circles), anti-CD19 (10 ?g/ml) (closed dia-
monds), or anti-IgM (25 ?g/ml) plus anti-CD19 (10 ?g/ml) (open
diamonds), after which [3H]inositol phosphates in cell extracts were
measured (left panel); data are from a representative experiment out
of three independent determinations. Alternatively, cells stimulated
for 1 min were subjected to immunoblot analysis with the indicated
VOL. 24, 2004 PHOSPHORYLATION AND ACTIVATION OF PLC-?29995
phates than that induced by anti-IgM alone (Fig. 8C). Al-
though CD19 stimulation alone has previously been shown to
induce Btk activation and subsequent PLC-?2 phosphorylation
in human and mouse B cells (5, 13), it induced neither mea-
surable PLC-?2 phosphorylation on Y759 nor inositol phos-
phate production under our experimental conditions (Fig. 8C).
The extent of costimulation-induced phosphorylation of
PLC-?2 on Y759 did not differ from that induced by BCR
engagement alone. The enhancement of PIP3production in-
duced by CD19 stimulation thus appeared to augment the
lipase activity of PLC-?2 (likely that of PLC-?1 also) without
affecting the state of PLC-?2 phosphorylation in BCR-stimu-
Identification of phosphorylation sites of PLC-?2. We have
prepared and established the specificity of antibodies that rec-
ognize individual tyrosine phosphorylation sites of PLC-?2.
Studies with these antibodies revealed that engagement of the
BCR induces phosphorylation of endogenous PLC-?2 on
Y753, Y759, and Y1217 in mammalian B-cell preparations. We
previously identified Y753 and Y759 as the sites of phosphor-
ylation of PLC-?2 in BCR-stimulated Ramos cells by two-
dimensional mapping of tryptic peptides followed by phos-
phoamino acid analysis; we failed, however, to detect a
pY1217-containing peptide by this approach, possibly because
pY1217 was distributed among several tryptic peptides as a
result of partial digestion (three consecutive Arg residues form
potential trypsin cleavage sites that would generate multiple
pY1217-containing peptides). Both Y753 and Y759 were also
proposed as sites of phosphorylation of PLC-?2 on the basis of
experiments with chicken DT-40 cells expressing human
PLC-?2 (35, 49). However, phosphorylation of Y1197, which
was proposed as a step necessary for PLC-?2 activation in the
BCR-stimulated chicken cells (49), did not occur to a measur-
able extent in Ramos lymphoma cells as well as in murine
splenic B cells.
Alignment of the amino acid sequences of PLC-?1 and
PLC-?2 reveals that Y753 and Y759 of PLC-?2 correspond to
Y775 and Y783, respectively, of PLC-?1. Y783 is the only
residue whose phosphorylation is critical for growth factor-
induced lipase activity of PLC-?1, and there is no evidence that
Y775 of this isozyme is phosphorylated in stimulated cells.
Residue Y1217 appears to be unique to PLC-?2.
Phosphorylation of PLC-?2 on Y753 and Y1217 depends on
cell type. We compared the patterns of PLC-?2 phosphoryla-
tion on Y753, Y759, Y1197, and Y1217 in BCR-stimulated
Ramos cells with those apparent in human platelets stimulated
via the collagen receptor, Jurkat human T-lymphoma cells
stimulated via the TCR, and PDGF-stimulated mouse Null
TV-1 fibroblasts expressing human PLC-?2. The results ob-
tained for Y759 were similar for all four cell types in that
phosphorylation of Y759 was induced by cell stimulation.
Phosphorylation of Y753 and Y1217, however, differed among
the cell types. Residue Y753 was constitutively phosphorylated
in both B and T cells and underwent further phosphorylation
in response to cell stimulation; pY753-containing molecules
were not detected in unstimulated platelets but were apparent
after cell stimulation, whereas Y753 phosphorylation was only
weakly induced compared to that of Y759 in fibroblasts. Phos-
phorylation of Y1217 was not detected in platelets, Jurkat cells,
or fibroblasts, and it did not appear to be an artifact of trans-
formation in Ramos cells because it was also apparent in
freshly isolated mouse splenic B cells. Phosphorylation on
Y1197 was undetectable in the four different cell types tested
after stimulation with their respective physiological agonists.
Although phosphorylation on Y1197 by Btk was observed in an
in vitro reaction using PLC-?2 fragments, it had not been
confirmed in the context of full-length protein either in vivo or
in vitro (49).
These cell type-dependent differences in PLC-?2 phosphor-
ylation may not be due to the specificities of kinases expressed
in the different cells. PLC-?1 is phosphorylated both at Y783
and Y1253 by various PTKs, including Src, ZAP-70, and the
EGF receptor in vitro. Y1253 phosphorylation is apparent in
fibroblasts stimulated with PDGF or EGF but undetectable in
lymphoma cells stimulated via immunoreceptors (39). The four
PLC-?2 Tyr residues (Y753, Y759, Y1197, and Y1217) could
be phosphorylated in vitro by Btk when peptides containing
these residues were used as substrates (49). It thus appears that
an aspect of the cellular environment other than the resident
kinases plays an important role in determining the pattern of
PLC-? isozyme phosphorylation. Activated receptor PTKs ini-
tiate phosphorylation of PLC-? isozymes through direct asso-
ciation with the latter’s SH2 domains. In contrast, in immuno-
receptor signaling, PLC-? isozymes do not associate directly
with the kinases responsible for their phosphorylation. Rather,
various adapter proteins provide a platform for such an inter-
action (23, 28, 36). Membrane-anchored proteins such as LAT
(linker for activation of T cells) constitute one class of such
adapters. LAT becomes phosphorylated on several Tyr resi-
dues in activated T cells and mediates the assembly of various
SH2 domain-containing proteins, including PLC-? and Itk, a
Tec family PTK (36). Mature B cells do not express LAT but
do express NTAL (non-T-cell activation linker), also known as
LAB (linker for activation of B cells), which possesses a basic
structural organization similar to that of LAT (2, 21). It re-
mains to be determined, however, whether NTAL is the func-
tional counterpart of LAT and links the BCR to PLC-? in B
cells. Another class of adapters implicated in PLC-? regulation
includes cytosolic proteins such as BLNK and SLP-76 (SH2
domain-containing leukocyte phosphoprotein of 76 kDa).
BLNK is expressed in B cells, whereas SLP-76 is found in T
cells and platelets (23, 28). Although both of these proteins
contain multiple Tyr residues to be phosphorylated, SLP-76
associates with PLC-? through interaction of its proline-rich
region with the SH3 domain of PLC-? (52, 53); BLNK binds
PLC-? as a result of interaction between its phosphotyrosine
residues and the SH2 domains of PLC-? (7, 20), however. A
possible simple explanation for the cell-type-specific pattern of
PLC-?2 phosphorylation therefore is that different adapters
present the lipase molecules to PTKs in different configura-
tions, with the result that the kinases may or may not have
access to Y753 or Y1217.
Distinct features of PLC-?2 phosphorylation at each site in
B cells. The BCR induces phosphorylation of only a small
fraction of PLC-?2 molecules: ?0.5% of molecules contained
pY753 and pY759 and ?1.5% of molecules contained pY1217
in Ramos cells stimulated in the absence of vanadate. Given
9996 KIM ET AL.MOL. CELL. BIOL.
that the pY753-containing enzyme was also present in resting
cells and that PLC activity was virtually undetectable in such
cells, phosphorylation of PLC-?2 on Y753 alone does not ap-
pear to be sufficient for lipase activity. The phosphorylation of
Y753 and that of Y759 occurred to similar extent, exhibited
similar time courses, and were enhanced by vanadate inhibi-
tion of PTPs in BCR-stimulated cells. In contrast, the charac-
teristics of Y1217 phosphorylation differed from those of phos-
phorylation of the linker region residues: The maximal level of
Y1217 phosphorylation attained in the absence of vanadate
was about three times that of Y753 or Y759 phosphorylation,
and Y1217 phosphorylation decayed more slowly and was not
enhanced by vanadate. In addition, phosphorylation of Y753
and Y759 seems likely to occur on the same PLC-?2 molecules,
which appeared to be different from those phosphorylated on
One study with chicken DT-40 cells expressing human
PLC-?2 suggested that phosphorylation of Y753 and Y759 is
sufficient for lipase activation (35), whereas another study with
the same cell line indicated that phosphorylation of not only
Y1217 but also Y1197 is as important as that of the linker
region residues (49). PLC-?2 null mammalian B cells have not
been established. Nevertheless, several lines of evidence from
the present study exclude a role for the COOH-terminal ty-
rosine residues in the activation of PLC-?2. BCR stimulation
thus did not elicit Y1197 phosphorylation in Ramos cells as
well as in mouse splenic B cells. Phosphorylation of Y1217 was
apparent in B cells but was virtually undetectable in other cell
types, including platelets, in which PLC-?2 is much more abun-
dant than is PLC-?1. Stimulation of the collagen receptor
induces PIP2hydrolysis in platelets through activation of
PLC-?2 (50). If the phosphorylation of Y1217 were required
for PLC-?2 activation, PIP2hydrolysis would not be expected
to occur in stimulated platelets. Moreover, we found that a
reduction in the amount of Btk or pharmacological interfer-
ence of Btk activity resulted in a reduction in the extent of
phosphorylation of Y753 and Y759 and in a concomitant re-
duction in PLC activity, whereas Y1217 phosphorylation was
not affected by this treatment. Also, treatment of Ramos cells
with the PKC inhibitor BIM resulted in up-regulation of
PLC-?2 phosphorylation on Y753 and Y759 as well as of PLC
activity, again without an effect on the level of Y1217 phos-
phorylation. These results indicate that phosphorylation of
Y1217 does not activate the lipase function of PLC-?2. Phos-
phorylation of this COOH-terminal residue, which occurs on
molecules distinct from those phosphorylated on Y753/Y759,
might have a function independent of lipase activity. PLC-?
isozymes exhibit several distinct lipase-independent biological
activities (17, 33, 40).
Btk-dependent and Btk-independent phosphorylation of
PLC-?2. Members of three distinct families (Src, Syk, and Tec)
of PTK have been implicated in immunoreceptor-mediated
phosphorylation of PLC-? (28, 46, 50). Lyn, Syk, and Btk have
been suggested to be the major PTKs of each family that
function in BCR signaling. We have now confirmed that these
three PTKs are necessary for normal induction of PLC-?2
phosphorylation by BCR stimulation. Inhibition of Src family
kinases by PP1 resulted in inhibition not only of PLC-?2 phos-
phorylation at each site but also of the phosphorylation of
many other proteins. Depletion of Syk by RNAi also inhibited
PLC-?2 phosphorylation at all sites, but it did not have a global
effect on protein tyrosine phosphorylation (data not shown).
However, given that Syk activity is required for the phosphor-
ylation of BLNK and subsequent recruitment of PLC-?2,
whether or not PLC-?2 is a direct substrate of this PTK re-
In contrast, Btk appears to phosphorylate certain residues of
PLC-?2 directly. Maximal inhibition of Btk function by
LY294002, wortmannin, or LFM-A13 resulted in 60 to 70%
inhibition of PLC-?2 phosphorylation on Y753 and Y759 with-
out an effect on that of Y1217. Moreover, RNAi-mediated
depletion of Btk by 70% and inhibition of the remaining en-
zyme molecules with a saturating concentration of LY294002
did not reduce the extent of phosphorylation of the linker
region residues by more than 60 to 70% and did not affect the
extent of Y1217 phosphorylation. These results indicate that
Btk is the kinase responsible for the phosphorylation of ?60%
of PLC-?2 molecules on Y753 and Y759, but that another
kinase (or kinases) also phosphorylates these linker region
residues. Phosphorylation of Y1217 appeared to be fully inde-
pendent of Btk, though an in vitro study by Watanabe et al.
(49) suggested this residue was a preferred Btk site. Chiu et al.
(7) recently showed that Btk and PLC-?2 must assemble on the
same BLNK molecules to achieve functional Ca2?signaling. It
is thus possible that PLC-?2 molecules either recruited to the
signalosome but not bound to Btk-occupied BLNK or re-
cruited by some other means might be phosphorylated by other
kinases such as Lyn or Syk.
Although we used PI 3-kinase inhibitors to downregulate
Btk function, the mechanism by which this is achieved is not
clear. There is a report suggesting that the tyrosine phosphor-
ylation and activation of Btk are not affected by the absence of
PI 3-kinase (43). Nevertheless, a large body of evidence includ-
ing our present study supports the involvement of PI 3-kinase
in PLC-?2 activation (8, 24). Therefore it is a possibility that
PIP3might be required for the proper localization of Btk
(likely in the BCR signalsome) necessary for PLC-?2 phos-
phorylation but not for the activation of Btk via tyrosine phos-
Immortalized cell lines established from B cells of XLA
patients manifest marked impairment of BCR-induced IP3
production and Ca2?signaling, whereas the extent of BCR-
induced tyrosine phosphorylation of PLC-?2 in these cells was
indistinguishable from that in wild-type B cells (10). These
results can now be explained by our finding that the Tyr resi-
dues essential for PLC-?2 activation are minor sites of phos-
phorylation. The sum of pY753 and pY759 thus constituted
only ?40% of total phosphorylated Tyr residues in PLC-?2 in
stimulated Ramos cells, and this percentage was further de-
creased to ?25% in cells in which Btk function was fully
inhibited. Moreover, given that phosphorylation of Y753 and
of Y759 decayed faster than did that of Y1217, the percentage
would be even smaller if the samples were not prepared at the
peak time. It is thus likely that the level of BCR-induced
tyrosine phosphorylation of PLC-?2 in XLA B cells could be
less than that in normal human B cells but that the difference
would not be sufficiently large to be detected with antibodies to
Feedback inhibition of PLC-?2 through Btk-dependent
phosphorylation. The activity of Btk is inhibited as a result of
VOL. 24, 2004 PHOSPHORYLATION AND ACTIVATION OF PLC-?2 9997
phosphorylation of the enzyme by PKC (25), suggesting the
existence of a link between PKC activation and PLC-?2 phos-
phorylation on Y753 and Y759. Indeed, activation of PKC by
PMA attenuated the BCR-induced phosphorylation of these
two residues of PLC-?2 without affecting that of Y1217. Con-
versely, inhibition of PKC by BIM augmented the BCR-in-
duced phosphorylation of Y753 and Y759 without altering that
of Y1217. Consistent with these observations, the BCR-in-
duced production of inositol phosphates was also increased in
cells exposed to BIM. Given that the activation of PKC occurs
immediately downstream of that of PLC, inhibition of Btk
activity and the consequent down-regulation of PLC-?2 activity
induced by PKC activation constitute a negative feedback loop
in BCR signaling.
Similarity and dissimilarity between the phosphorylation
and activation of PLC-?2 and those of PLC-?1. PLC-?1 is
phosphorylated on Y783 and Y1253 in response to stimulation
of cells with PDGF or EGF, and the level of Y783 phosphor-
ylation is 1.5- to 2-fold greater than that of Y1253 phosphor-
ylation. Unlike PLC-?2 phosphorylation in B cells, a single
kinase, the receptor PTK, appears to phosphorylate both Y783
and Y1253 of PLC-?1, mostly on the same molecules. Similar
to PLC-?2, however, phosphorylation of the linker region res-
idue (Y783), but not that of the COOH-terminal residue
(Y1253), is required for activation of the lipase function of
PLC-?1. Also similar to PLC-?2, phosphorylation of the acti-
vating residue (Y783) appears to occur independently of cell
type, whereas that of the nonactivating site (Y1253) was not
detected in Ramos and Jurkat cells stimulated via their im-
mune receptors (39).
Activation of both PLC-?1 and PLC-?2 is enhanced by PIP3,
but the corresponding modes of action of this lipid might be
cell type dependent. Whereas in immunoreceptor signaling
PIP3promotes the activation of PLC-? by enhancing both its
tyrosine phosphorylation by Tec family PTKs (38, 46) and the
catalytic activity of the resulting phosphorylated enzyme (this
study), the lipid promotion of PLC-? activation in growth fac-
tor signaling is limited to the effect on the phosphorylated
Y.S.K. is a recipient of the Brain Korea 21 fellowship from the
Ministry of Education, South Korea.
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