©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of p72 with the src Homology-2 (SH2) Domains of PLC1 in B Lymphocytes (*)

Amy L. Sillman (§) , John G. Monroe (¶)

From the (1) Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase C-catalyzed inositol phospholipid hydrolysis, a critical step in B cell antigen receptor signaling leading to second messenger generation and proliferation, depends upon tyrosine kinase activation. The B cell antigen receptor-associated tyrosine kinases p53/56, p59, p55, and p72 are assumed to participate in receptor-initiated signaling. It is unknown, however, which of these kinases is involved in the tyrosine phosphorylation and resulting activation of phospholipase C in response to antigen receptor cross-linking. We have used a fusion protein containing the tandem src homology-2 (SH2) domains of phospholipase C1 (PLC1) to identify B cell kinases which associate with PLC1. Using an in vitro kinase assay, we demonstrate SH2-dependent association of tyrosine kinase activity from anti-µ-stimulated B cells. The PLC1 SH2 domains associate with a prominent 70-72-kDa tyrosine phosphoprotein from anti-µ-stimulated, but not resting, B cells. Immunoblotting and secondary immunoprecipitation studies definitively identify this protein as p72. These results imply a physical interaction between PLC1 and p72 in antigen receptor-stimulated B cells. This conclusion is confirmed by our ability to co-immunoprecipitate p72 and PLC1 from lysates of anti-µ-stimulated B cells. These results implicate p72 in the activation of phospholipase C1 during B cell antigen receptor signaling.


INTRODUCTION

The B cell antigen receptor (BCR)() -associated src family protein tyrosine kinases (PTKs) p53/56, p59, and p55, and the non-src family PTK p72, become activated in B cells within seconds following BCR cross-linking (1, 2, 3) . It is assumed, although not formally proven, that each of these PTKs plays an important role in transducing BCR-initiated signals for B cell activation. Studies using PTK inhibitors have shown that tyrosine kinase activity is a requirement for phosphatidylinositol 4,5-bisphosphate hydrolysis in B cells following BCR cross-linking (4-6). This critical step in the pathway generates the second messengers diacylglycerol and inositol 1,4,5-trisphosphate, which are responsible for protein kinase C activation and a component of the increase in levels of intracellular calcium, respectively (7, 8, 9) . Hydrolysis of phosphatidylinositol 4,5-bisphosphate is catalyzed by phosphatidylinositol-specific phospholipase C (PLC) (10) .

B cells express two isozymes of PLC, 1 and 2 (11) . Both isozymes are phosphorylated on tyrosine following BCR stimulation (4, 11, 12, 13) . Tyrosine phosphorylation increases the activity of PLC1, and may be the principal means of its activation in vivo(14, 15) . In B cells, it has been assumed, although not demonstrated, that this phosphorylation is mediated by one or more of the receptor-associated PTKs which are activated immediately following BCR cross-linking. Recent studies in a p72 -deficient avian B cell line suggest that p72 is required for BCR-coupled tyrosine phosphorylation of PLC2 and inositol 1,4,5-trisphosphate production (16). However, these cells may not accurately model normal B cells as they also lacked expression of the src family PTKs p55 and p59. Studies to determine PLC-PTK associations at the molecular level are necessary to establish the direct involvement of particular PTK(s) in this important BCR signaling pathway.

PLC1 activation by the platelet-derived growth factor and epidermal growth factor receptor PTKs is known to depend upon an interaction between the SH2 domains of PLC1 and phosphorylated tyrosine residues of the activated receptor PTK (17, 18, 19, 20, 21) . Extending this model to the BCR, which is noncovalently coupled to several cytoplasmic PTKs, PLC1 interaction with the PTK(s) involved in its regulation is predicted to be dependent upon a physical interaction between one or both of the src homology-2 (SH2) domains of PLC1 and phosphorylated tyrosine residue(s) on the BCR-activated kinase. Exploiting this model of PLC-PTK interaction, we have used a glutathione S-transferase (GST) fusion protein containing the two tandem SH2 domains of PLC1 to identify PTK(s) which associate with PLC1 following B cell activation through the BCR.


MATERIALS AND METHODS

Mice

Balb/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in our animal colony.

Preparation of Splenic B Cells

Mice were killed by cervical dislocation and spleens were removed aseptically. Cell suspensions were made by grinding spleens between the frosted ends of two glass microscope slides. The suspension was depleted of T cells by anti-Thy-1.2 antibody (HO-13-4) and lysis in rabbit complement (Pel-Freez Biologicals, Rogers, AR); erythrocytes were removed by osmotic shock. Finally, B cells were purified by centrifuging over a step gradient of 50/75% Percoll (Pharmacia, Piscataway, NJ). This preparation is routinely 85-95% B cells (IgM B220).

Purification of Bacterial GST Fusion Proteins

500 ml of LB bacterial growth medium containing ampicillin was inoculated 1:10 with an overnight culture of Escherichia coli DH5F` harboring either the pGEXKG vector (22) (a derivative of the pGEX2T vector, Pharmacia, Piscataway, NJ) encoding only GST, or the pGEXKG-PLC plasmid encoding GST and amino acids 549-755 of bovine PLC1, which comprise the tandem SH2 domains of PLC1 (a gift of Dr. Tony Pawson, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto). Following 1 h of shaking at 37 °C (A = 0.4), fusion protein production was induced by addition of isopropyl--D-thio-galactopyranoside (Stratagene Cloning Systems, La Jolla, CA) to 0.5 mM. Cells were grown an additional 3 h at 37 °C with shaking, and then harvested by centrifugation. Cell pellets were lysed in 10 ml of phosphate-buffered saline, 1% Triton X-100 with 0.2 mg/ml lysozyme, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 50 mM EDTA. Following sonication to reduce viscosity, cell debris was pelleted by centrifugation in a Sorvall SS34 rotor at 15,000 rpm for 20 min. Glycerol was added to the supernatant to a concentration of 20%; the supernatant was aliquoted and stored at -80 °C. Fusion proteins were purified from supernatants by affinity chromatography on glutathione-Sepharose 4B (Pharmacia, Piscataway, NJ). The amounts of GST and PLC1 SH2-GST bacterial lysate used to coat the Sepharose beads were adjusted so that similar amounts of the two proteins would be present in precipitation experiments. Fusion protein-coated beads were washed four times in phosphate-buffered saline, 2% Triton X-100 with 2 mM PMSF and 50 mM EDTA, and then twice in 0.5% Nonidet P-40 (Calbiochem) kinase assay lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris, pH 7.3, 0.4 mM EDTA, 10.8 µg/ml aprotinin, 1.5 µg/ml each of leupeptin, pepstatin A, chymostatin, and antipain, 2 mM PMSF, 2 mM sodium orthovanadate, 10 mM NaF). Washed, fusion protein-coated glutathione-Sepharose beads were used to precipitate B cell lysates as described below.

Preparation of B Cell Lysates

Following purification, B cells were resuspended in Hank's balanced salt solution without fetal calf serum at a density of 2 10 cells/ml. Following a 15-min equilibration in a 37 °C water bath, cells were stimulated for the time points indicated in the figures using 30 µg/ml goat anti-mouse µ heavy chain antibodies (Jackson Immunoresearch, West Grove, PA) or 20 µg/ml F(ab`) fragments of goat anti-mouse µ heavy chain antibodies (Jackson Immunoresearch, West Grove, PA) as a polyclonal activator. Cells were pelleted and lysed with 0.5% Nonidet P-40 kinase assay lysis buffer on ice for 10 min. Lysates were cleared by microcentrifugation at 14,000 rpm for 10 min at 4 °C. Cleared lysates were precipitated with fusion protein-coated glutathione-Sepharose beads (30-40 µl of a 50% slurry per sample) on a rotator overnight at 4 °C. Lysate-adsorbed, fusion protein-coated beads were washed four times with ice-cold 0.5% Nonidet P-40 kinase assay lysis buffer, boiled in 2 reducing sample buffer (125 mM Tris pH 6.8, 20% (w/v) glycerol, 10% (v/v) 2-mercaptoethanol, 4.6% SDS), and then eluted proteins were fractionated using SDS-7.5% polyacrylamide gel electrophoresis (PAGE). Fractionated proteins were electroblotted onto Hybond-ECL nitrocellulose filters (Amersham Corp.) for use in immunoblot analysis. Alternatively, lysate-adsorbed fusion protein-coated beads were washed as stated above and then subjected to the in vitro kinase assay or the thrombin cleavage procedure.

In Vitro Kinase Assay

The in vitro kinase reaction was performed essentially as in Ref. 23. Washed lysate-adsorbed fusion protein-coated beads (40 µl of a 50% slurry per sample) were washed once with ice-cold kinase assay wash buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.3, 2 mM PMSF, 2 mM sodium orthovanadate), once with kinase assay buffer (10 mM MgCl, 10 mM HEPES, pH 7.0, 2 mM PMSF, 2 mM sodium orthovanadate), and then resuspended in 40 µl of kinase assay buffer containing 10 µCi of [-P]ATP (6,000 Ci/mmol, 10 mCi/ml; DuPont NEN). Beads were incubated for 10 min at 30 °C, and then washed three times with ice-cold 0.5% Nonidet P-40 kinase assay lysis buffer. Proteins were eluted from the beads by boiling in 2 reducing sample buffer, and then fractionated using SDS-10% PAGE. Gels were fixed for 2 h in 20% methanol, 10% acetic acid, dried, and then subjected to autoradiography. In one experiment, the gel was fixed, treated with 1 N KOH for 2 h at 55 °C (to selectively hydrolyze phosphoserine and phosphothreonine), neutralized in fixative, dried, and then subjected to autoradiography (24) .

Immunoblot Analysis

Membranes were incubated overnight in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris, pH 8, 137 mM NaCl, 0.05% Tween 20) containing 2% bovine serum albumin to block nonspecific binding. To detect tyrosine-phosphorylated substrates, blocked membranes were incubated for 90 min with the monoclonal antibody 4G10 (Upstate Biotechnology Inc., Lake Placid, NY), followed by horseradish peroxidase-conjugated sheep anti-mouse Ig secondary antibody (Amersham) for 90 min. To detect p72, membranes were incubated with rabbit IgG directed against a bacterial fusion protein containing the COOH-terminal SH2 domain of p72 (a gift of Drs. Ellen Pure and Mark Forman, The Wistar Institute, Philadelphia, PA) for 90 min, washed, and then incubated with horseradish peroxidase-conjugated F(ab`) fragments of donkey anti-rabbit Ig antibodies (Jackson Immunoresearch). PLC1 was detected using a mixture of monoclonal anti-PLC1 antibodies (Upstate Biotechnology Inc.) followed by the horseradish peroxidase-conjugated sheep anti-mouse Ig secondary antibody listed above. p59, p55, and p53/56 were detected in a similar manner using antibodies given to us by Dr. Joseph Bolen (Bristol-Myers Squibb, Princeton, NJ). Following three 15-min washes in TBST, membranes were developed using the enhanced chemiluminescence (ECL) system (Amersham) followed by autoradiography. Membranes were stripped of bound antibodies by incubating in stripping buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS) for 30 min in a 50 °C shaking water bath, followed by three 15-min washes in TBST.

Thrombin Cleavage and Immunoprecipitation of p72

The thrombin cleavage procedure was modified from Ref. 22. Washed lysate-adsorbed fusion protein-coated beads (40 µl of a 50% slurry per sample) were washed once with thrombin cleavage buffer (150 mM NaCl, 50 mM Tris, pH 8, 2.5 mM CaCl, 0.1% 2-mercaptoethanol, 2 mM sodium orthovanadate, 100 ng/ml okadaic acid), and then incubated in 1 ml/sample of thrombin cleavage buffer containing 12 µg of human thrombin (Sigma) for 20 min at ambient temperature. Samples were placed on ice, and PMSF, aprotinin, leupeptin, pepstatin A, chymostatin, and antipain were added to the final concentrations listed previously. Samples were centrifuged to pellet the glutathione-Sepharose beads. The supernatant was removed and incubated with 5 µl/sample of anti-p72 rabbit antiserum (Upstate Biotechnology Inc.) overnight at 4 °C, followed by 50 µl of a 50% slurry of protein A-Sepharose (Sigma) for 3 h at 4 °C. Immunoprecipitations were washed 4 times in 0.5% Nonidet P-40 immunoprecipitate wash buffer (10 mM Tris, pH 8, 0.5% Nonidet P-40, 0.5% SDS) with protease and phosphatase inhibitors added to the final concentrations listed previously. Immunoprecipitated proteins were eluted by boiling in 2 reducing sample buffer. SDS-PAGE, electroblotting, and anti-phosphotyrosine immunoblot analysis were performed as stated above.

Immunoprecipitation of PLC1

2 10 B cells were stimulated as before and lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 10 mM Tris, pH 8) with protease and phosphatase inhibitors added. Lysates were precleared by rotating for 1 h at 4 °C with 30 µl of a 50% slurry of protein A-Sepharose (Sigma). Following centrifugation to pellet the protein A-Sepharose beads, lysates were incubated with 6 µg of a mixture of monoclonal anti-PLC1 antibodies (Upstate Biotechnology Inc.) on ice for 30 min. 30 µl of a 50% slurry of protein A-Sepharose was added and lysates were rotated at 4 °C overnight. Immunoprecipitates were washed 4 times with 0.5% Nonidet P-40 immunoprecipitate wash buffer and then eluted by boiling in 2 reducing sample buffer. SDS-PAGE, electroblotting, and anti-p72 and anti-PLC1 immunoblot analysis were performed as stated above.

RESULTS

The PLC1 SH2 Domain Fusion Protein Precipitates Tyrosine Kinase Activity from B Cell Lysates

To determine whether the PLC1 SH2 domains associate with PTKs from BCR-stimulated B cells, we used the PLC1 SH2-GST fusion protein to precipitate proteins from lysates of anti-receptor (anti-µ heavy chain) antibody-stimulated splenic B cells. Precipitated proteins were then assayed for kinase activity in an in vitro kinase assay in the presence of [-P]ATP. Evidence for SH2-associated tyrosine kinase activity was determined by the presence of phosphoprotein bands in the SDS-PAGE gel after treatment with potassium hydroxide to eliminate phosphoserine and phosphothreonine (not shown). As shown in Fig. 1, demonstrable kinase activity was observed in lanes containing PLC1 SH2-GST fusion protein-precipitated material from B cells stimulated for 30 and 60 s with anti-µ antibody. Several substrates are present in both lanes, presumably representing autophosphorylated kinase(s) and/or B cell proteins co-precipitated with these kinases as well as proteins contained within the bacterial lysates from which the fusion protein was isolated. Importantly, no kinase activity was precipitated by the GST protein alone (Fig. 1), or in the absence of B cell lysate (not shown), indicating that the kinase activity present in these precipitates is derived from the B cell lysates, and that its association with the fusion protein is dependent upon the presence of the PLC1 SH2 domains. The PLC1 SH2 Domain Fusion Protein Precipitates a 70-72-kDa Tyrosine-phosphorylated Protein from Lysates of Anti-µ-stimulated B Cells-To identify the PTK(s) that associate with the PLC1 SH2-GST fusion protein, we took advantage of the fact that the BCR-associated PTKs are themselves activated by tyrosine phosphorylation. Exploiting this characteristic, we determined whether the PLC1 SH2 domains bind to tyrosine-phosphorylated proteins following BCR signaling in vivo. We performed immunoblot analysis with the monoclonal anti-phosphotyrosine antibody 4G10 on proteins precipitated from B cell lysates with the PLC1 SH2 fusion protein (Fig. 2). A prominent tyrosine-phosphorylated protein with an approximate molecular mass of 70-72 kDa was observed in lysates of anti-µ-stimulated, but not resting, B cells. The intensity of this protein band is at its highest level in precipitates from B cells that have been stimulated with anti-µ antibodies for 1 min. By 2 min post-stimulation, the intensity of this band has decreased. Other, higher molecular weight tyrosine-phosphorylated proteins are also precipitated from anti-µ-stimulated B cell lysates by the PLC1 SH2-GST fusion protein. The intensity of these higher molecular mass bands, relative to the 70-72 kDa band, suggest that they represent tyrosine-phosphorylated proteins which have a lower affinity for the PLC1 SH2-GST fusion protein. Alternatively, they may represent tyrosine-phosphorylated proteins which co-associate with the 70-72-kDa phosphoprotein, rather than bind directly to the SH2 domains of PLC1. The GST-only fusion protein did not precipitate any tyrosine-phosphorylated proteins from the B cell lysates, and PLC1 SH2-GST fusion protein in the absence of B cell lysate did not contain detectable tyrosine-phosphorylated proteins. Whole cell lysates from unstimulated and anti-µ-stimulated B cells were included on the blot as a positive control, to show that protein tyrosine phosphorylation was induced upon BCR stimulation. The 70-72-kDa Tyrosine-phosphorylated Protein Precipitated by the PLC1 SH2 Domain Fusion Protein from Lysates of Anti-µ-stimulated B Cells Reacts with Anti-p72Antibodies on Immunoblots-Our studies to this point indicated association of the PLC1 SH2 domains with both tyrosine kinase activity and an inducible tyrosine phosphoprotein of 70-72 kDa. Because one possible mechanism of the physical interaction of PLC1 with its activating tyrosine kinase is between the SH2 domains of PLC1 and a phosphotyrosine residue on the activated kinase, we considered that the 70-72-kDa tyrosine phosphoprotein represented the PTK which activates PLC1. In this regard, among the PTKs known to be activated as a consequence of anti-µ-induced BCR cross-linking, p72 was an obvious candidate for the PLC1 SH2-associated kinase. To test this possibility, we used the PLC1 SH2-GST fusion protein to precipitate proteins from B cell lysates as before, and then performed anti-p72 immunoblot analysis on these proteins (Fig. 3A). The anti-p72 antibody detected a 72-kDa protein in the PLC1 SH2-GST-precipitated lysates from B cells stimulated with anti-µ for 2 min. A number of other proteins were detected, but they are also present in the fusion protein-only (no B cell lysate) precipitate. Importantly, the 72-kDa band is not present in the fusion protein-only (no B cell lysate) lane, indicating that it is of B cell, and not bacterial, origin. Stripping and reprobing this blot with anti-phosphotyrosine antibodies allowed us to determine that the p72 band overlays exactly with the 72-kDa tyrosine phosphoprotein (not shown). Finally, immunoblot analysis using antibodies to p55, p53/56, and p59 failed to detect any PLC1 SH2-specific binding of these PTKs, indicating specificity of the PLC1 SH2 domains for p72 (not shown).


Figure 1: In vitro kinase assay of proteins precipitated from B cell lysates by the SH2 domains of PLC1. Splenic B cells (2 10/lane) were stimulated with 30 µg/ml goat anti-mouse µ antibodies for 30 or 60 s, and then lysed in 0.5% Nonidet P-40 kinase assay lysis buffer. Lysates were precipitated with fusion protein-coated Sepharose beads: either a fusion protein comprised of GST alone (GST lanes) or of GST and the tandem SH2 domains of PLC1 (SH2-GST lanes). Following precipitation, the beads were washed, and an in vitro kinase assay was performed in the presence of [-P]ATP. Proteins were eluted from the beads with 2 reducing sample buffer, fractionated by SDS-10% PAGE, and then detected by autoradiography. The apparent sizes (in kilodaltons) and positions of prestained molecular weight standards are indicated on the right; time points (in seconds) of anti-µ stimulation are indicated at the top. Evidence for SH2-associated tyrosine kinase activity was determined by the presence of phosphoprotein bands in the SDS-PAGE gel after treatment with potassium hydroxide to eliminate phosphoserine and phosphothreonine (not shown).




Figure 2: Detection of a 70-72-kDa tyrosine-phosphorylated protein in PLC1 SH2 domain precipitates from lysates of anti-µ-stimulated B cells. Murine splenic B cells were stimulated, lysed, and precipitated with GST fusion proteins as in Fig. 1 (GST-SH2-SH2 lanes contain precipitations done with the PLC1 SH2 domain-containing fusion protein.) Precipitated proteins were eluted from the beads with 2 reducing sample buffer, fractionated by SDS-7.5% PAGE, and electroblotted onto nitrocellulose filters. Filters were probed with the anti-phosphotyrosine monoclonal antibody 4G10 followed by horseradish peroxidase-conjugated sheep anti-mouse Ig antibodies. Detection was by ECL followed by autoradiography. Whole cell lysate lanes contain lysates from 5 10 unstimulated or anti-µ-stimulated B cells. Time points (in minutes) of anti-µ stimulation are indicated at the top of the figure; n.l., fusion protein-coated beads only (no lysate). The apparent sizes (in kilodaltons) and positions of prestained molecular weight standards are indicated on the right.




Figure 3: Detection of p72 in PLC1 SH2 domain precipitates from lysates of anti-µ-stimulated B cells. A, PLC1 GST-SH2 fusion protein precipitations from anti-µ-stimulated B cell lysates followed by SDS-7.5% PAGE and electroblotting were done as described in the legend to Fig. 2. Blots were probed with rabbit anti-p72 antibodies followed by horseradish peroxidase-conjugated F(ab`) fragments of donkey anti-rabbit Ig antibodies. Detection was by ECL followed by autoradiography. p72 is indicated by an arrow. B, murine splenic B cells were stimulated, lysed, and precipitated with the PLC1 GST-SH2 fusion protein-coated Sepharose beads as described in the legend to Fig. 1. Following several washes, the beads were treated with thrombin, which cleaves the fusion protein between the GST domain and the SH2 domains. The supernatant from this treatment, which contained the PLC1 SH2 domains and any proteins bound to them, was then immunoprecipitated with anti-p72 antibodies and protein A-Sepharose beads. Precipitated proteins were eluted from the beads with 2 reducing sample buffer, fractionated by SDS-7.5% PAGE, and electroblotted onto a nitrocellulose filter. The filter was probed with the anti-phosphotyrosine monoclonal antibody 4G10 followed by horseradish peroxidase-conjugated sheep anti-mouse Ig antibodies. Detection was by ECL followed by autoradiography. Time points (in minutes) of anti-µ stimulation are indicated at the top of the figure; n.l., fusion protein-coated beads only (no lysate). The apparent sizes (in kilodaltons) and positions of prestained molecular weight standards are indicated on the right of both panels; p72 is indicated by an arrow.



p72Can be Immunoprecipitated from B Cell Proteins Which Bind to the PLC1 SH2 Domains

We also evaluated whether we could immunoprecipitate p72 from the B cell proteins which bind to the PLC1 SH2-GST fusion protein. We took advantage of a proteolytic cleavage site in the PLC1 SH2-GST fusion protein. Thrombin cleaves between the GST domain and the amino-terminal SH2 domain of the PLC1 SH2-GST fusion protein, releasing the SH2 domains into the supernatant, along with any bound proteins. The GST domains remain bound to the glutathione-Sepharose beads, and can be removed. We immunoprecipitated p72 from this supernatant, and subjected the immunoprecipitated proteins to anti-phosphotyrosine immunoblot analysis with 4G10 (Fig. 3B). We observed a tyrosine-phosphorylated protein of approximately 72 kDa that is immunoprecipitated by anti-p72 antiserum from PLC1 SH2 domain bound proteins. This tyrosine-phosphorylated protein is immunoprecipitated by anti-p72 antibodies from lysates of B cells stimulated with anti-µ antibodies for 1 min, indicating that the major protein that associates with the SH2 domains of PLC1 in this system is the PTK p72.

p72Can Be Co-immunoprecipitated with PLC1 from Lysates of Anti-µ-stimulated B Cells

These data indicate that p72 from anti-µ-stimulated B cells is able to bind to the PLC1 SH2-GST fusion protein. In order to determine whether the interaction between p72 from anti-µ-stimulated B cells and the PLC1 SH2-GST fusion protein predicts what occurs in vivo during B cell antigen receptor signal transduction, we wished to establish whether p72 is associated with PLC1 in lysates of antigen receptor-stimulated B cells. PLC1 was immunoprecipitated from lysates of anti-µ-stimulated B cells, and anti-p72 immunoblot analysis was performed on the immunoprecipitated proteins (Fig. 4). Anti-p72 immunoblot analysis of anti-PLC1 immunoprecipitates shows that p72 is associated with PLC1 in lysates of anti-µ-stimulated B cells (Fig. 4, right panel, lane 3). p72 is not present in lanes containing proteins pre-cleared by protein A-Sepharose beads (Fig. 4, right panel, lane 2). This blot was stripped and re-probed with anti-PLC1 antibodies to show the efficiency of the anti-PLC1 immunoprecipitation in this experiment (Fig. 4, left panel). In summary, the ability to co-immunoprecipitate p72 and PLC1 from lysates of BCR-stimulated B cells supports the physiological relevance of the p72 -PLC1 association defined by these studies.


Figure 4: Detection of p72 in anti-PLC1 immunoprecipitates from lysates of anti-µ-stimulated B cells. Lysates from 2 10 resting or anti-µ-stimulated B cells were precleared by incubation with protein A-Sepharose (pre-clear lanes) and then incubated with 6 µg of anti-PLC1 monoclonal antibodies and protein A-Sepharose (anti-PLC1 immunoppt. lanes). Precipitated proteins were eluted by boiling in 2 reducing sample buffer; SDS-10% PAGE and electroblotting were done as described in the legend to Fig. 3. Filters were probed with anti-p72 antibodies (right panel) as described in the legend to Fig. 3, stripped, and re-probed with anti-PLC1 monoclonal antibodies followed by horseradish peroxidase-conjugated F(ab`) fragments of donkey anti-mouse Ig antibodies (left panel). Detection was by ECL followed by autoradiography. Whole cell lysates from 5 10 anti-µ-stimulated B cells is shown (whole lysate lanes) as a control. The apparent sizes (in kilodaltons) and positions of prestained molecular weight standards are indicated on the right; p72 is indicated by an arrow.



DISCUSSION

We have shown that a fusion protein containing the SH2 domains of PLC1 can precipitate tyrosine kinase activity from lysates of BCR-stimulated murine splenic B cells. We have also shown that the PLC1 SH2 domains precipitate a 72-kDa tyrosine phosphoprotein which is identified as p72 based upon reactivity with anti-p72 antibodies both on immunoblots and in secondary immunoprecipitation experiments. These results imply that p72 is the tyrosine kinase responsible for PLC1 phosphorylation following BCR stimulation in vivo. Consistent with this possibility is our demonstration of p72 -PLC1 association in vivo. Specifically, we have demonstrated co-immunoprecipitation of p72 and PLC1 from lysates of anti-µ-stimulated B cells. Reciprocal experiments in which immunoprecipitates of p72 were tested for PLC1 co-immunoprecipitation were negative. We believe that this inconsistency with the anti-PLC1 immunoprecipitations shown here is due to the amount of p72 immunoprecipitated under these conditions. In our hands, the anti-p72 antibody immunoprecipitates a relatively small proportion of the total p72 protein present in B cells. Because PLC1 may be only one of the substrates of p72 in B cells, only a fraction of the p72 protein would be found associated with PLC1. Therefore, the relative inefficiency of the anti-p72 antibody in immunoprecipitation experiments makes this approach to the experiment less efficient than testing anti-PLC1 immunoprecipitates for the presence of p72.

The fact that p72 can interact with the SH2 domains of PLC1 in this in vitro system and is found complexed with PLC1 in lysates of anti-µ-stimulated B cells suggests that p72 interacts with PLC1 in vivo during BCR-initiated signal transduction. This leads us to postulate that p72, and not one of the src family PTKs, is the PTK which directly activates PLC1 by phosphorylating it in B cells following BCR cross-linking. Thus, we conclude that PLC1 interacts with activated p72 during BCR-initiated signal transduction, and that this interaction involves PLC1 SH2 domains and activation-associated phosphotyrosine residues of p72. This interaction then leads to the phosphorylation and activation of PLC1, and subsequent signaling events.

Three observations further support our model of the BCR-initiated signaling cascade. First, the peak of p72 association with the PLC1 SH2 domains (at 1 min post-BCR stimulation, Fig. 2) occurs before the peak of PLC1 tyrosine phosphorylation (2-5 min post-BCR stimulation).() Second, a recent study demonstrated that a p72 -deficient avian B cell line could not hydrolyze inositol phospholipids in response to anti-IgM stimulation. These cells were also unable to phosphorylate PLC2 in response to anti-IgM stimulation. Transfection of a kinase-deficient form of p72 was unable to restore anti-IgM-stimulated inositol phospholipid hydrolysis, indicating that this signaling event depended directly upon the kinase activity of p72(16) . Third, another study showed that aggregation of a CD16/syk, but not a CD16/fyn, chimeric cell surface molecule could induce tyrosine phosphorylation of PLC1 in TCR Jurkat T cells (25) . Our results extend these studies by showing that this interaction can be mediated via the PLC1 SH2 domains and occurs in the more physiological context of non-transformed B cells in which the levels of BCR-associated PTKs are under normal cellular control. Taken together, these observations substantiate our conclusion that p72 is directly responsible for the phosphorylation of PLC and the initiation of phosphatidylinositol 4,5-bisphosphate hydrolysis in response to stimulation through BCR in vivo.

We cannot, however, state unequivocally that this interaction is direct rather than mediated via an as yet unidentified intermediary protein. If involved, this protein is unlikely to be one of the other receptor-associated PTKs, as the lack of tyrosine phosphoproteins with molecular weights consistent with src family PTKs in PLC1 SH2-GST precipitations shows (Fig. 2). Also, the inability of our immunoblot studies to show p59, p53/56, or p55 association with the PLC1 SH2 domains argues against this possibility (not shown). The exact nature of the PLC1 SH2 domain interaction with p72 with regards to the possibility of intermediary proteins remains to be evaluated.

The relative contributions of PLC1 and PLC2 to signaling through BCR is not known. Other groups have reported that PLC2 is the predominant isoform expressed in B cells (11, 13, 26) . However, we observe equal or higher expression of PLC1 in primary murine B cells by immunoblot analysis.() Because both isoforms are phosphorylated on tyrosine following BCR cross-linking, both are presumed to participate in the signaling pathway (4, 11, 12, 13) . However, the SH2 domains of PLC1 and PLC2 are not identical. The NH- and COOH-terminal SH2 domains are 70 and 76% homologous, respectively, between the two isoforms (27) . This leaves open the possibility that PLC2 may associate with p72 but at a different affinity than PLC1 does, or that it may associate with a different kinase altogether. This could be another example of redundancy in the BCR signaling pathway, or it could be a means of regulating the response to antigen receptor stimulation, depending on the levels of the different PTKs and PLC isoforms expressed.

The precise mechanism of p72 activation, or the activation of any of the BCR-associated PTKs, is currently unknown, although there is some evidence that the src family PTKs (p53/56 in particular) may be able to phosphorylate and activate p72 (28, 29). Whether all of the BCR-associated PTKs are activated simultaneously by clustering of BCR during cross-linking, or whether they are activated in some sort of BCR-initiated sequence is an area under study.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants AI23568 and AI23592. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a predoctoral training grant from the National Cancer Institute.

Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 538a Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-2873; Fax: 215-573-2014.

The abbreviations used are: BCR, B cell receptor; PTK, protein tyrosine kinase; PLC, phospholipase C; SH2, src homology-2; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence.

A. L. Sillman and J. G. Monroe, unpublished observations.

A. L. Sillman and J. G. Monroe, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Tony Pawson for the generous gift of the PLC1 SH2 domain fusion protein construct, Dr. Bill Dougall for his advice on fusion protein expression, and Drs. Ellen Pure and Mark Forman for providing us with the anti-p72 antiserum which we used for the immunoblot analysis. Our gratitude also to Drs. Marian Birkeland and Mark Forman for their critical reading of this manuscript.


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