tec kinases mediate sustained calcium influx via site ... · 07/06/2004 · 4 specific...

Btk regulates PLCγ2 via site-specific phosphorylation

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Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of

the PLCγ SH2-SH3 linker

Lisa A. Humphries1, Carol Dangelmaier4, , Karen Sommer3, Kevin Kipp3, Roberta M. Kato3, Natasha

Griffith6, Irene Bakman7, Christoph W. Turk5, James L. Daniel4, and David J. Rawlings2,3,*

1Molecular Biology Institute, University of California, Los Angeles, CA 90095-1752, 2Department of

Immunology and 3Department of Pediatrics, University of Washington School of Medicine, Seattle,

WA 98195, 4Department of Pharmacology and Sol Sherry Thrombosis Research Center, Temple

University School of Medicine, Philadelphia, PA, 5Max Plank Institute of Psychiatry, Molecular,

Cellular, Clinical Proteomics, Munich, Germany, 6Department of Microbiology and Immunology,

7Department of Chemical Engineering, University of California, Los Angeles, 90095

*Corresponding author e-mail: [email protected], phone: 206-543-3207, FAX: 206-221-

5469

Running Title: Btk regulates PLCγ2 via site-specific phosphorylation

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Summary

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates,

and maintains, intracellular calcium mobilization in response to B cell antigen receptor (BCR)

engagement. Although members from three distinct families of non-receptor tyrosine kinases can

phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ-activation in

vivo remains unknown. Bruton’s tyrosine kinase (Btk) deficient human B cells exhibit diminished IP3

production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine

phosphorylation. This suggested that Btk might modify a critical subset of residues essential for

PLCγ2 activity. To evaluate this hypothesis, we generated phosphotyrosine-site specific antibodies

recognizing four putative regulatory residues within PLCγ2. While all four sites were rapidly

modified in response to BCR engagement in normal B cells, Btk deficient B cells exhibited a marked

reduction in phosphorylation of the SH2-SH3 linker region sites, Y753 and Y759. Phosphorylation of

both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation

of the PLCγ2 carboxyl-terminal sites, Y1197 and Y1217, was unaffected by the absence of functional

Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium

signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker.

Keywords: Phospholipase Cγ2/BCR Signaling/Bruton’s tyrosine kinase/Calcium Signaling/Site

Specific Phosphorylation

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Introduction

Signals generated by the pre-B and mature B cell receptors are essential for B cell

development, activation, and maintenance of mature B cell populations (1). Engagement of the BCR

initiates the formation of a lipid-raft associated signaling complex, or “signalosome”, containing

tyrosine and serine/threonine kinases, adapter molecules, and lipid hydrolases including phospholipase

Cγ isoforms. Together these events promote a series of downstream signals including a sustained

increase in intracellular calcium concentrations.

PLCγ is essential for antigen receptor-mediated calcium mobilization (2, 3, 4). Activated

PLCγ hydrolyzes its substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2 , PIP2),

generating the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (5, 6).

IP3 acts to open intracellular calcium stores promoting an initial, transient, rise in intracellular

calcium. Depletion of intracellular calcium stores triggers the opening of plasma membrane store

operated calcium (SOC) channels (7) resulting in an influx of extracellular calcium and a secondary,

sustained calcium signal. The amplitude and duration of this sustained calcium signal is a key

determinant of the specific transcription program initiated in response to antigen receptor engagement

(8, 9, 10).

The most abundantly expressed PLCγ isoform in B lineage cells is PLCγ2. Chicken B

lymphoma cells lacking PLCγ2 are unable to generate IP3 in response to BCR engagement resulting in

the abrogation of receptor-mediated calcium mobilization (11). Similarly, mice deficient in PLCγ2

have a defective response to BCR engagement and exhibit a block in B cell development (12, 13, 14).

Despite its crucial role in BCR dependent calcium mobilization, the molecular events regulating

PLCγ2 activity remain incompletely defined.

Activation of PLCγ isoforms correlates with an increase in protein tyrosine phosphorylation

(15, 16), and inhibition of tyrosine kinases abolishes BCR-dependent IP3 production and calcium

signaling (17). Consistent with this, members of the Src, Syk/Zap70, and Tec/Btk kinase families are

each capable of phosphorylating PLCγ isoforms and peptide fragments in vitro (18, 19, 20, 21). The

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specific contribution of these kinases to BCR-mediated phosphorylation and activation of PLCγ in

vivo, however, remains unknown (4).

Btk/Tec kinases specifically regulate the BCR-dependent sustained phase of calcium

signaling (21, 22, 23). Deficient function of Btk leads to the related immunodeficiencies, X-linked

agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (XID) in mice (24).

IgM++/IgD+ B cells derived from XLA patients exhibit reduced IP3 levels and fail to generate a

sustained calcium signal in response to BCR engagement (21). Reconstitution of XLA B cells with

increasing doses of wild-type Btk can restore and specifically enhance the sustained calcium signal

(21). Despite the marked reduction in peak IP3 levels and calcium influx in XLA B cells, BCR-

dependent PLCγ2 tyrosine phosphorylation is indistinguishable from that present in normal B cells

(21,25). Similarly, Btk deficient murine B-lymphocytes, mast cells, and platelets also exhibit

diminished calcium mobilization and phosphoinositide hydrolysis despite normal receptor-mediated

PLCγ2 tyrosine phosphorylation (26, 27, 28). One potential explanation for these contrasting

observations is that Btk may regulate the sustained calcium signal via phosphorylation of a subset of

key tyrosine residues essential for PLCγ2 activity.

Recently two groups identified up to four putative regulatory phosphorylation sites within

PLCγ2 using in vitro kinase assays and reconstitution studies (20, 29). These sites included tyrosine

residues within the SH2-SH3 linker region (Y753 and Y759) (20, 29) and at the carboxyl-terminus

(Y1197 and Y1217) (29). Notably, the Y753 and Y759 PLCγ2 sites appeared analogous in location to

defined PLCγ1 SH2-SH3 linker regulatory sites (30). Reconstitution of PLCγ2 deficient cells with

PLCγ2 containing mutations in the SH2-SH3 linker sites alone (20), or all four sites concurrently (29),

led to the near ablation of BCR dependent calcium signaling. While these studies demonstrate a key

role for these sites in regulating PLCγ2 functional activity, the kinases responsible for mediating site-

specific modification in the context of receptor dependent signaling remain undefined.

In the current study receptor-mediated PLCγ2 activation was directly evaluated within the

context of an intact cellular system through the use of antibodies specific for distinct PLCγ2 phospho-

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tyrosyl regulatory residues. Our data demonstrate that at least four regulatory sites are phosphorylated

in response to BCR engagement and that phosphorylation of the PLCγ2 SH2-SH3 linker region is

entirely dependent upon Btk/Tec family kinases. This specific property provides a coherent

explanation for the unique role of Btk/Tec kinases in immunoreceptor dependent sustained calcium

signaling.

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Experimental Procedures

Cells and Reagents

The human B cell lines, WT (LDN-1), XLA-1 (LDX-1; 7), and Ramos were cultured in RPMI 1640

with L-Glutamine (CellGro) plus 5% fetal calf serum (FCS) and 50µM 2-mercaptoethanol. Total

splenocytes were obtained by harvesting whole spleens from age-matched Balb/c and Balb/Xid mice.

Splenocytes were prepared by mechanical disruption followed by depletion of erythrocytes by lysis

with ammonium chloride solution and resuspension in serum-free RPMI plus supplement. Antibody

reagents included: F(ab’)2 goat anti-human IgM Fc5µ specific and F(ab’)2 goat anti-murine IgM µ

chain specific (Jackson Laboratories); rabbit anti-murine Btk (31); anti-phosphotyrosine mAb 4G10,

anti-Itk, anti-Tec, and anti-Zap-70 (Upstate Biotechnology); anti-PLCγ2 (Q20), anti-PLCγ1 (1249),

and anti-Syk (C-20) (Santa Cruz Biotechnology); anti-phosphotyrosine mAb PY20 (Becton

Dickenson Pharmingen); anti-P-Y783-PLCγ1(Biosource International). For analysis by flow

cytometry, primary splenocytes were surfaced stained by incubating cells with anti-B220 (CD45R) PE

(1:200) and anti-IgM FITC (1:100) (Pharmingen) at 40C for 20 minutes. WT and XLA-1 human B

cells were surface stained with human anti-IgM FITC (1:100) (Southern Biotechnology). Data was

collected on a FACScalibur flow cytometer (Becton- Dickenson- BD) and analyzed with CELLQuest

software (BD).

Cell Stimulation, Lysis, Immunoprecipitation and Immunoblotting

Prior to stimulation, B cells were incubated in serum-free RPMI plus glutamine for 45 minutes at

370 C. 1 x 107 cells were then stimulated with either pervanadate (150 µM) or human or murine

specific IgM F(ab’)2 antibodies (10 µg/ml) for the indicated times. Pervanadate was prepared as

previously described (32). Cells were lysed for 10 minutes on ice in a lysis buffer consisting of

200mM boric acid pH 8.0, 150mM NaCl, 0.5% Triton X-100, 5mM NaF, 5mM EDTA, and 1mM

sodium orthovanadate plus protease inhibitors leupeptin and aprotinin (10µg/ml each) and

phenylmethylsulfonyl fluride (1mM). Cellular debris were spun down at 14,000 rpm at 40C for 15

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minutes and cleared lysates were used as whole cell lysate or for immunoprecipitation. PLCγ2 was

immunoprecipitated from cells stimulated with pervanadate due to increased stability and efficiency of

detection of specifically phosphorylated PLCγ2. In contrast, site-specific phosphorylation was more

reliably detected via analysis of whole cell lysate from IgM stimulated primary and transformed B

cells. PLCγ2 was immunoprecipitated by using 2µg of anti-PLCγ2 antibody (Q-20, Santa Cruz

Biotechnology) followed by incubation with protein sepharose A for 2 hr at 40C. All samples were

resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane. Pervanadate studies

represent 5 x 105 cell equivalents per lane whereas IgM studies represent 5 x 106 cell equivalents per

lane. Western blot analysis was performed using standard procedures as previously described (21).

Enhanced chemi-luminesence (ECL) was used for antibody detection according to manufacturer’s

instructions (Amersham).

Generation of Phosphospecific Antibodies and ELISA

PLCγ2 Y753, Y759, Y1197, and Y1217 phosphospecific antibodies were generated by inoculating

rabbits with 100µg of phosphopeptide conjugated to KLH as immunogen with Fruend’s complete

adjuvant. Phosphopeptide sequences used were as follows: PLCγ2 Y753 and Y759 see Fig. 2, Y1197

PVLESEEELpYSSCRQLRRRQ, and Y1217 CELNNQLFLpYDTHQNLRNAN. Initial injection was

followed by 4 booster injections every 2-3 weeks in Fruend’s incomplete adjuvant. To obtain affinity-

purified antibodies sera were first absorbed to a non-phosphorylated peptide column. Column flow-

through was then run over a phosphopeptide column to isolate phosphospecific antibodies for use in

our assays (31). To evaluate antibody reactivity by ELISA microtiter wells were coated with non-

phosphorylated or phosphorylated peptide species.

Site-Directed Mutagenesis and Generation of Recombinant Vaccinia Virus

Mutagenesis of full-length human PLCγ2 cDNA was carried out in the pBlue-Script (pBS) vector

using the QuickChange Site-directed mutagenesis kit (Stratagene). Primers were designed in which a

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single nucleotide change resulted in a single amino acid change (tyrosine to phenylalanine). The

Y753F/Y759F double mutant was generated by sequential mutagenesis. Mutagenized constructs were

digested and purified for cloning into the pSC66 vaccinia virus vector. Sequence analysis of pBS and

pSC66 PLCγ2 WT and mutant constructs was performed to verify each mutation. Recombinant

vaccinia virus expressing WT or mutant human PLCγ2 were generated as previously described (33).

Preparation of recombinant human PLCγ2

Full length PLCγ2 was expressed in bacteria using the pCAL expression and purification system.

Briefly, Escherichia coli BL21 were transformed with WT or mutant pCAL-PLCγ2 expression

constructs. Transformed bacteria were grown at room-temperature with constant agitation until

reaching an optical density at 550nm of 0.8-1.0. PLCγ2 expression was induced by the addition of 0.1

mM of “the inducer” (Molecular Research Laboratories, LLC). Bacteria were harvested and

recombinant PLCγ2 purified using a calmodulin column as previously described (19).

PLCγ2 in vitro IP3 assay for activity

Recombinant purified WT or mutant PLCγ2 was added to a reaction mixture containing 200 µM

Ptd[3H] Ins 4,5-P2 (25,000 dpm), 35mM NaH2 PO4 (pH 6.8), 70mM KCl, 2mM MgCl2, 1mM EDTA,

5µg/ml BSA, 5mM DTT, and 0.6 mM CaCl2 in the presence of recombinant, purified Lck (purified

recombinant human Gst-Lck provided by Alexander Y. Tsygankov, Temple Univeristy, Philadelphia,

PA). ATP (25uM final concentration) was added to selected reactions and mixtures were incubated

for 10 minutes at 250C. Reactions were stopped by transferring mixtures to an ice bath and adding of

0.5 ml chloroform/methanol/HCl (100:100:0.6) followed by 150µl 1N HCl with 5mM EDTA.

Aqueous and organic phases were separated by centrifugation and a portion of the upper aqueous

phase (200µl) was removed for liquid scintillation counting. All experiments were performed in

triplicate.

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PLCγ2 in vitro kinase assay

NIH-3T3 cells (5 x 106) were infected with vaccinia virus expressing recombinant wild-type or mutant

PLCγ2 at an MOI of 10 for 12 hrs at 370C. Cells were harvested and whole cell lysates prepared as

previously described in the experimental procedures. PLCγ2 was immunoprecipitated by using 2µg of

anti-PLCγ2 antibody (Q-20, Santa Cruz Biotechnology) followed by incubation with protein

sepharose A for 2 hours. Sepharose beads were then incubated in a reaction mixture containing

200mM Tris HCl pH 7.5, 0.4 mM EDTA, and 0.4 mM Na3VO4 in the presence or absence of 0.25 µg

active Lck kinase (Upstate Biotechnology) for 10 minutes at 300C. All other reagents including

enzyme dilution buffer and Magnesium/ATP cocktail were prepared per the manufacturer’s

instructions. Reactions were stopped by adding SDS sample buffer and boiling samples for 10

minutes. Alternatively, recombinant PLCγ2 was incubated with 0.25 µg of active Lck in the presence

or absence of 25µM ATP in a kinase buffer (50mM MOPS, pH7.4, 5mM MnCl2, 5mM MgCl2, 5mM

DTT) at 240C for 5 minutes. Reactions were stopped by the addition of SDS sample buffer and

boiling.

Calcium Mobilization Assays

A20 B cells (5 x 106) were loaded with 1mM indo-1 acetoxymethylester (indo-1 AM; Molecular

Probes) for 30 minutes at 370C in loading media (RPMI, 2% FCS, 10mM HEPES). Indo-1 AM

loaded cells were washed and resuspended in a buffer of Hank’s balanced salt solution (Sigma HBSS

with calcium) plus 10mM HEPES pH 7.0 prior to analysis. Cells were stimulated with either 10µg/ml

anti-human IgM (Jackson Laboratories), 10µg/ml anti-murine IgG (Jackson Laboratories) or 150µM

pervanadate and monitored for 3 minutes followed by the addition Ionomycin (1µM) as a positive

control. Intracellular calcium was measured using a bulk spectofluorimeter (Photon Techonology

International, PTI) and calculated as a ratio of 400:488nm emission following 350 nm excitation.

Data was analyzed with FELIX software (PTI).

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Results

Total tyrosine phosphorylation of PLCγ2 is not indicative of its signaling function in XLA B

cells.

To determine the role of Btk in PLCγ2 activation we compared the calcium response of wild-

type (WT) and XLA-1 human B cell lines (21) stimulated with anti-IgM or the protein-tyrosine

phosphatase (PTP) inhibitor, pervanadate. Consistent with our previously published data, XLA-1 B

cells exhibited reduced calcium mobilization in response to IgM engagement (Fig. 1A) Signaling via

pervanadate requires the presence of a functional BCR transducer complex and promotes

phosphorylation, recruitment, and activation of BCR associated molecules (32, 34). Pervanadate,

however, represents a more potent mimic of BCR engagement due to irreversible oxidation of key

cysteine residues on PTPs generating a more sustained phosphotyrosine signal (35). Notably, despite

the ability of pervanadate to produce a marked enhancement in the phosphotyrosine signal of total

cellular proteins compared to anti-IgM (Fig. 1C), these events were not sufficient to rescue the

calcium signaling defect in Btk deficient cells (Fig. 1B).

Tyrosine phosphorylation of PLCγ2 is commonly used as a measure of its activation status.

We compared the total PLCγ2 phosphotyrosine signal in IgM or pervanadate stimulated WT and Btk

deficient B cells. Consistent with previous results (21), tyrosine phosphorylation of PLCγ2 in

response to BCR engagement (as assessed by the pan-specific anti-phosphotyrosine antibody 4G10)

was indistinguishable between XLA-1 and WT B cells (Fig. 1D). Similarly, stimulation with

pervanadate resulted in equivalent levels of PLCγ2 tyrosine phosphorylation in XLA-1 and WT B

cells and no significant differences were observed over an extended activation time course (Fig. 1E

and data not shown). Similar results were obtained using an alternative, pan-specific anti-

phosphotyrosine antibody (PY20) and in studies using an independently derived XLA B cell line (data

not shown). Thus, while the reduction in BCR dependent IP3 production and calcium flux in Btk

deficient B cells clearly suggests a defect in the PLCγ2 pathway, these events do not correlate with the

phosphotyrosine content of PLCγ2 as measured by conventional anti-phosphotyrosine antibodies.

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Generation of site-specific antibodies to candidate regulatory PLCγ2 phosphorylation sites.

One explanation for these data is that Btk regulates PLCγ2 activity via phosphorylation of a

subset of tyrosine residues and that a loss of these specific phosphorylation events is not readily

detectable using antibodies that recognize global tyrosine phosphorylation. Initial attempts to identify

PLCγ2 regulatory sites by mass spectrometry were problematic due to low stoichiometric

concentrations and the generation of multiple phospho-peptide fragments. Other groups have reported

similar difficulties using this technique (20). We therefore chose to evaluate site-specific

phosphorylation using phosphospecific antibodies generated against individual candidate sites.

Potential phosphorylation sites were initially identified by comparing PLCγ2 to the better-

characterized, ubiquitously expressed PLCγ isoform, PLCγ1 (Fig. 2A). PLCγ1 contains three

regulatory tyrosines: Y771 and Y783 within its SH2-SH3 linker region and Y1254 at the carboxyl

terminus. These sites are inducibly phosphorylated in response to stimulation by growth factors (36,

37) or CD3 cross-linking in T cells (38) and mutation of any one of these residues leads to

dysregulated phospholipase activity (30). Specifically, mutation of Y783 (to F) results in the

complete abrogation of PLCγ1 function in fibroblasts and in reconstituted PLCγ1 deficient Jurkat T

cells (30, 39).

Although the PLCγ isoforms share a similar domain structure, they share only ~60% overall

amino-acid sequence homology, including less than 30% homology within the SH2-SH3 linker.

Comparison with PLCγ1 revealed three potential, similarly located, residues within the SH2-SH3

linker including Y743, Y753, and Y759 (Fig. 2B), however there was no distinct homology at the

carboxyl terminus. Among the initial candidate residues, location and spacing suggested that PLCγ2

Y759 was most homologous to PLCγ1 Y783. When examined for sequence homology with Btk

substrates (including WASP, BAP135, and the major Btk autophosphorylation site, Y223) several

sites including PLCγ2 Y753, Y759, Y1197, and 1217 were identified. Recently Rodiguez et al. also

identified the Y753, Y759 sites using similar methods (20) and Watanabe et al. identified these and

two additional sites (Y1197, 1217) using PLCγ2 GST-peptide fragments (29). Taken together these

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finding suggested that PLCγ2 Y753, Y759, Y1197, and Y1217 might be functionally homologous to

regulatory phosphorylation sites in PLCγ1.

To determine if the candidate residues within the SH2-SH3 linker region were receptor

dependent phosphorylation sites, we generated polyclonal phospho-site specific antibodies

recognizing either phosphorylated Y753 or Y759. Because of the proximity of the candidate residues,

the peptide immunogens overlapped by three residues but neither contained the alternative tyrosine

residue (Fig. 2B). Analysis by ELISA confirmed antibody specificity for the respective

phosphorylated PLCγ2 peptide immunogen (data not shown).

Antibody specificity was next evaluated in the context of a native protein species by analysis

of recombinant WT, single mutant (Y753F or Y759F), or double mutant (Y753FY759F) PLCγ2

phosphorylated in vitro using the purified src family kinase, Lck. Recombinant Lck was utilized

because of ease of expression, retained activity in bacterial cultures, and recent work demonstrating its

ability to phosphorylate PLCγ2 Y753 and Y759 in vitro (19). While each antibody recognized

phosphorylated WT PLCγ2 or PLCγ2 with a Y to F mutation at the alternate residue, mutation of the

original tyrosyl epitope resulted in the complete loss of antibody reactivity. There was no reactivity

with the unphosphorylated PLCγ2 species nor with PLCγ2 mutated at both Y753 and Y759 (Fig. 2C).

In addition, each antibody fully recognized PLCγ2 mutated at the putative carboxyl residues (Fig. 7A

right panel). Neither antibody exhibited reactivity with overexpressed or immunoprecipitated PLCγ1

(data not shown). The specificity of each of these antibodies within the context of a complex mixture

of phosphorylated B lineage signaling proteins was also evaluated by sequential western blotting.

Using whole cell lysates from unstimulated and pervanadate stimulated Ramos B cells (Fig. 2D), we

found each antibody predominantly recognized a phospho-protein species that co-migrated precisely

with PLCγ2.

We next determined if PLCγ2 Y753 and Y759 were modified in response to BCR

engagement. Whole cell lysates were prepared from Ramos B cells stimulated with anti-IgM and

sequentially blotted with each phosphospecific antibody followed by blotting with an anti-PLCγ2

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specific antibody. Phosphorylation at both sites was readily detectable within 30 seconds of IgM

receptor engagement and remained above the basal level of phosphorylation for up to 40 minutes (Fig.

2E and data not shown).

Y753 and Y759 are crucial for inducible phospholipase activity and for PLCγ2 mediated

calcium signaling.

To evaluate the role for PLCγ2 Y753/Y759 site-specific phosphorylation in PLCγ2 function,

we examined the consequences of site-directed mutagenesis of these residues. Using recombinant

PLCγ2 and Lck, the basal and inducible activity of WT, Y753F, Y759F, and Y753F/Y759F PLCγ2

were determined as measured by IP3 production (in the presence or absence of ATP). While WT

PLCγ2 exhibited an ~8 fold increase in IP3 production in the presence of Lck and ATP, PLCγ2

Y753F and PLCγ2 Y759F single-mutant proteins yielded only a 2-3 fold increase. Further mutation

of both residues (PLCγ2 Y753F/Y759F) effectively abrogated inducible phospholipase activity in all

experiments (Fig. 3A). We observed variation in the basal catalytic activity of the PLCγ2

Y753/Y759F mutant (slightly above or below WT in different experiments) and this has precluded

speculation as to the effect of this mutation on basal PLCγ2 activity. In contrast to Y753 and Y759,

mutation of an alternate tyrosine within the SH2-SH3 linker, Y743, resulted in basal and inducible

lipase activity that was indistinguishable from WT PLCγ2. This indicates that, unlike Y753 and

Y759, Y743 did not function as a regulatory tyrosine.

We next assayed the functional effect of the Y753/Y759 mutation within the context of

antigen receptor signaling in mammalian cells. A20 murine B cells were mock infected or infected

with vaccinia viruses expressing WT or the Y753F/Y759F mutant PLCγ2 and calcium mobilization

evaluated. While overexpression of WT protein had no appreciable effect even at very high

expression levels (5-10 fold above endogenous), overexpression of PLCγ2 Y753F/Y759F led to a dose

dependent reduction in both peak and sustained calcium levels (Fig. 3B). Notably, the calcium signal

in cells expressing the highest dosage of the Y753F/Y759F mutant was similar to that observed in Btk

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deficient murine and human B cells. These data, in conjunction with previous studies (20, 29), indicate

that phosphorylation of Y753 and Y759 is integral to the activation PLCγ2 and generation of the

BCR-dependent sustained calcium signal.

BCR dependent phosphorylation of PLCγ2 Y753 and Y759 requires Btk kinase activity.

If phosphorylation at PLCγ2 Y753 and Y759 is required for receptor-mediated sustained

calcium mobilization, then the diminished calcium signal in Btk deficient B cells might be attributable

to a loss of phosphorylation at these sites. To test this possibility, we first examined phosphorylation

of PLCγ2 in primary B cells from WT (Balb/c) and Btk-mutant (Balb/XID) mice. Total splenocytes

from each strain were stimulated with anti-IgM for the indicated times and assessed for site-specific

phosphorylation by sequential western blotting. Western blotting of lysates from both strains

demonstrated a marked, but equivalent, increase in overall tyrosine phosphorylation of multiple BCR

dependent substrates (Fig. 4A bottom panel). Phosphorylation of both Y753 and Y759 was readily

identified following stimulation of WT splenocytes, peaking between 2-5 minutes and diminishing to

near basal levels by ~20 minutes (Fig. 4A, left). Conversely, BCR stimulated Xid cells exhibited a

minimal, transient increase in Y759 phosphorylation and no detectable increase in Y753

phosphorylation (Fig. 4A, right). Densitometry analysis (corrected for the ~1.3 fold larger number of

WT IgM+ B cells) indicated an ~3-fold decrease in Y759 phosphorylation in Btk deficient vs. WT

cells. (Fig. 4B). Similar data were also obtained using purified CD43+ depleted, splenic B cells

(containing >90% B220+ cells; data not shown).

We next determined whether Y753 and Y759 phosphorylation was also altered in human

XLA B cells. WT and XLA-1 B cells were stimulated with anti-IgM for the indicated times and

evaluated using serial western blotting. While total PLCγ2 tyrosine phosphorylation was

indistinguishable (Fig. 1D), XLA-1 B cells exhibited a striking reduction in Y759 phosphorylation as

assessed over a 30 minute time period (Fig. 4C). Western blotting of the same lysates using the anti-

P-Y753 antibody did not yield a significant signal in either WT or XLA-1 lines. This finding most

likely reflects the relative weaker antibody reactivity of anti-P-Y753 and less robust BCR signaling

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activity in these LMP2-deficient EBV transformed B cell lines. To overcome the limited signal

intensity, we examined site-specific phosphorylation using the more potent BCR mimetic,

pervanadate. Pervanadate activation of WT cells resulted in rapid phosphorylation at both Y753 and

Y759 (Fig. 4D). In contrast, despite an equivalent overall phosphotyrosine signal (Fig. 4D, 3rd panel),

pervanadate stimulated XLA-1 B cells exhibited minimal phosphorylation at Y753 and Y759 (Fig.

4D, panels 1 and 2). Relative to WT B cells, phosphorylation at Y753 and Y759 was reduced 4- and

3-fold, respectively. Similar results were obtained using an alternative LMP2-deficient EBV-

transformed XLA B cell line (data not shown). Thus, phosphorylation of Y753 and Y759 required the

presence of functional Btk.

Since calcium signaling in XLA-1 B cells can be restored by exogenous expression of wild-

type Btk (21, 40), we next investigated whether WT Btk expression would restore PLCγ2 site-specific

tyrosine phosphorylation. XLA-1 B cells were mock-infected or infected with vaccinia virus

expressing Btk. Expression of WT Btk restored PLCγ2 Y759 phosphorylation in both IgM and

pervanadate stimulated cells and also restored Y753 phosphorylation in pervanadate stimulated XLA

B cells (Fig. 5A and B). In contrast, a kinase inactive Btk mutant (Btk K430R) had no effect (data not

shown). Together, these results demonstrate that Btk mediates BCR dependent site specific

phosphorylation of the PLCγ2 SH2-SH3 linker and is the major kinase responsible for this activity.

PLCγ2 Y753 and Y759 phosphorylation is specifically restored by Tec, but not Syk, family

kinases

The Tec family of non-receptor tyrosine kinases includes Tec, Btk, Itk (Tsk/Emt), Rlk (Txk),

and Bmx (41, 42). Previous work has demonstrated that alternative Tec kinases can functionally

reconstitute the calcium signal in Btk deficient B cell models (21, 40). Additionally, Syk can mediate

site-specific phosphorylation of PLCγ1 in vitro (18) and BCR-dependent PLCγ2 phosphorylation is

markedly reduced in Syk deficient B cell lines (43). To identify kinases capable of regulating PLCγ2

we evaluated the ability of alternative Tec and Syk/Zap-70 family kinases to restore PLCγ2 site-

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specific phosphorylation in Btk deficient B cells. XLA-1 B cells were mock infected, or infected with

vaccinia viruses expressing candidate Tec or Syk family kinases, and stimulated with pervanadate.

While expression of either Tec or Itk fully restored phosphorylation at Y753 and Y759 in XLA-1 B

cells (Fig. 5C), over-expression of Syk or Zap-70 had no appreciable effect (Fig. 5D). These findings

correlate with previous data demonstrating reconstitution of calcium signaling in Btk deficient cells

with the addition of Tec, but not Syk, kinases (21; and data not shown).

BCR dependent phosphorylation of PLCγ1 Y783 also requires Btk kinase activity.

The specificity of Btk in PLCγ2 regulatory site phosphorylation suggested that Tec kinases

might play an analogous role in the regulation of other PLCγ isoforms. While PLCγ2 is the

predominant isoform expressed in B cells, PLCγ1 is also expressed at low levels (2, 3) and is

inducibly phosphorylated in response to receptor engagement (5). Similar to our studies with PLCγ2,

preliminary experiments demonstrated equivalent levels of BCR-inducible total PLCγ1

phosphorylation in WT and XLA cells. We therefore we examined phosphorylation of the key

regulatory site, Y783, in the PLCγ1 SH2-SH3 linker. Following stimulation with pervanadate, PLCγ1

was immunoprecipitated from WT and XLA-1 B cells and western blotted using a commercially

available, Y783 phosphospecific antibody. Y783 phosphorylation was reduced ~4 fold in XLA-1 cells

compared with WT cells (Fig. 6A). Expression of Btk, or Itk, fully restored Y783 phosphorylation

(Fig. 6B) whereas, similar to PLCγ2, neither Syk nor Zap-70 could restore PLCγ1 SH2-SH3

regulatory site-specific phosphorylation.

The PLCγ2 carboxyl-terminal residues, Y1197 and Y1217, are not Btk dependent

phosphorylation sites.

Despite the loss of phosphorylation at two key residues, the high level of PLCγ2 total tyrosine

phosphorylation in Btk deficient cells signified the presence of additional phosphorylation sites.

Indeed, our initial sequence analysis of PLCγ2 suggested at least two additional candidate

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phosphorylation sites in the carboxyl terminus. Watanabe and colleagues recently identified these

residues (Y1197 and Y1217) as potential Btk-dependent phosphorylation sites using an in vitro kinase

assay with PLCγ2 GST-peptide fragments and purified Btk (29).

To determine the events regulating BCR dependent PLCγ2 carboxyl phosphorylation and the

specific role for Btk in this process, we generated phosphospecific antibodies against Y1197 and

Y1217. Individual antibody specificity was evaluated by ELISA (data not shown) and by full-length

wild type, Y1197F, Y1217F, and Y1197F/Y1217F PLCγ2 phosphorylated in vitro by Lck. Both

antibodies specifically recognized WT PLCγ2 or PLCγ2 mutated at the alternate carboxyl tyrosine

residue (Fig. 7A, left panel). Individual phospho-antibodies were then further examined for reactivity

among all four inducible sites. Analysis demonstrated the absence of antibody cross-reactivity

between the Btk phosphorylation consensus site sequences located in the SH2-SH3 linker and

carboxyl terminus (Fig. 7A, right panel).

Initial analysis revealed that both residues were inducibly phosphorylated in response to BCR

engagement in a B cell line as well as in primary B cells (Fig.7B and C). We next evaluated the

kinetics of Y1197 and Y1217 phosphorylation in IgM stimulated Balb and Balb/Xid primary B cells.

In contrast to Y753 and Y759, BCR engagement led to an equivalent level of inducible

phosphorylation at both Y1197 and Y1217 in splenocytes from both strains, at all time points (Fig.

7C). Similarly, WT and XLA-1 B cells exhibited indistinguishable levels of Y1197 and Y1217

phosphorylation at all time points following pervanadate stimulation (Fig. 7D). Consistent with these

data, reconstitution of XLA-1 cells with WT Btk had no effect on Y1197 phosphorylation. Btk

overexpression did lead to a modest, but consistent, increase in phosphorylation at Y1217 (Fig. 7E).

Together, these data demonstrate that Btk is not required for phosphorylation at the PLCγ2 carboxyl

terminus. However, it remains possible that, under some activation conditions, Btk may contribute to

phosphorylation at Y1217.

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Discussion

The amplitude and duration of the intracellular calcium signal is a key determinant of the

cellular response to immunoreceptor engagement. In particular, a sustained calcium signal is crucial

for the selective activation, or inhibition, of specific subsets of transcription factors (9, 10, 44) and for

the DAG associated NF-κB dependent survival signal (45). Although Btk is clearly required for

sustained calcium signaling, the mechanisms by which it exerts this unique effect remain incompletely

defined. The current work offers important insight into these events by providing in vivo evidence that

a key, non-redundant function of Btk/Tec kinases is to phosphorylate PLCγ2 at two major regulatory

sites, Y753 and Y759, crucial for immunoreceptor-dependent PLCγ2 activation.

Our studies complement previous work demonstrating an essential role for the SH2-SH3

linker region in regulating PLCγ function (19, 20, 29, 30, 36). Characterization of PLCγ1 identified

key inducibly phosphorylated regulatory sites, Y771 and Y783, within the PLCγ1 SH2-SH3 linker (5,

37, 38). Mutation of either residue resulted in altered PDGF-induced PLCγ1 activity. In these studies,

PLCγ1 Y783 served a positive regulatory role whereas Y771 appeared to be a negative regulator (30).

Although the PLCγ1 and PLCγ2 SH2-SH3 linker sites are unlikely to be wholly analogous in function,

this region is clearly pivotal in the regulation of PLCγ activity. Using phospho-specific antibodies we

demonstrate that BCR engagement leads to a rapid and sustained phosphorylation at two PLCγ2 SH2-

SH3 linker residues, Y753 and Y759. Consistent with a predicted regulatory function, phosphorylation

of Y753 and Y759 (but not a similarly located linker tyrosine, Y743) was required for the optimal

inducible enzymatic activity of PLCγ2 in vitro. Analogous to results in T cells overexpressing the

PLCγ1 linker mutant Y783F (39), overexpression of PLCγ2 linker mutants promoted a dose-

dependent inhibition of BCR-mediated calcium mobilization. These results are consistent with studies

in PLCγ2 deficient avian B lymphoma cells that demonstrate a markedly reduced calcium response in

cells reconstituted with PLCγ2 Y753/Y759 double mutant proteins (20, 29).

Most notably, our data demonstrate that BCR-dependent PLCγ2 activation is mediated

almost entirely via Tec/Btk kinase site-specific tyrosine phosphorylation within the PLCγ SH2-SH3

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linker. While the relative total level of PLCγ2 tyrosine phosphorylation appears intact in IgM and

pervanadate stimulated Btk deficient human B cells, there was a striking reduction in linker site-

specific phosphorylation. Similarly, BCR-dependent PLCγ2 Y753 and Y759 phosphorylation was

severely reduced in splenic B cells from Btk mutant, XID mice. The limited residual, inducible Y759

phosphorylation signal present in XID cells declined much more rapidly (peaking at 2 minutes) than

the coordinate signal in WT cells where phospho-Y759 was detectable for >15 minutes following

receptor engagement. These kinetic data directly parallel previous functional data in which Btk

deficient B cells were unable to maintain IP3 production in response to receptor engagement (21, 28).

Strikingly, the addition of Btk or other Tec family kinase members fully restored receptor mediated

PLCγ2 site-specific phosphorylation at both residues in XLA B cells. This correlates with previous

data demonstrating functional reconstitution of the sustained calcium signal in Btk deficient cells by

Tec family kinases (21, 40). In addition, reconstitution of site specific phosphorylation required the

kinase activity of Btk, as overexpression of kinase inactive Btk failed to restore linker phosphorylation

(data not shown).

Despite the marked reduction in Y753 and Y759 phosphorylation in both XLA-1 and XID B

cells, a low level of inducible phosphorylation remained detectable at both sites. Although Syk/Zap70

kinases have been implicated in PLCγ regulation (11, 43) several lines of evidence suggest that

endogenous Syk is unlikely to modify these regulatory sites. Interpretation of the potential role for

Syk in PLCγ2 regulation is complicated by its multiple functions including phosphorylation of BLNK

and activation of PI3K, events that could directly or indirectly lead to reduced PLCγ2 phosphorylation

(43, 46,47, 48). These Syk/BLNK dependent events, however, remain unaffected in Btk deficient cells

where a marked loss in Y753 and Y759 phosphorylation is observed (11, 49). Furthermore, while Tec

kinases rescued PLCγ2 site-specific phosphorylation, Syk/Zap-70 kinases failed to do so even at

supra-endogenous expression levels. Finally, a previous study clearly indicated that PLCγ2 is a

relatively poor substrate for purified recombinant Syk in vitro (20).

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The most likely explanation for residual linker phosphorylation in XLA B cells is redundancy

provided by alternative Tec kinase(s) (41, 50). All hematopoietic cell lineages express at least two

distinct Tec family kinases. B lineage cells, including the XLA and XID B cell populations studied

here, express both Btk and Tec. Consistent with this idea, the developmental differences between Btk

deficient humans and mice are largely attributable to variances in Tec activity and/or expression levels

(51). Indeed, over-expressed Tec can fully compensate for Btk-dependent PLCγ2 site specific

phosphorylation and calcium mobilization (Fig.5C, 21, 40). We have attempted to determine the

relative contribution of Tec in BCR dependent signaling using Btk/Tec double deficient mice.

Unfortunately, the severe reduction in peripheral B cell numbers has made this approach difficult (51;

and data not shown). While our current data cannot rule out a role for non-Tec family kinases, these

combined observations strongly suggest that PLCγ2 SH2-SH3 linker phosphorylation is

predominantly, if not entirely, dependent on Tec family kinases.

Our data also suggest Tec kinase-dependent SH2-SH3 linker phosphorylation represents a

key control point for BCR dependent activation of PLCγ1. Correlating with our PLCγ2 data,

phosphorylation of the PLCγ1 SH2-SH3 linker regulatory site Y783 was also markedly reduced in

XLA-1 cells and this phosphorylation could be restored by the expression of Tec, but not Syk, family

kinases. These data are consistent with observations in T cells expressing raft-targeted PLCγ1. In

those studies PLCγ1 phosphorylation and calcium dependent gene expression occurred independently

of Zap-70 or the adaptors LAT and Slp-76. Furthermore, membrane targeted PLCγ1 was readily

phosphorylated by the raft associated Tec kinase, Rlk, but not by Zap-70 (52). Because PLCγ1 is

activated in response to multiple alternative immunoreceptors, this Tec kinase-dependent regulatory

mechanism is likely to be broadly conserved.

The level of receptor dependent global tyrosine phosphorylation in Btk deficient B cells

suggested the presence of additional tyrosine phosphorylation sites within PLCγ2. A recent study

using PLCγ2 GST-peptide fragments identified two carboxyl-terminal phosphorylation sites, Y1197

and Y1217, and suggested that both sites were targets for Btk (29). Our data clearly demonstrate that,

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in the context of BCR dependent signaling, phosphorylation of Y1197 and Y1217 occurs

independently of endogenous Btk activity. However, the ability of over-expressed Btk to enhance

Y1217 phosphorylation suggested that, under some circumstances, Btk might participate with an

alternative kinase(s) in the optimal phosphorylation of this site. Primary phosphorylation of the PLCγ2

carboxyl regulatory sites is most likely mediated by a Src family kinase(s) as recent studies, including

the current work, demonstrate that Src family kinases can directly phosphorylate PLCγ2 in vitro (19,

20). In contrast, Syk overexpression had no additive effect on Y1197 and Y1217 phosphorylation

(Fig. 7E) and Syk does not readily modify purified PLCγ2 (20). Notably though, phosphorylation of

these additional BCR-dependent sites, in the absence of Btk activity, is clearly insufficient to mediate

the sustained activation of PLCγ2.

Our data indicate the need for caution in utilizing global phosphotyrosine content as a

measure of PLCγ2 activity. A lack of correlation between receptor mediated PLCγ phosphorylation

and calcium mobilization has been reported in Btk deficient cell models including signaling via the

calnexin/surrogate light-chain/CD79b complex on pro-B cells (53), the BCR on B cells (21, 26), and

the FcεRI receptor on mast cells (28). Similar observations have also been reported with respect to

oxidative stress signaling in B cells (54) and collagen receptor mediated calcium signaling in platelets

(27). These results have lead to confusion regarding interpretation of the role for Btk in downstream

signals. Use of phosphospecific antibodies should clarify the events modulating activation of this

pivotal enzymatic pathway in these and additional receptor systems.

Current observations, in association with previously published data, suggest the following

working model for regulation of PLCγ2 activity and BCR-dependent calcium signaling: Upon receptor

engagement, raft associated Src kinases are activated and Syk is recruited to the receptor complex and

activated. Syk then phosphorylates the adaptor molecule, BLNK resulting in the recruitment of a

BLNK/ PLCγ2 complex to the plasma membrane (46, 55). Concurrently, Btk is recruited to the

membrane via its PH domain and activated by src family kinases (23, 33, 56)(Fig. 8A). Once co-

localized in the BCR signalosome, activated Btk is within proximity to PLCγ2 (through a direct

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association with BLNK (46), a direct interaction with PLCγ2 (57), or an alternative, Btk SH2

mediated interaction (58)). These combined events promote regulatory phosphorylation of the PLCγ2

SH2-SH3 linker and carboxyl terminus by Btk/Tec and src kinases, respectively. Activation of

PLCγ2, specifically following phosphorylation of Y753 and Y759, leads to the generation of threshold

IP3 levels, required for depletion of intracellular calcium stores and induction of store-operated

calcium (SOC) influx. Recent collaborative work from our laboratory has also demonstrated that Btk

is constitutively associated with phosphatidylinositol-4-phosphate 5-kinase (PIP5K) isoforms. By

means of this interaction, Btk and PIP5K are coordinately recruited into the BCR signalosome

promoting an increase in the local levels of PtdIns-4,5-P2 and PtdIns-3,4,5-P2 (59). Retention of this

Btk/PLCγ2/PIP5K complex at the membrane therefore allows for continued activation of PLCγ2 and

generation of peak and sustained IP3 levels to mediate the store-operated calcium channel dependent,

sustained calcium signal (Fig. 8B).

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Acknowledgements

We thank Drs. A. Scharenberg, Martin Turner, Owen Witte, Ed Clark, and Sharon Matthews for

critical reading of the manuscript and members of the Rawlings lab including Keun Chae, Kevin Kipp,

and Ruby Tabuchi for technical assistance and Phyllis Yu and Thomas Su for thoughtful discussions.

D.J.R. is the recipient of a McDonnell Scholar Award, a Leukemia and Lymphoma Society Scholar

Award, and the Joan J. Drake Grant for Excellence in Cancer Research. This work was supported, in

part, by NIH grants HD37091 and CA81140.

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Figure Legends Figure 1. XLA-1 B cells exhibit defective anti-IgM and pervandate induced calcium

mobilization despite induction of an equivalent level of total PLCγ2 tyrosine phosphorylation.

Indo-1AM loaded WT (WT) and XLA-1 cells were stimulated with either 10µg/ml anti-human IgM

(A) or 150µM pervanadate (PV) (B) and monitored for changes in intracellular calcium by

spectroflourimetry. (C) Serum starved WT and XLA-1 B cells were stimulated with either anti-human

IgM or pervanadate for the indicated times. Whole cell lysates (WCL) were assessed by western

blotting with the 4G10 anti-phosphotyrosine antibody (PY). (D & E) PLCγ2 was immunoprecipitated

(IP) from whole cell lysate prepared as described in (C) following 3 minutes stimulation with anti-IgM

or pervanadate and analyzed for total tyrosine phosphorylation. The same blot was stripped and re-

probed using anti-PLCγ2 to evaluate protein loading. Representative data from more than 5

independent experiments are shown.

Figure 2. PLCγ2 P-Y753 and PLCγ2 P-Y759 antibodies are specific for induced

phosphorylation at individual sites.

(A) Domain structure of PLCγ isoforms and location of established and potential regulatory tyrosine

residues within the SH2-SH3 linker and carboxyl-terminus regions of PLCγ2 and PLCγ1. Putative

sites in PLCγ2 were chosen based similarity of location compared with known PLCγ1 regulatory sites,

Y771, Y783, and Y1254 and/or representing potential Btk phosphorylation sites. (B) Alignment of the

SH2-SH3 linker regions of human PLCγ isoforms. Phosphospecific antibodies were generated against

predicted phosphorylation sites, Y753 and Y759, using the indicated peptide sequences. (C) Left

panels: Bacterially expressed, full-length recombinant WT, Y753F, and Y759F mutant human PLCγ2

were purified and phosphorylated in vitro using purified recombinant Lck in the absence (-) or

presence (+) of ATP as described in materials and methods. Right panel: Virally expressed

recombinant WT and Y753F/Y759F PLCγ2 were purified and incubated with Mg+/ATP in the

presence or absence of Lck. Samples were analyzed by western blotting with each PLCγ2

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phosphospecific antibody. Blots were subsequently stripped and re-blotted with an anti-PLCγ2

antibody. (D) Ramos B cells were serum starved for 45 minutes and stimulated with pervanadate for

3 minutes at 370C. Whole cell lysates from unstimulated and stimulated cells were assessed by

sequential immunoblotting with the indicated antibodies in the order shown (left to right). (E) Ramos

cells were serum starved for 45 minutes and stimulated with anti-IgM for the times indicated. Whole

cell lysates were analyzed by sequential immunoblotting with the antibodies noted (top to bottom).

Experiments utilizing an alternative order of sequential blotting yielded identical results.

Representative data from one of at least three independent experiments are shown.

Figure 3. Y753 andY759 are crucial for PLCγ2 function in vitro and in response to BCR

engagement. (A) Recombinant wild type, Y753F, Y759F, Y743F, and Y753F/Y759F PLCγ2 were

subjected to in vitro phosphorylation using recombinant Lck in the absence (white bars) or presence

(shaded bars) of ATP and assayed for phospholipase activity. Activity was measured by [3H]IP3

production in the presence of excess Ptd [3H]Ins 4,5-P2 substrate and quantified as µM IP3/mg PLCγ2.

(B) A20 cells were mock infected, or infected with vaccinia viruses expressing WT (WT) or

Y753F/Y759F mutant PLCγ2 at the indicated multiplicity of infection (MOI) for 8 hours at 370C.

Protein expression levels were controlled by viral titer and normalized by MOI. Cells were loaded

with Indo-1 AM for 30 minutes and stimulated through the B cell receptor using 10µg/ml anti-murine

IgG F(ab)2 at 20 seconds. Calcium flux was monitored using spectroflourimetry. Traces are marked:

open diamonds, mock infected cells MOI=10; closed squares, WT PLCγ2, MOI =10; open circles,

Y753F/Y759F, MOI = 2; open triangles, Y753F/Y759F, MOI = 5; and open squares, Y753F/Y759F,

MOI = 10. Addition of ionomycin demonstrated equivalent peak calcium responses in all infected

populations (data not shown). Whole cell lysates from each population were blotted with anti-PLCγ2

to evaluate the relative protein expression levels (bottom panel). These data are representative of at

least 4 independent experiments.

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Figure 4. Btk deficient B cells exhibit markedly reduced site-specific PLCγ2 tyrosine

phosphorylation in response to BCR engagement. (A) Total splenocytes were isolated from Balb/c

and Balb/XID mice, serum starved for 10 minutes, and activated by 10µg/ml anti-murine IgM cross-

linking for the times indicated. Whole cell lysates were analyzed by sequential immunoblotting with

P-Y759 and P-Y753 phosphospecific antibodies. The membrane was also stripped and sequentially

blotted with anti-PLCγ2, anti-Btk, and anti-phosphotyrosine antibodies to assess protein loading, and

cellular activation. Densitometry was performed to measure the relative phosphorylation of Y753 and

Y759 between Balb and Balb/Xid splenocytes (data under respective blot). Data were normalized to

PLCγ2 protein content. (B) Relative phosphorylation of PLCγ2 Y759 was quantified by densitometry

for each time point and normalized to both PLCγ2 protein content and to reflect the reduced number

of IgM+ cells in Xid (25% fewer based on B220 staining and FACS analysis) in the experiment

shown. Representative data from one of three experiments are shown. (C) Serum starved WT and

XLA-1 B cells were stimulated with anti-IgM for the times indicated. Whole cell lysates were blotted

with the anti-P-Y759 antibody followed by anti-PLCγ2 blotting to evaluate protein loading.

Densitometry data (normalized to total PLCγ2 content) indicating the relative signal intensity are

displayed below the anti-P-Y759 phosphospecific blot. (D) Serum starved WT and XLA-1 B cells

were stimulated with pervanadate for 3 minutes, lysed, and PLCγ2 was immunoprecipitated and

immunoblotted sequentially with anti-P-Y753, anti-P-Y759, anti-PLCγ2, and anti-phosphotyrosine

antibodies, respectively. Representative data from one of more than 5 independent experiments are

shown.

Figure 5. BCR dependent, site-specific phosphorylation of PLCγ2 in XLA-1 B cells can be

restored by Tec, but not Syk/Zap-70, family kinases. (A) XLA-1 B cells were mock infected or

infected using recombinant vaccinia virus expressing wild type Btk (MOI of 10 for 12 hrs.) Cells

were serum starved and stimulated with anti-IgM for 5 minutes and lysed. Whole cell lysates were

prepared as described in materials and methods and sequentially blotted for specific phosphorylation

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and expression. (B) XLA-1 B cells were infected as in (A). Cells were serum starved, stimulated with

pervanadate for 3 minutes, and PLCγ2 was immunoprecipitated. Site-specific phosphorylation was

evaluated by sequential western blotting with the antibodies indicated (top to bottom). (C- D) WT Btk,

Itk, Tec, Syk, and Zap-70 proteins were expressed in XLA-1 cells using recombinant vaccinia viruses

followed by stimulation as described in (B). PLCγ2 was immunoprecipitated and analyzed by

sequential immunoblotting with the indicated antibodies. Representative data from one of more than 3

independent experiments are shown. Whole cell lysates were blotted with antibodies to Btk, Syk, and

Zap-70 to evaluate protein expression (D, bottom panels).

Figure 6. BCR dependent phosphorylation of PLCγ1 Y783 requires Btk and is restored by Tec

but not Syk family kinases. (A) WT and XLA-1 B cells were serum-starved, stimulated with

pervanadate for 3 minutes, and PLCγ1 was immunoprecipitated. Immunoprecipitates were

immunoblotted with an anti-P-Y783 antibody followed by anti-PLCγ1. Densitometry data

(normalized to total PLCγ1 content) indicating the relative signal intensity are displayed below the

anti-P-Y783 phosphospecific blot. (B) WT Btk, Itk, Tec, Syk and Zap-70 proteins were expressed in

XLA-1 cells using recombinant vaccinia. PLCγ1 immunoprecipitates were sequentially blotted as in

(A). Whole cell lysates were blotted with antibodies to Btk, Itk, Syk, and Zap-70 to evaluate protein

expression (bottom panels). Representative data from one of three independent experiments are

shown.

Figure 7. PLCγ2 Y1197 and Y1217 inducible phosphorylation is not Btk dependent. (A) Full-

length recombinant WT, Y1197F, Y1217F, Y1197F/Y1217F, and Y753F/Y759F PLCγ2 were

immunoprecipitated and phosphorylated by Lck in vitro as described in materials and methods.

Individual samples were divided equally and analyzed by western blotting by sequential stripping and

reprobing with the antibodies noted. (B) Ramos B cells were serum starved for 45 minutes and

stimulated with anti-IgM for the indicated times. Whole cell lysates were analyzed by sequential

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western blotting with the antibodies listed. (C) Total splenocytes from Balb/c and Balb/Xid mice were

serum starved for 10 minutes and activated by anti-IgM per the time course. Whole cell lysates were

equally divided and blotted with the stated antibodies. (D) Serum starved LDN and XLA-1 cells were

stimulated with pervanadate for the indicated time points. Immunoprecipitated PLCγ2 was divided

into three equal samples, run in triplicate, and individually blotted. (E) XLA-1 B cells were mock-

infected or infected with vaccinia virus for expression of Btk and Syk as described in Fig. 6A. Cells

were serum starved for 45 minutes prior to stimulation with pervanadate for 3 minutes. PLCγ2 was

immunoprecipitated and evaluated by sequential western blotting with the indicated antibodies. All

data shown are representative of at least three independent experiments.

Figure 8. A working model for PLCγ2 mediated, BCR-dependent calcium signaling. (A)

Engagement of the BCR initiates formation of a lipid-raft associated signalosome providing access to

substrates for recruitment and activation. (B) BLNK mediated recruitment of PLCγ2 to the

signalosome promotes phosphorylation of the PLCγ2 carboxyl residues by a src-family kinase (most

likely Lyn) and of the PLCγ2 SH2-SH3 linker by Btk/Tec kinases. This latter event is essential for

full PLCγ2 enzymatic activity. Btk dependent recruitment of PIP5K serves to increase local levels of

both PIP2 and PIP3. Thus, localization of PLCγ2 with the Btk/PIP5K complex functions coordinately

to regulate PLCγ2 activity through the phosphorylation of four key tyrosine residues and through

enhanced accessibility of PLCγ2 to its enzymatic substrate. See discussion for details.

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Fig. 1Humphries, et al.

A

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

0 50 100 150 200 2500

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 200 400 600 800

Time (sec)Time (sec)

Indo

-1 R

ati o

I nd o

-1 R

atio

WT

XLA

WT

XLA

+ αIgM + PervanadateBA

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

0 50 100 150 200 2500

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 200 400 600 800

Time (sec)Time (sec)

Indo

-1 R

ati o

I nd o

-1 R

atio

WT

XLA

WT

XLA

+ αIgM + PervanadateB

D

αPY

αPLCγ2

IP: αPLCγ2

WT XLAαIgM: - + - + Blot:

αPY

αPLCγ2

IP: αPLCγ2



E

αPY

αPLCγ2

IP: αPLCγ2

WT XLA- +PV: - + Blot:

αPY

αPLCγ2

IP: αPLCγ2

WT XLA- +PV: - + Blot:WT XLA- +PV: - + Blot:

C

αPY

WT XLA

0 5 15 30 3 Blot:WCL 3αIgMPVαIgM PV PV PV

0 5 15 30 3 3

WT XLA

Exp: 2 min 30s

62

83

115

179αPY

WT XLA

0 5 15 30 3 Blot:WCL 3αIgMPVαIgM PV PV PV

0 5 15 30 3 3

WT XLA

Exp: 2 min 30s

62

83

115

179

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A

B Y753 peptide Y759 peptide

L E R Y N - M E R D I N S L Y D V S R - - M Y V D P S E I740 743 753 759 765

PLCγ2

L E K I G T A E P D Y G A L Y E G R N P G F Y V E A N P M771 783 789

PLCγ1761

Y753 peptide Y759 peptideY753 peptide Y759 peptide


PLCγ2


PLCγ1761


PLCγ2


PLCγ1761

PLCγ2

753Y-P 759Y-P

PH X SPH YSH2 SH2 SH3 SPH

1197Y-P 1217Y-P

PLCγ1

771Y-P 783Y-P 1254Y-P


PLCγ2

753Y-P 759Y-P


1197Y-P 1217Y-P

PLCγ2

753Y-P 759Y-P


1197Y-P 1217Y-P

PLCγ1

771Y-P 783Y-P 1254Y-P

PH X SPH YSH2 SH2 SH3 SPHPLCγ1

771Y-P 783Y-P 1254Y-P


D

WCL

αPLCγ2 αP-Y759

PV:

αP-Y753

- +Ramos B Cells

αPY

- + - + - +

Blot:

WCL

αPLCγ2 αP-Y759

PV:

αP-Y753

- +Ramos B Cells

αPY

- + - + - +

Blot:

E

αIgM:

αP-Y753

0 30s 1’ 2’ 5’

WCL

Ramos B Cells

αPLCγ2

αP-Y759

Blot:αIgM:

αP-Y753

0 30s 1’ 2’ 5’

WCL

Ramos B Cells

αPLCγ2

αP-Y759

Blot:

C

αP-Y753- + - + - +

αPLCγ2

ATP:WT Y753F Y759F

Blot:PLCγ2:Lck+

αP-Y753- + - + - +

αPLCγ2

ATP:WT Y753F Y759F

Blot:PLCγ2:Lck+PLCγ2:Lck+

αP-Y759

αPLCγ2

WT- + - + - +

Y753F Y759FATP: Blot:PLCγ2:Lck+

αP-Y759

αPLCγ2

WT- + - + - +

Y753F Y759FATP: Blot:PLCγ2:Lck+PLCγ2:Lck+

PLCγ2:

αP-Y753

- + - +

αPLCγ2

Lck:WT Y53/59F

Blot:αP-Y759

PLCγ2:

αP-Y753

- + - +

αPLCγ2

Lck:WT Y53/59F

Blot:αP-Y759

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A

0

1

2

3

4

5

6

WT Y753F Y759F YY Y743F

∠m

ol IP

3/m

g PL

C∠2

VacciniaConstruct:

PLCγ2YY 5

PLCγ2YY 10

PLCγ2WT 10M.O.I.:

Blot: αPLCγ2

PLCγ2

PLCγ2YY2

A20Mock

10

VacciniaConstruct:

PLCγ2YY 5

PLCγ2YY 10

PLCγ2WT 10M.O.I.:

Blot: αPLCγ2Blot: αPLCγ2

PLCγ2

PLCγ2YY2

A20Mock

10

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

0 20 40 60 80 100 120 140 160 180 200

B

YY (5)

WT (10)YY (2)

YY (10)

Mock (10)

Time (sec)

Indo

-1 R

atio

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

0 20 40 60 80 100 120 140 160 180 200

B

YY (5)

WT (10)YY (2)

YY (10)

Mock (10)

Time (sec)

Indo

-1 R

atio

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C

B


DIP: αPLCγ2

- + - +WT XLA

PV:αP-Y753

αP-Y759

αPY

αPLCγ2

1 12 1 3

0

1

2

3

4

5

6

7

0 2' 5' 10' 20' 0 2' 5' 10' 20'

Balb Balb/Xid

Rel

ativ

e Y7

59 P

hosp

hory

latio

n

0

1

2

3

4

5

6

7

0 2' 5' 10' 20' 0 2' 5' 10' 20'

Balb Balb/Xid

Rel

ativ

e Y7

59 P

hosp

hory

latio

n

WT XLA

αP-Y759

αPLCγ2

0 2’ 5’ 15’ 30’ 0 2’ 5’ 15’ 30’αIgM:

WCLBlot:

1 5 4.6 3.4 1.7 1 2.5 3.0 2.5 1.8

WT XLA

αP-Y759

αPLCγ2

0 2’ 5’ 15’ 30’ 0 2’ 5’ 15’ 30’αIgM:

WCLBlot:

1 5 4.6 3.4 1.7 1 2.5 3.0 2.5 1.8

A

αPLCγ2

αP-Y753

αBtk

αP-Y

82

62

50

αP-Y759

0 2’ 5’ 10’ 20’ 0 2’ 5’ 10’ 20’BALB BALB/XID

αIgM:

WCL

1 6.9 4.7 3.3 1.6 1 1.7 0.8 0.4 0.6

Blot:

1 2.1 5.2 1.1 1 1 1 1 1 0.9

αPLCγ2

αP-Y753

αBtk

αP-Y

82

62

50

αP-Y759

0 2’ 5’ 10’ 20’ 0 2’ 5’ 10’ 20’BALB BALB/XID

αIgM:

WCL

1 6.9 4.7 3.3 1.6 1 1.7 0.8 0.4 0.6

Blot:

1 2.1 5.2 1.1 1 1 1 1 1 0.9

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C

IP: PLCγ2

αPLCγ2

αP-Y759

αPY

αP-Y753

- + - +

XLA+Itk

XLA+Tec

- +PV: - + - +WT XLA

XLA+Btk

Blot:

C

IP: PLCγ2

αPLCγ2

αP-Y759

αPY

αP-Y753

- + - +

XLA+Itk

XLA+Tec

- +PV: - + - +WT XLA

XLA+Btk

Blot:

BWT XLA XLA + Btk

IP: PLCγ2

- + - + - + PV:

αP-Y753

αP-Y759

αPY

αPLCγ2

Blot:


XLA+Syk

PV:WT XLA

XLA+Btk

IP: PLCγ2

- + - + - + - +- +

XLA+Zap-70

αPLCγ2

αP-Y759

αPY

αP-Y753

DBlot:

αSyk

αZap-70

WCL αBtk

XLA+Syk

PV:WT XLA

XLA+Btk

IP: PLCγ2

- + - + - + - +- +

XLA+Zap-70

αPLCγ2

αP-Y759

αPY

αP-Y753

DBlot:

αSyk

αZap-70

WCL αBtk

AWT XLA XLA + Btk

WCL- + - + - + αIgM:

αP-Y759

αPLCγ2

Blot:

αBtk

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A


B

αPLCγ1

αP-Y783IP: PLCγ1

XLA+Syk

XLA+Zap-70

- +

XLA+Itk

- +PV: - + - +WT XLA

XLA+Btk- + - + Blot:

WCL αBtk

αItk

αSyk

αZap-70

αPLCγ1

αP-Y783IP: PLCγ1

XLA+Syk

XLA+Zap-70

- +

XLA+Itk

- +PV: - + - +WT XLA

XLA+Btk- + - + Blot:

WCL αBtk

αItk

αSyk

αZap-70

IP: PLCγ1 αP-Y783

αPLCγ1

PV: - +WT XLA

- + Blot:

1 11.9 1 2.9

IP: PLCγ1 αP-Y783

αPLCγ1

PV: - +WT XLA

- + Blot:

1 11.9 1 2.9

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B

A

C

αPLCγ2

αP-Y1217

αP-Y1197

0 2’ 5’ 10’ 20’ 0 2’ 5’ 10’ 20’BALB BALB/XID

αIgM:

WCL

Blot:

αPLCγ2

αP-Y1217

αP-Y1197

0 2’ 5’ 10’ 20’ 0 2’ 5’ 10’ 20’BALB BALB/XID

αIgM:

WCL

Blot:

D

αPLCγ2

αP-Y1217

αP-Y759

0 30s 1’ 2’ 5’ 0 30s 1’ 2’ 5’LDN XLA

PV:

IP:PLCγ2

Blot:

αPY1197

αPLCγ2

αP-Y1217

αP-Y759

0 30s 1’ 2’ 5’ 0 30s 1’ 2’ 5’LDN XLA

PV:

IP:PLCγ2

Blot:

αPY1197

αIgM:

αP-Y1197WCL

Ramos B Cells

αP-Y759

αP-Y1217

Blot:0 30s. 2’ 5’ 10’ 30’

αPLCγ2

αIgM:

αP-Y1197WCL

Ramos B Cells

αP-Y759

αP-Y1217

Blot:0 30s. 2’ 5’ 10’ 30’

αPLCγ2

E

αPLCγ2

IP: PLCγ2PV:

αP-Y1197

αP-Y759

αPY

αP-Y1217

WT- +

XLA- +

XLA + Btk- + Blot:

XLA + Syk- +

αPLCγ2

IP: PLCγ2PV:

αP-Y1197

αP-Y759

αPY

αP-Y1217

WT- +

XLA- +

XLA + Btk- + Blot:

XLA + Syk- +

WT

αP-Y1197

αPLCγ2

- + - + - +Y1197F Y1217FPLCγ2:

Lck: Blot:

αP-Y1217

WT

αP-Y1197

αPLCγ2

- + - + - +Y1197F Y1217FPLCγ2:

Lck: Blot:

αP-Y1217

αP-Y753

αPLCγ2

WT- +

Y53/59FPLCγ2:Lck: Blot:

Y11/12F

αP-Y759

αP-Y1197

αP-Y1217

- + - +

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A

PKCβ

PIP2Syk

Btk

PIP3LynSOC Channel

SOC ControlCalcium Store

BLNKP

PBLNK

P

P

Y1217Y1197

PLCγ2PH

SPH

X Y

SPH SH3SH2SH2 Y753

Y759

Y1217Y1197

PLCγ2PH

SPH

X Y

SPH SH3SH2SH2

PLCγ2PLCγ2PH

SPH

X Y

SPH SH3SH2SH2

PH

SPH

X YX Y

SPH SH3SH2SH2

SH3SH2SH2 Y753

Y759

IP3

DAG

Ca++

Sustained Ca++ PKCβSyk

BLNKP Btk

PIP3LynSOC Channel

SOC ControlCalcium Store

P

Ca++

P

Y1217

Y1197

PLCγ2PH

SPH

X Y

SPH SH3SH2SH2

PLCγ2PLCγ2PH

SPH

X Y

SPH SH3SH2SH2

PH

SPH

X YX Y

SPH SH3SH2SH2

SH3SH2SH2

P

PP

Y753

Y759

B

PIP5

KPI

P5K

PIP5

KPI

P5K

PI3KPI3K

PI3KPI3K PIP2

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RawlingsNatasha Griffith, Irene Bakman, Christoph W. Turk, James L. Daniel and David J.

Lisa A. Humphries, Carol Dangelmaier, Karen Sommer, Kevin Kipp, Roberta M. Kato, SH2-SH3 linkerγof the PLC

Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation

published online June 7, 2004J. Biol. Chem.

10.1074/jbc.M311985200Access the most updated version of this article at doi:

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tec kinases mediate sustained calcium influx via site ... · 07/06/2004 · 4 specific...

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