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The Journal of Immunology, 1999, 163: 1746-1749.
Copyright © 1999 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Association of Phospholipase C-{gamma}2 Src Homology 2 Domains with BLNK Is Critical for B Cell Antigen Receptor Signaling1

Masamichi Ishiai*, Hitoshi Sugawara{dagger}, Mari Kurosaki* and Tomohiro Kurosaki2,*

* Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan; and {dagger} Department of Integrated Medicine, Omiya Medical Center, Jichi Medical School, Omiya, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
To explore the mechanism(s) by which phospholipase C (PLC)-{gamma}2 participates in B cell Ag receptor (BCR) signaling, we have studied the function of PLC-{gamma}2 mutants in B cells deficient in PLC-{gamma}2. Mutation of the N-terminal Src homology 2 domain [SH2(N)] resulted in the complete loss of inositol 1,4,5-trisphosphate generation upon BCR engagement. A possible explanation for the SH2(N) requirement was provided by findings that this mutation abrogates the association of PLC-{gamma}2 with an adaptor protein BLNK. Moreover, expression of a membrane-associated form (CD16/PLC-{gamma}2) with SH2(N) mutation required coligation of BCR and CD16 for inositol 1,4,5-trisphosphate generation. Together, our results suggest a central role for the SH2(N) domain in directing PLC-{gamma}2 into the close proximity of BCR signaling complex by its association with BLNK, whereby PLC-{gamma}2 becomes tyrosine phosphorylated and thereby activated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Stimulating the B cell Ag receptor (BCR)3 initiates a cascade of signal transduction events in which cytoplasmic protein tyrosine kinase (PTK) activation is the earliest known event. Three distinct types of nonreceptor PTKs, Src-PTK (including Lyn, Fyn, and Blk), Syk, and Btk have been found to be activated upon BCR engagement. These three types of PTKs are likely to contribute to the phosphorylation and activation of downstream effectors, leading to generation of multiple second messengers (1, 2, 3, 4). Critical downstream signaling events include the activation of phospholipase C (PLC)-{gamma}2, tyrosine phosphorylation of which is mediated by Syk and Btk (5, 6, 7). The activated PLC-{gamma}2 converts phosphatidylinositol 4,5-bisphosphate (PIP2) into the two messengers diacylglycerol and inositol 1,4,5-trisphosphate (IP3), which results in activation of protein kinase C and increased calcium mobilization, respectively (8, 9, 10, 11).

Structurally, PLC-{gamma}2 has a putative pleckstrin homology domain at its amino terminus, followed by the two conserved parts of the catalytic domain separated by two tandem Src homology 2 (SH2) domains and an SH3 domain (12, 13, 14, 15). Here, we focus on the functions of SH2 domains in BCR-mediated PLC-{gamma}2 activation. Although both N-terminal SH2 (SH2(N)) and C-terminal SH2 (SH2(C)) domains were required for efficient IP3 production, the SH2(N) domain was more critically required for PLC-{gamma}2 activation. Furthermore, a membrane-associated CD16/PLC-{gamma}2 chimera with SH2(N) mutation did not generate IP3 by BCR ligation alone, whereas coligation of the BCR with this mutant chimera was capable of generating IP3, demonstrating that the function of the SH2(N) domain could not be accounted for by the simple membrane translocation. Instead, the failure of the PLC-{gamma}2 SH2(N) mutant to bind to BLNK, together with our previous evidence that BLNK is essential for PLC-{gamma}2 activation (16), suggests that directing PLC-{gamma}2 into the close proximity of the BCR signaling complex by the interaction between its SH2(N) domain and BLNK is essential for coupling BCR to PLC-{gamma}2 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells, expression constructs, and Abs

PLC-{gamma}2 deficient DT40 B cell (17) and various transfectants were cultured as described previously (16). All PLC-{gamma}2 constructs used in this study contained the T7 epitope tag (MASMTGGQQMGR) at the C terminus of rat PLC-{gamma}2. The SH2(N) and SH2(C) mutants (Arg564 to Ala and Arg672 to Ala, respectively) and membrane PLC-{gamma}2 chimeras (CD16/PLC-{gamma}2 in Fig. 3GoA) were created by the PCR method and cloned into expression vector pApuro (5). These cDNAs were transfected as described (5), and expression of transfected cDNA was confirmed by Western blot analysis (T7-tagged PLC-{gamma}2) or FACS analysis (CD16/PLC-{gamma}2 chimeras). Cell-surface expression of BCR or CD16 was analyzed by FACScan (Becton Dickinson, Mountain View, CA). The following Abs were used: anti-BLNK Ab (16), anti-T7 epitope mAb (Novagen, Madison, WI), anti-chicken IgM mAb (M4) (18), anti-phosphotyrosine mAb (4G10) (Upstate Biotechnology, Lake Placid, NY), rabbit anti-mouse IgM Ab (Zymed, San Francisco, CA), FITC-conjugated anti-chicken IgM (Bethyl Laboratories, Montgomery, TX), and FITC conjugated anti-human CD16 Ab (PharMingen, San Diego, CA).



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  		FIGURE 3. Analysis of CD16/PLC-{gamma}2 chimeras in PLC-{gamma}2-deficient DT40 cells. A, Schematic diagram of membrane PLC-{gamma}2 chimeras with the T7 epitope at the C terminus. These are composed of the human CD16 extracellular domain, the human TCR {zeta}-chain transmembrane domain, and the complete or mutated rat PLC-{gamma}2 as a cytoplasmic domain (16 ). The SH2(N) mutant changed Arg564 to Ala of rat PLC-{gamma}2. B, Surface expression level of CD16 by FACS analysis. Unstained cells were used as a negative control (dot lines). C, IP3 production in PLC-{gamma}2-deficient DT40 cells expressing both chimeras were directly compared in the same experiments. For coligation (BCR+CD16), rabbit anti-mouse IgM (7.5 µg) was added before stimulation with M4 (2 µg). Results are expressed as the mean from three independent experiments. Error bars represent the SD from the mean.

 
Immunoprecipitation and Western blot analysis

For immunoprecipitation, cells were solubilized in Nonidet P-40 lysis buffer, and precleared lysates were sequentially incubated with proper Abs and protein A-agarose as described previously (5). Lysates or immunoprecipitates were separated by 7% SDS-PAGE, transferred to nitrocellulose membranes, and detected by appropriate Abs and enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

IP3 generation assay

Cells (2 x 106) were stimulated with mAb M4 (2 µg) at 37°C for indicated times. For CD16/PLC-{gamma}2 chimera (Fig. 3GoC), coligation of BCR and the chimera was conducted by adding rabbit anti-mouse IgM (7.5 µg) before stimulation with M4 (2 µg). Kinetic analysis of IP3 production was performed using BIOTRAK IP3 assay system (Amersham) as described previously (16).

In vitro PLC assay

PIP2 hydrosis activity was measured by quantitating IP3 production (11, 19). Briefly, PLC-{gamma}2 protein was immunoprecipitated from PLC-{gamma}2-deficient DT40 cells expressing transfected PLC-{gamma}2 (1.5 x 107) using anti-T7 mAb and was resuspended in PLC assay buffer (35 mM Na2PO4, pH 6.8, 70 mM KCl, 0.15% n-octyl-ß-D-glucopyranoside, 0.8 mM EGTA, 0.8 mM CaCl2) with the substrate [3H]PIP2 (NEN, Boston, MA). The reaction mixture was incubated at 37°C for 15 min and was stopped by adding TCA and BSA. After centrifugation, radioactivity in the supernatant was measured. Relative in vitro PLC activity of PLC-{gamma}2 mutants were as follows (average of two experiments): wild-type (wt), 100%; SH2(N), 108%; and SH2(C), 106%.

Subcellular analysis of PLC-{gamma}2

Subcellular fractionation was performed as previously described (16). PLC-{gamma}2-deficient DT40 cells expressing PLC-{gamma}2 mutants (1 x 108) were homogenized in hypotonic lysis buffer (16). After removal of nuclei by centrifugation, supernatants were further centrifuged at 100,000 x g for 30 min. The resulting supernatants were saved as cytosolic fractions, while precipitates were resuspended in lysis buffer containing 1% Triton X-100. These resuspensions were centrifuged at 12,000 x g for 20 min, and final supernatants were saved as particulate fractions.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
To study the functional importance of the two SH2 domains of PLC-{gamma}2 in BCR signaling, we introduced mutations within the highly conserved residues of SH2(N) and SH2(C) domains of T7-tagged PLC-{gamma}2 (see Materials and Methods). DNA was transfected into PLC-{gamma}2-deficient DT40 B cells, and the expression of mutated PLC-{gamma}2 was monitored by immunoblotting with anti-T7 mAb (Fig. 1GoA). Cell surface expression of BCR on these transfected cells was assayed by flow cytometric analysis and demonstrated essentially the same level as parental PLC-{gamma}2-deficient DT40 cells (data not shown).



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  		FIGURE 1. IP3 analysis of PLC-{gamma}2-deficient DT40 B cells expressing PLC-{gamma}2 mutants. A, Protein expression analysis of various PLC-{gamma}2 mutants in PLC-{gamma}2-deficient cells. Whole-cell lysates prepared from 2 x 106 cells were analyzed by Western blotting using anti-T7 mAb. B, BCR-induced IP3 generation assay. Cells (2 x 106) were stimulated with M4 (2 µg) for indicated times, and IP3 productions were measured. Data are shown as fold increase of the value before stimulation with M4. Results are expressed as the mean from three independent experiments. Error bars represent the SD from the mean.

 
In previous studies, we have shown that DT40 B cells express exclusively PLC-{gamma}2 and that this enzyme is essential for coupling the BCR to IP3 generation (17). Therefore, as a functional complementation assay, we analyzed whether PLC-{gamma}2 mutants were able to restore IP3 generation upon BCR stimulation. As shown in Fig. 1GoB, PLC-{gamma}2-deficient DT40 cells expressing the SH2(N) mutant did not evoke the IP3 generation following BCR stimulation. In contrast to no IP3 generation by the SH2(N) mutation, PLC-{gamma}2-deficient DT40 cells expressing the SH2(C) mutant exhibited a transient IP3 generation at 0.5 min, after which generation declined at 3 min, suggesting a requirement for the SH2(C) domain in sustaining IP3 generation. These observations demonstrate that both SH2 domains of PLC-{gamma}2 are required for proper coupling the BCR to IP3 generation and that the SH2(N) domain is more dominantly required than the SH2(C) domain.

One possible explanation for the failure of these SH2 mutants to restore the IP3 generation is that these SH2 mutants have reduced lipid hydrolyzing enzymatic activity presumably by their conformational changes. However, in vitro PLC activities were not significantly affected by these mutations (see Materials and Methods). These results suggest the alternative possibility that both SH2 domains are required for recruitment of PLC-{gamma}2 to the membrane fraction upon BCR ligation, leading to its subsequent activation. This possibility was explored by cell fractionation experiments. As shown in Fig. 2GoA, an increased association of wild-type PLC-{gamma}2 with the membrane-enriched particulate fraction was observed upon BCR engagement, while this translocation was abolished or decreased in the particulate fraction from PLC-{gamma}2-deficient DT40 cells expressing the PLC-{gamma}2 SH2(N) or SH2(C) mutant, respectively. The extent of the BCR-induced membrane association of various PLC-{gamma}2 mutants was well correlated with the BCR-induced tyrosine phosphorylation status of these mutants (Fig. 2GoB).



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  		FIGURE 2. Subcellular analysis and tyrosine phosphorylation of various PLC-{gamma}2 mutants upon BCR stimulation. A, Fractions were prepared from cells stimulated by M4 for 1 min (+) or left unstimulated (-). Equivalent amounts of each fraction were analyzed by Western blotting with anti-T7 mAb. Particulate fractions (top) and cytosolic fractions (bottom) were shown. B, BCR-induced tyrosine phosphorylation of various PLC-{gamma}2 mutants. After stimulation by M4 for 1 min, immunoprecipitates with anti-T7 mAb (1 x 107 cells/lane) were prepared. Samples were divided into half and analyzed by Western blotting with 4G10 (top) and anti-T7 mAb (bottom).

 
Assuming that Syk and Btk, both of which are involved in PLC-{gamma}2 tyrosine phosphorylation, are associated with the BCR complex after receptor ligation (5, 6, 7), the above observations may suggest that the PLC-{gamma}2 SH2 domains, particularly the SH2(N) domain, are required not only for simple PLC-{gamma}2 membrane translocation, but also for bringing this enzyme into the proximity of the BCR signaling complex. To examine this possibility, chimeras expressing the wt and SH2(N) mutant of PLC-{gamma}2 in place of the cytoplasmic domain of CD16 (Fig. 3GoA) were expressed in PLC-{gamma}2-deficient DT40 B cells (Fig. 3GoB). Both chimeras retained an almost identical level of catalytic activity as measured by in vitro PIP2 hydrolysis, despite about 5-fold less efficiency than nonchimeric PLC-{gamma}2 (data not shown). Stimulation of BCR alone on PLC-{gamma}2-deficient DT40 cells expressing wt CD16/PLC-{gamma}2 evoked IP3 generation, albeit less efficiently than that in PLC-{gamma}2-deficient DT40 cells expressing wt PLC-{gamma}2. This weak activation is presumably due to the structural constraints by the chimeric construct. When BCR and wt CD16/PLC-{gamma}2 were coligated, the IP3 generation was much greater than that by BCR ligation alone (Fig. 3GoC), presumably by enforced recruiting PLC-{gamma}2 to the BCR complex.

In contrast to significant IP3 generation by wt CD16/PLC-{gamma}2 after BCR cross-linking alone, the CD16/PLC-{gamma}2 SH2(N) mutant was not able to generate IP3 (Fig. 3GoC). These results demonstrate that the function of the SH2(N) domain cannot be replaced by the simple membrane localization. When BCR and CD16/PLC-{gamma}2 SH2(N) mutant chimeric receptor were coligated, an almost identical level of IP3 generation was seen compared with that by coligation of BCR and wt CD16/PLC-{gamma}2 (Fig. 3GoC).

Given the evidence that BLNK is required for PLC-{gamma}2 activation (16), the above data suggest the possibility that the PLC-{gamma}2 SH2(N) domain is essential for binding to BLNK, thereby leading to recruitment of this enzyme into the BCR signaling complex. Supporting this idea, as shown in Fig. 4Go, wt PLC-{gamma}2 was recruited to BLNK after BCR ligation, whereas this association was abolished or reduced by the SH2(N) or SH2(C) mutation, respectively.



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  		FIGURE 4. Association of PLC-{gamma}2 mutants with BLNK. After stimulation by M4 for 1 min, PLC-{gamma}2 proteins were immunoprecipitated with anti-T7 mAb from PLC-{gamma}2-deficient DT40 cells expressing PLC-{gamma}2 mutants (5 x 107). Samples were divided and analyzed by Western blotting with anti-BLNK Ab (top) and anti-T7 mAb (bottom).

 
Based on the data that substitution of Tyr348 and Tyr352 in the Syk linker region eliminated both its binding to PLC-{gamma}1 SH2(C) domain and its ability to induce tyrosine phosphorylation of PLC-{gamma}1, a direct association model between Syk and PLC-{gamma}1 in an SH2-phosphotyrosine-dependent manner has been proposed (20). Although this model well explains the necessity of Syk in BCR-induced PLC-{gamma}1/2 activation, the experiments presented in this study, together with previous genetic evidence that Syk, Btk, and BLNK are required for activation of PLC-{gamma}2 (5, 6, 16), support the following multimolecular signaling complex model for BCR-induced PLC-{gamma}2 activation: i) after Syk phosphorylates multiple tyrosines on BLNK (21), BLNK provides docking sites for PLC-{gamma}2 SH2 domains, leading to recruitment of PLC-{gamma}2 to the BCR signaling complex; ii) because Btk SH2 domain is able to bind to the phosphorylated BLNK (22), the phosphorylated BLNK might form a complex with Btk in addition to PLC-{gamma}2; iii) subsequently, PLC-{gamma}2 is tyrosine phophorylated by Syk and Btk, and thereby activated.


   Acknowledgments
 
We thank Dr. Yoshimi Homma for advising the in vitro PLC assay.


   Footnotes
 
1 This work was supported by grants to M.I. and T.K. from the Ministry of Education, Science, Sports, and Culture of Japan and to T. K. from Toray Science Foundation, Takeda Science Foundation, Hoh-ansha Foundation, and Human Frontier Science Program. Back

2 Address correspondence and reprint requests to Dr. Tomohiro Kurosaki, Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi 570-8506, Japan. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; PLC, phospholipase C; SH, Src homology; SH2(N), N-terminal SH2 domain; SH2(C), C-terminal SH2 domain; IP3, inositol 1,4,5-trisphosphate; PTK, protein tyrosine kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; wt, wild-type. Back

Received for publication April 27, 1999. Accepted for publication June 11, 1999.


   References
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[Abstract] [Full Text] [PDF]

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S. I. Gringhuis, E. A. M. Papendrecht-van der Voort, A. Leow, E. W. N. Levarht, F. C. Breedveld, and C. L. Verweij
Effect of Redox Balance Alterations on Cellular Localization of LAT and Downstream T-Cell Receptor Signaling Pathways
Mol. Cell. Biol., January 15, 2002; 22(2): 400 - 411.
[Abstract] [Full Text] [PDF]

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D. Watanabe, S. Hashimoto, M. Ishiai, M. Matsushita, Y. Baba, T. Kishimoto, T. Kurosaki, and S. Tsukada
Four Tyrosine Residues in Phospholipase C-gamma 2, Identified as Btk-dependent Phosphorylation Sites, Are Required for B Cell Antigen Receptor-coupled Calcium Signaling
J. Biol. Chem., October 12, 2001; 276(42): 38595 - 38601.
[Abstract] [Full Text] [PDF]

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N. Engels, M. Merchant, R. Pappu, A. C. Chan, R. Longnecker, and J. Wienands
Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) Employs the SLP-65 Signaling Module
J. Exp. Med., July 30, 2001; 194(3): 255 - 264.
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D. Yablonski, T. Kadlecek, and A. Weiss
Identification of a Phospholipase C-{gamma}1 (PLC-{gamma}1) SH3 Domain-Binding Site in SLP-76 Required for T-Cell Receptor-Mediated Activation of PLC-{gamma}1 and NFAT
Mol. Cell. Biol., July 1, 2001; 21(13): 4208 - 4218.
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M. Matsuda, H. F. Paterson, R. Rodriguez, A. C. Fensome, M. V. Ellis, K. Swann, and M. Katan
Real Time Fluorescence Imaging of PLC{gamma} Translocation and Its Interaction with the Epidermal Growth Factor Receptor
J. Cell Biol., April 30, 2001; 153(3): 599 - 612.
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M. J. Rebecchi and S. N. Pentyala
Structure, Function, and Control of Phosphoinositide-Specific Phospholipase C
Physiol Rev, October 1, 2000; 80(4): 1291 - 1335.
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S. R. Brooks, X. Li, E. J. Volanakis, and R. H. Carter
Systematic Analysis of the Role of CD19 Cytoplasmic Tyrosines in Enhancement of Activation in Daudi Human B Cells: Clustering of Phospholipase C and Vav and of Grb2 and Sos with Different CD19 Tyrosines
J. Immunol., March 15, 2000; 164(6): 3123 - 3131.
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T. Yasuda, A. Maeda, M. Kurosaki, T. Tezuka, K. Hironaka, T. Yamamoto, and T. Kurosaki
Cbl Suppresses B Cell Receptor-mediated Phospholipase C (PLC)-{gamma}2 Activation by Regulating B Cell Linker Protein-PLC-{gamma}2 Binding
J. Exp. Med., February 21, 2000; 191(4): 641 - 650.
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Y. Kawakami, J. Kitaura, S. E. Hartman, C. A. Lowell, R. P. Siraganian, and T. Kawakami
Regulation of protein kinase Cbeta I by two protein-tyrosine kinases, Btk and Syk
PNAS, June 20, 2000; 97(13): 7423 - 7428.
[Abstract] [Full Text] [PDF]

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