The role of BLNK, DOK 3 DIP in BCR signaling 1

THE ROLE OF BLNK, DOK-3 & DIP IN BCR SIGNALING JOY EN-LIN TAN (B.Biotech.(Hons.)) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGMENT ACKNOWLEDGMENT I would like to thank the following people who made this thesis possible and an enjoyable experience for me. I am indebted to my supervisor A/P Lam Kong Peng, for his support and guidance as well as giving me an opportunity to work in his laboratory. Without his help, this work would not have been possible. I would also like to thank the members of my committee, A/P Benjamin Li and Assist. Prof. Robert Qi for their constructive suggestions and discussion. I am also grateful to Assist. Prof. Tang Bor Luen, who so kindly provided invaluable suggestions and gave immense support and encouragement. Special thanks goes to my past and present colleagues in the Molecular and Cellular Immunology Laboratory of the Institute of Molecular and Cell Biology (IMCB). In particular, Dr. Xu Shengli, who generated the BLNK-deficient mice, together with his advice, friendship and constant encouragements. Dr. Wong Siew Cheng, who taught me how to perform western analysis and gel-shift experiments and provided valuable suggestions and discussions. Mr. Chew Weng Keong, Mr. Lee Koon Guan, Ms. Tan Su-Li, Ms. Tan Ai-Tee for their excellent help through the various years in maintaining the laboratory. Fellow comrades in the laboratory Mr. Ng Chee Hoe, Mr. Andy Tan, Ms. Valerie Chew for making the laboratory a friendlier place to be in. I I ACKNOWLEDGMENT would also like to acknowledge the staff of the In Vivo Model Systems Unit of IMCB for taking care of the mice. I would like to thank my friends at IMCB especially Darren, Boon Tin, Zhihong and Esther for those fun times studying and working together. I am also grateful to the Administrative Staff, the COMIT Staff, the Lab-Supply Staff, the Maintenance Staff and the Glass-Ware Staff who tirelessly helped made my work much easier at IMCB. I would like to thank my family for their constant support. I am greatly indebted to my parents who helped me each day with encouragements and advices and my brother who never stop believing in me. Above all, my husband, John who encouraged me to persevere on even though there were times when things were tough. Thank you for your constant love, unfailing support and everlasting understanding. Lastly, I would like to attribute all glory to God for this was only made possible because of Him. I dedicate this thesis to my mother and father. Joy En-Lin Tan July 2004 II TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGMENT I TABLE OF CONTENTS III LIST OF FIGURES IX ABBREVIATIONS .XI SUMMARY . XIII LIST OF PUBLICATIONS XVI CHAPTER 1: INTRODUCTION . 1.1 THE IMMUNE SYSTEM . 1.2 DEVELOPMENT OF B LYMPHOCYTES . 1.2.1 1.3 Immunoglobulin gene rearrangement MATURATION OF B LYMPHOCYTE REGULATED BY BCR AND THEIR SURROGATES 1.3.1 Development of pro- and pre-B lymphocytes .8 1.3.2 Immature B lymphocytes undergo negative selection 1.3.3 Transitional B lymphocytes develop into mature B lymphocytes 11 1.3.4 Functional maturation of periphery B lymphocytes 12 1.3.4.1 1.3.4.2 1.4 T cell-dependent immune response . 13 T cell-independent immune response 14 ANTIGEN RECEPTORS SIGNALING IN B LYMPHOCYTES . 15 1.4.1 Structure of B cell receptor 15 1.4.2 Signal transduction through the B cell receptor 17 1.4.2.1 1.4.2.1.1 1.4.2.1.2 1.4.2.1.3 Protein tyrosine kinases . 18 Activation of src protein tyrosine kinases . 18 Membrane recruitment and activation of syk 20 Activation of Bruton’s tec kinase 21 III TABLE OF CONTENTS 1.4.2.2 1.4.2.2.1 1.4.2.2.2 1.4.3 Protein tyrosine phosphatases 22 CD45 required for BCR signaling . 22 Lipid phosphatase 5’ inositol phosphatase (SHIP-1) . 23 Structure of co-BCR receptor FcγRIIB 23 1.4.3.1 FcγRIIB mediated inhibition . 24 1.4.4 Major downstream signaling pathways of BCR 26 1.4.4.1 1.4.4.2 1.4.4.3 1.4.4.4 Activation of the PI3-K pathway . 26 Activation of phospholipase C (PLCγ) pathway 30 Activation of the Rho-family GTPase . 32 Activation of the Ras signaling pathway . 33 1.5 LIPID RAFTS IN BCR SIGNALING 34 1.6 REGULATION OF BCR SIGNALING BY ADAPTOR PROTEINS 37 1.6.1 Domains and motifs found in adaptor proteins .37 1.6.2 Adaptor proteins in BCR signaling 38 1.6.2.1 1.6.2.1.1 1.6.2.1.2 1.6.2.1.3 1.6.2.2 1.6.2.2.1 1.6.2.2.2 Adaptors involved in positive regulation of BCR signaling 39 Bam32 39 BCAP . 40 BLNK . 41 Adaptors involved in negative regulation of BCR signaling . 43 c-Cbl . 43 Dok . 43 1.7 APOPTOSIS IN B-LYMPHOCYTE DEVELOPMENT 45 1.8 AIMS AND RATIONALE OF CURRENT RESEARCH 47 CHAPTER 2: MATERIAL AND METHODS . 48 2.1 LIST OF ANTIBODIES . 49 2.2 LIST OF PRIMERS 51 2.3 RNA/DNA METHODOLOGY . 53 2.3.1 Extraction of RNA 53 2.3.2 Northern analysis .54 2.3.3 First strand cDNA synthesis 55 2.3.4 Amplification of cDNA .56 2.3.5 Polymerase chain reaction (PCR) .57 2.3.6 Agarose gel electrophoresis .59 2.3.7 Elution of DNA from agarose gel 59 IV TABLE OF CONTENTS 2.3.8 Restriction enzymes (RE) digestion of plasmid DNA .60 2.3.9 Dephosphorylation of plasmid DNA 60 2.3.10 Ligation 61 2.3.11 Preparation of competent cells, DH5α 61 2.3.12 Bacterial transformation by the heat shock protocol 62 2.3.13 Mini-preparation of plasmid DNA by alkaline lysis 62 2.3.14 Maxi-preparation of plasmid DNA 63 2.3.15 Sequencing of DNA 64 2.4 PROTEIN METHODOLOGY . 65 2.4.1 Yeast-two-hybrid screen .65 2.4.1.1 Synthetic dropout (SD) solution 65 2.4.1.2 Small-scale yeast transformation using LiAc 66 2.4.1.3 Preparation of DNA-BD-GAL4 bait for mating 68 2.4.1.4 Mating of pre-transformed library with bait 68 2.4.1.5 β-galactosidase colony-lift filter assay 69 2.4.2 Protein concentration determination by BCA protein assay .69 2.4.3 Western blot analysis .70 2.4.4 Alkaline phosphatase assay .71 2.4.5 Immunoprecipitation 71 2.4.6 Indirect immunofluorescent labelling 71 2.4.6 Transient transfection methods 72 2.4.6.1 Lipofectamine transfection 72 2.4.6.2 Effectene transfection 73 2.4.6.3 Amaxa transfection 73 2.4.7 Lipid raft separation 74 2.4.8 NF-κB assays .74 2.4.8.1 2.4.8.2 2.4.8.3 2.5 Stimulation of cells for NF-κB assays . 74 Preparation of nuclear extracts . 75 Electrophoretic mobility shift assays . 75 MAMMALIAN CELL CULTURE AND ASSAYS 76 2.5.1 Cell culture .76 2.5.2 Apoptosis assay 76 2.5.3 Preparation of primary B cells from mouse spleen .77 2.5.4 Cell proliferation assay 77 V TABLE OF CONTENTS 2.5.5 Cell cycle and cell death analyses .78 CHAPTER 3: THE ROLE OF ADAPTOR PROTEIN BLNK IN BCR SIGNALING OF CELL CYCLE PROGRESSION AND SURVIVAL IN B LYMPHOCYTES 79 3.1 INTRODUCTION . 80 3.2 ANTI-IGM STIMULATED BLNK-/- B CELLS FAIL TO ENTER THE CELL CYCLE 81 3.3 IMPAIRED INDUCTION OF CELL CYCLE REGULATORY PROTEINS -/- IN ANTI-IGM STIMULATED BLNK 3.4 B CELLS 86 BLNK-/- B CELLS DO NOT EXPRESS BCL-XL UPON ANTI-IGM STIMULATION 88 3.5 BLNK-/- B CELLS EXHIBIT A HIGH RATE OF SPONTANEOUS APOPTOSIS IN CULTURE 89 3.6 NORMAL ACTIVATION OF MAPKS AND AKT IN MOUSE BLNK-/B CELLS . 94 3.7 BLNK IS REQUIRED FOR THE ACTIVATION OF NF-κB IN RESPONSE TO BCR ENGAGEMENT . 96 3.8 THE ACTIVATION OF BRUTON’S TYROSINE KINASE IS NORMAL BUT THAT OF PLC-γ2 IS IMPAIRED IN BCR-STIMULATED BLNK-/- B CELLS 102 3.9 DISCUSSION . 105 CHAPTER 4: THE ROLE OF DOK-3 IN NEGATIVE REGULATION OF BCR SIGNALING . 113 4.1 INTRODUCTION . 114 VI TABLE OF CONTENTS 4.2 PHOSPHORYLATION OF DOK-3 UPON BCR AND FCγRIIB COLIGATION 117 4.3 LIPID RAFT LOCALIZATION OF DOK-3 UPON BCR + FCγRIIB CO-LIGATION 119 4.4 LIPID RAFT LOCALIZATION OF DOK-3 IS PERTURBED BY BLOCKING FCγRIIB . 126 4.5 DISCUSSION . 126 CHAPTER 5: CHARACTERIZATION OF A DOK-3INTERACTING PROTEIN, DIP 130 5.1 INTRODUCTION . 131 5.2 CLONING OF FULL LENGTH DOK-3 CDNA AND IDENTIFICATION OF A DOK-3-INTERACTING PROTEIN, DIP . 132 5.3 INTERACTION OF DOK-3 AND DIP ANALYSED BY COTRANSFECTION STUDIES . 135 5.4 TISSUE EXPRESSION PROFILE OF DIP 139 5.5 SUBCELLULAR LOCALIZATION OF DIP IN RESTING AND ACTIVATED CELLS 143 5.6 CO-LOCALIZATION OF DIP AND DOK-3 TO LIPID RAFTS UPON BCR + FCR CO-LIGATION . 146 5.7 DIP BINDS TO DOK-3 THROUGH ITS C-TERMINAL DOMAIN IN A PHOSPHORYLATION-INDEPENDENT MANNER . 150 5.8 INTERACTION OF DIP WITH SPECIFIC MEMBERS OF THE DOK FAMILY ADAPTORS 151 5.9 DIP MEDIATES APOPTOSIS THROUGH A CASPASE-3 DEPENDENT MECHANISM 154 VII TABLE OF CONTENTS 5.10 DOK-3 AND DOK-1 INHIBITS DIP-MEDIATED APOPTOSIS . 159 5.11 DISCUSSION . 162 CHAPTER GENERAL CONCLUSION 166 6.1 IMPORTANCE OF POSITIVE REGULATOR ADAPTOR BLNK IN BCR SIGNALING . 167 6.2 THE ROLE OF DOK-3 IN NEGATIVE REGULATION OF BCR SIGNALING 168 REFERENCES . 170 PUBLICATIONS 199 VIII LIST OF FIGURES LIST OF FIGURES Figure 1.1 B lymphocyte development. . Figure 1.2 Structure of the BCR complex . 16 Figure 1.3 Activation of proximal PTKs upon BCR engagement . 19 Figure 1.4 Signaling of B lymphocyte inhibitory receptor, FcγRIIB 25 Figure 1.5 Major downstream signaling pathways of BCR. . 28 Figure 1.6 A schematic view of BLNK protein domains . 42 Figure 3.1 Defective proliferation of BLNK-/- B cells in response to anti-IgM but not LPS stimulation 83 Figure 3.2 Anti-IgM-stimulated BLNK-/- B cells failed to enter the cell cycle. . 84 Figure 3.3 Lack of induction of cell cycle regulatory proteins in anti-IgMstimulated BLNK-/- B cells 87 Figure 3.4 Absence of Bcl-xL expression in anti-IgM-stimulated BLNK-/- B cells. 90 Figure 3.5 The expression of Bcl-2 is not altered in wild-type and BLNK-/- B cells treated with various stimuli. 91 Figure 3.6 High rate of spontaneous apoptosis of BLNK-/- B cells in culture.93 Figure 3.7 BLNK-/- B cells exhibited normal activation of MAPKs and Akt upon BCR engagement. 95 Figure 3.8 Imparied NF-κB activation in BCR-stimulated BLNK-/- B cells. 100 Figure 3.9 Expression and activation of Btk and PLCγ2 in anti-IgMstimulated BLNK-/-B cells . 104 Figure 3.10 A model for the BCR-induced activation of NF-κB . 112 Figure 4.1 Phosphorylation of Dok-3 upon BCR and BCR+FcR co-ligation. . 118 Figure 4.2 PLCγ2 and BCR are present in the lipid raft upon BCR engagement. . 120 Figure 4.3 Dok-3 is recruited to the lipid rafts only upon BCR + FcR coligation 122 Figure 4.4 Association of Dok-3 with lipid rafts seen upon BCR + FcR coligation 124 Figure 4.5 Localization of Dok-3 to lipid rafts upon BCR + FcR co-ligation . 125 IX Role of BLNK in Cell Cycle Entry and NF-␬B Activation 20057 FIG. 1. Defective proliferation of BLNK؊/؊ B cells in response to antiIgM but not LPS stimulation. Purified splenic wild-type and BLNKϪ/Ϫ B cells were stimulated for 48 h with increasing concentrations of goat anti-mouse IgM F(abЈ)2 fragment (A) or LPS (B). Cell proliferation was quantified in an MTT colorimetric assay. Figure shown is representative of five independent experiments and reproducible with B cells obtained from mice of different ages. O.D., optical density. cells in the resting G0/G1 phase, cells in the S phase of the cell cycle are actively synthesizing DNA and will incorporate BrdUrd. They will also exhibit a higher level of PI staining due to their increased cellular DNA content. As seen in Fig. 2, analysis of anti-IgM-stimulated wild-type B cells clearly reveals a population of cells in the S phase of the cell cycle. In contrast, such S phase cycling cells are absent in anti-IgMtreated samples of BLNKϪ/Ϫ B cells, suggesting that the mutant cells are arrested at the G0/G1 phase. Again as control, BLNKϪ/Ϫ B cells are able to enter the cell cycle upon treatment with LPS, and they show a pattern of BrdUrd incorporation indistinguishable from that of LPS-treated wild-type B cells (Fig. 2). This is consistent with the proliferation data shown in Fig. 1B, which indicate the ability of BLNKϪ/Ϫ B cells to respond to LPS stimulation. Thus, the data presented in Figs. and together show that the failure of BLNKϪ/Ϫ B cells to proliferate in respond to anti-IgM stimulation is due to their inability to enter the cell cycle. Impaired Induction of Cell Cycle Regulatory Proteins in AntiIgM-stimulated BLNKϪ/Ϫ B Cells—The entry of cells into the cell cycle is regulated by the activity of specific proteins such as the cyclins and the cyclin-dependent kinases (cdks) (27). The D-type cyclins and their kinase partners, cdk4 and cdk6, are the earliest cell cycle regulatory protein complexes to be expressed when cells leave quiescence and enter the cell cycle (28). Normal proliferating B cells express cyclins D2 and D3 but not D1 (25). Cyclin D2 and its kinase partner, cdk4, are upregulated from mid to late-G1 phase of the cell cycle and are readily detected in cycling B cells (29). To determine if cyclin D2 and cdk4 can be induced in BLNKϪ/Ϫ B cells, we treated the wild-type and mutant cells with anti-IgM antibodies for various times. As shown in Fig. 3, cyclin D2 is maximally induced in wild-type B cells 24 h after BCR cross-linking and its expression can be detected even after 48 h of treatment. Similarly, cdk4, which is expressed at basal level in normal resting B cells, is also up-regulated upon anti-IgM stimulation. In contrast, both cyclin D2 and cdk4 are not expressed or upregulated in BLNKϪ/Ϫ B cells regardless of the duration of their BCR cross-linking. As control, we examined the induction of cell cycle regulatory proteins in BLNKϪ/Ϫ B cells after LPS treatment. As expected, LPS induces the up-regulation of cyclin D2 and cdk4 in wildtype B cells (Fig. 3). LPS is also able to induce the expression of cyclin D2 and cdk4 in BLNKϪ/Ϫ B cells, consistent with the fact that LPS-treated mutant B cells proliferate (Fig. 1), incorporate BrdUrd (Fig. 2), and enter the cell cycle. This suggests that the failure to express the cell cycle regulatory proteins in FIG. 2. Anti-IgM-stimulated BLNK؊/؊ B cells failed to enter the cell cycle. Purified splenic wild-type and BLNKϪ/Ϫ B cells were cultured in the presence of 40 ␮M BrdUrd (BrdU) for 48 h without stimulus or treated with 20 ␮g/ml goat anti-mouse IgM F(ab)Ј2 fragment or 25 ␮g/ml LPS. BrdUrd incorporation and PI staining of total cellular DNA content are used to reveal cells at various stages of the cell cycle. Arrows indicate apoptotic cells (A) or cells in the G0/G1 and S phase of the cell cycle. BLNKϪ/Ϫ B cells is specific to anti-IgM stimulation. Thus, the failure of anti-IgM-stimulated BLNKϪ/Ϫ B cells to proliferate may be due to the inability of these cells to transduce the signal for the induction of specific regulatory proteins that are critical for the entry into cell cycle. BLNKϪ/Ϫ B Cells Do Not Express Bcl-xL upon Anti-IgM Stimulation—In addition to the induction of cell cycle regulatory proteins, the cross-linking of the BCR on normal B cells also leads to the expression of the cell survival protein, Bcl-xL (30, 31). The induction of Bcl-xL has been correlated with the ability of activated B cells to undergo cellular proliferation (30) and may be required for them to complete the cell cycle (11, 13). 20058 Role of BLNK in Cell Cycle Entry and NF-␬B Activation FIG. 3. Lack of induction of cell cycle regulatory proteins in anti-IgM-stimulated BLNK؊/؊ B cells. Purified splenic wild-type and BLNKϪ/Ϫ B cells were treated with goat anti-mouse IgM F(ab)Ј2 fragment or LPS for various times and examined for the expression of cell cycle regulatory proteins in Western blot analyses. The anti-tubulin blot served as a control for the loading of whole cell lysate. Figure shown is representative of three separate analyses. As shown in Fig. 4A, this protein is expressed in anti-IgMtreated wild-type or BLNKϩ/Ϫ B cells within 24 h of stimulation and its continued expression can be detected up to 72 h of culture (data not shown). In contrast, Bcl-xL is absent in BLNKϪ/Ϫ B cells treated with anti-IgM for the same time duration (Fig. 4A and data not shown). Again as control, Bcl-xL expression can be detected within 24 h in both LPS-treated wild-type and BLNKϪ/Ϫ B cells (Fig. 4B). Thus, the data indicate that anti-IgM-treated BLNKϪ/Ϫ B cells may also abort the cell cycle due to their inability to express the cell survival Bcl-xL protein upon activation. Normal B lymphocytes also express the anti-apoptotic protein, Bcl-2 (32). To examine if BLNK deficiency affects the expression of Bcl-2, as is the case for Bcl-xL above, we examined the expression level of this protein in normal and BLNKϪ/Ϫ B cells before and after various stimulations. As shown in Fig. (A and B), Bcl-2 is expressed at equivalent level in wild-type and BLNKϪ/Ϫ B cells ex vivo and its expression level remains relatively unchanged upon stimulation with anti-IgM antibodies or LPS. Thus, a BLNK deficiency specifically affects the expression of Bcl-xL but not that of Bcl-2 in B lymphocytes. BLNKϪ/Ϫ B Cells Exhibit a High Rate of Spontaneous Apoptosis in Culture—In the course of the cell cycle analysis as shown in Fig. 2, it was noticed that BLNKϪ/Ϫ B cells cultured for 48 h in medium alone or in the presence of anti-IgM antibodies show a higher degree of apoptosis compared with similarly treated wild-type B cells. To determine if BLNKϪ/Ϫ B cells indeed exhibit a higher propensity to undergo spontaneous apoptosis in vitro, we examined these cells after overnight culture, either non-stimulated or treated with the various stimuli. Apoptotic cells can be distinguished in FACS analyses by their reduced cellular DNA content, as revealed by PI staining, and by their smaller size, as shown by a reduction in the forward and side scatter profiles. As shown in Fig. 6, BLNKϪ/Ϫ B cells are twice as likely (50 – 65%) to undergo spontaneous apoptosis in medium after overnight culture as compared with their normal counterparts (33–38%). Anti-IgM treatment can reduce the fraction of dying wild-type (20 –23%) but not BLNKϪ/Ϫ (55– 63%) B cells. This can be explained by the ability of wild-type but not BLNKϪ/Ϫ B cells to express Bcl-xL upon activation (see Fig. 4). Finally, LPS treatment substantially reduced the fraction of dying cells in both the wild-type and BLNKϪ/Ϫ B cell samples, although the population of apoptotic cells is still higher in the latter compared with the former. This is consistent with the fact that LPS can induce the expression of Bcl-xL in both the wild-type and BLNKϪ/Ϫ B cells (see Fig. 4). Normal Activation of MAPKs and Akt in Mouse BLNKϪ/Ϫ B Cells—The various analyses above describe the cellular defects of BLNKϪ/Ϫ B cells in response to BCR signaling. Given the fact that BLNK is an adaptor protein involved in signal trans- FIG. 4. Absence of Bcl-xL expression in anti-IgM-stimulated BLNK؊/؊ B cells. Western blot analysis of Bcl-xL expression in wildtype, BLNKϩ/Ϫ and BLNKϪ/Ϫ B cells stimulated with 10 ␮g/ml goat anti-mouse IgM F(ab)Ј2 fragment for 48 h (A) or 10 ␮g/ml LPS for 24 – 48 h (B). The A431 cell line provides the specificity control for the anti-Bcl-xL antibody. The anti-tubulin blot serves as a control for the loading of whole cell lysate. FIG. 5. The expression of Bcl-2 is not altered in wild-type and BLNK؊/؊ B cells treated with various stimuli. Western blot analysis of Bcl-2 expression in wild-type and BLNKϪ/Ϫ B cells stimulated for 24 – 48 h with either 10 ␮g/ml goat anti-mouse IgM F(ab)Ј2 fragment (A) or 10 ␮g/ml LPS (B). The anti-tubulin blot serves as a control for the loading of whole cell lysate. duction, it would be of interest to correlate the above cellular defects with disruption of distinct signaling pathways. BCR engagement is known to activate the three different classes of MAPKs: ERK, JNK, and p38 MAPK, that have been shown to regulate proliferation, survival, and differentiation in various cellular systems (33). In the DT40 chicken B cell line, BLNK is required for the activation of both JNK and p38 MAPK and for the sustained activation of ERK in response to BCR signaling (8). We therefore re-examined if the activation of MAPKs was similarly affected in mouse primary B cells lacking BLNK and if these signaling defects (if any) could plausibly explain the cellular defects that we have observed above. As shown in Fig. 7A, the phosphorylation of the p42 and p44 forms of ERK occurs within 30 s and is sustained for as long as 10 (data not shown) after BCR cross-linking in wild-type B cells. ERK phosphorylation can also be detected with the same kinetics after BCR stimulation in BLNKϪ/Ϫ B cells, suggesting that the activation of ERK is normal in these mutant B cells. Similarly, Western blot analyses of whole cell lysates derived from non-treated and anti-IgM-stimulated wild-type and Role of BLNK in Cell Cycle Entry and NF-␬B Activation 20059 FIG. 6. High rate of spontaneous apoptosis of BLNK؊/؊ B cells in culture. Purified splenic wild-type and BLNKϪ/Ϫ B cells were cultured overnight in media alone or with 20 ␮g/ml goat anti-mouse IgM F(ab)Ј2 fragment or 25 ␮g/ml LPS. A, apoptotic cells with sub-G0 amount of DNA as revealed by PI staining in FACS analyses were marked and expressed as a percentage of total cells analyzed. B, apoptotic cells as revealed by their reduced forward and side scatter profiles in FACS analyses were gated and expressed as a percentage of total cells examined. BLNKϪ/Ϫ B cells reveals that JNK (Fig. 7B) and p38 MAPK (Fig. 7C) are also phosphorylated with the same kinetics in both the samples tested. The intact activation of the three classes of MAPKs in BLNKϪ/Ϫ mouse primary B cells is in contrast to the impaired activation of these kinases in the DT40 chicken B cell line (8). Other than the MAPKs, BCR engagement also activates the Akt signaling pathway that is known to regulate cell survival (34). As such, we examine if this pathway is compromised in BLNKϪ/Ϫ B cells. As shown in Fig. 7D, the kinetics of Akt activation is again similar in both the wild-type and BLNKϪ/Ϫ B cells stimulated by anti-IgM antibodies. Thus, the overall data suggest that BLNK plays no role in transducing the BCR signal that activates the three different classes of MAPKs and Akt in mouse primary B lymphocytes and that these signaling pathways are not likely responsible for the cellular defects described earlier. BLNK Is Required for the Activation of NF-␬B in Response to BCR Engagement—The engagement of the antigen receptor on B lymphocyte also activates the transcription factor NF-␬B that is known to regulate genes involved in cell proliferation and survival (35). Several groups have shown that the predominant form of NF-␬B in B cells is largely the p50-c-Rel heterodimer (36); in particular, c-Rel was shown to be essential for B cell proliferation after BCR engagement (35, 36). To investigate the role of BLNK in BCR-induced NF-␬B activation, nuclear extracts were prepared from non-treated and anti-IgM-stimulated wild-type and BLNKϪ/Ϫ mouse primary B cells. As shown in the EMSA in Fig. 8A, BCR stimula- tion of wild-type B cells leads to a marked increase in nuclear NF-␬B activity (lanes and 2) as evidenced by the binding of a radiolabeled probe that contains two consensus NF-␬B binding sequences. In contrast, there was no increase in BCR-induced NF-␬B activity above the background levels in the nuclear extract obtained from BLNKϪ/Ϫ B cells (lanes and 5). As control, the treatment of wild-type and BLNKϪ/Ϫ B cells with phorbol ester (PMA) leads to a corresponding increase in nuclear NF-␬B activity in both the samples tested (compare lanes and and lanes and 6). This suggests that BLNKϪ/Ϫ B cells can activate NF-␬B in response to other stimulus but not to anti-IgM stimulation. Thus, the data indicate that BLNK is specifically required for BCR-induced activation of NF-␬B. The immediate events regulating NF-␬B activation have been elucidated. In non-stimulated cells, NF-␬B/Rel factors are sequestered in the cytoplasm in complexes with the I␬ B family of proteins (35, 36). Upon treatment of cells with specific stimulus, the I␬B kinases are activated and this results in the serine/threonine phosphorylation of I␬B proteins and their subsequent degradation. The NF-␬B/Rel proteins are released from the inhibitory complexes and localized to the nucleus to effect gene transcription (35, 36). The lack of nuclear NF-␬B binding activity in BCR-stimulated BLNKϪ/Ϫ B cells could therefore be due to reduced NF-␬B proteins in the nucleus. To determine if this is indeed the case, we examined the amount of c-Rel and the p50 subunit in the nucleus of mutant cells. As seen in the Western blot analysis in Fig. 8B, PMA or anti-IgM stimulation led to an accumulation of c-Rel in the nucleus of wild-type B cells (lanes and 3). How- 20060 Role of BLNK in Cell Cycle Entry and NF-␬B Activation FIG. 7. Normal activation of MAPKs and Akt in BCR-stimulated BLNK؊/؊ B cells. Wild-type and BLNKϪ/Ϫ B cells were treated with anti-IgM antibodies and the expression and activation of ERK (A), JNK (B), p38 MAPK (C), and Akt (D) were examined in Western blot analyses. The whole cell lysates were first probed with an antibody that recognizes the phosphorylated form and subsequently with an antibody that binds the non-phosphorylated form of the protein that is being studied. ever, in contrast, the accumulation of nuclear c-Rel was effected only with PMA but not with anti-IgM treatment of BLNKϪ/Ϫ B cells (lanes and 6). These data are consistent with the NF-␬B binding assay shown in Fig. 8A. The reduced level of nuclear c-Rel in the BCR-induced BLNKϪ/Ϫ B cell sample was not due to variation in the integrity of the nuclear extracts, as control Western blot analysis revealed the presence of similar amount of nuclear transcription factor IID in all the samples tested (Fig. 8B, middle panel). Examination of the p50 subunit also reveals that it is not translocated into the nucleus of BLNKϪ/Ϫ B cells after anti-IgM stimulation (Fig. 8B, lower panel) similar to the situation found with c-Rel. It is conceivable that BLNKϪ/Ϫ B cells may produce reduced amount of total c-Rel/p50 proteins and this in turn affects the amount found in the nucleus. To explore this possibility, we examine the amount of c-Rel in the whole cell lysates of wildtype and BLNKϪ/Ϫ B cells. Western blot analysis shown in Fig. 8C reveals that untreated, PMA-stimulated, or anti-IgM-stimulated BLNKϪ/Ϫ B cells produced amounts of c-Rel equivalent to that produced by similarly treated wild-type B cells. Thus, the data suggest that BLNKϪ/Ϫ B cells have specific impairment in the nuclear translocation of NF-␬B factors in response to BCR engagement. As mentioned above, NF-␬B/Rel transcription factors are sequestered in the cytoplasm by I␬B factors (35, 36). The failure of NF-␬B/Rel transcription factors to translocate into the nucleus of BCR-stimulated BLNKϪ/Ϫ B cells could be due to impairment in the degradation of the I␬B proteins. To test this possibility, we examine the degradation of I␬B␣ in these cells in response to the different stimuli. Both the wild-type and BLNKϪ/Ϫ splenic B cells were cultured in the presence of cycloheximide to prevent the de novo synthesis of I␬B␣. As seen in Fig. 8D and consistent with the data presented earlier, I␬B␣ was degraded in wild-type B cells stimulated with either PMA or anti-IgM antibodies (lanes and 3). In comparison, the degradation of I␬B␣ was normal in BLNKϪ/Ϫ B cells treated FIG. 8. Impaired NF-␬B activation in BCR-stimulated BLNK؊/؊ B cells. A, lack of NF-␬B binding activity in the nuclear extract of BLNKϪ/Ϫ B cells. EMSA analysis of nuclear NF-␬B activity in wild-type (lanes 1–3) and BLNKϪ/Ϫ (lanes – 6) splenic B cells that were either non-treated or stimulated with PMA (2 ␮g/ml) or anti-IgM antibodies (50 ␮g/ml) for h. B, impaired nuclear translocation of c-Rel and the p50 subunit in BCR-stimulated BLNKϪ/Ϫ B cells. Western blot analysis of nuclear extracts (5 ␮g) obtained from wild-type and BLNKϪ/Ϫ B cells that were non-treated or stimulated with PMA or anti-IgM antibodies. The blot was first probed with anti-c-Rel antibody and subsequently re-probed with anti-transcription factor IID (anti-TFIID) antibody to control for the amount and integrity of the nuclear extracts used. In a separate blot, the amount of nuclear p50 (bottom panel) was examined. C, equivalent amount of cytoplasmic c-Rel is found in wild-type and BLNKϪ/Ϫ B cells. Western blot analysis of whole cell lysates (5 ϫ 106 cells) obtained from wild-type and BLNKϪ/Ϫ B cells that were treated as above. The blot was initially probed with the anti-c-Rel antibody and re-probed with the anti-p38 MAPK antibody as control. D, degradation of I␬B␣ is impaired in BCR-stimulated BLNKϪ/Ϫ B cells. Splenic wildtype and BLNKϪ/Ϫ B cells were pre-cultured and later stimulated with PMA or anti-IgM antibodies for h in the continuous presence of cycloheximide. Anti-I␬B␣ antibody was used to determine for the presence of the I␬B␣ proteins, whereas anti-p38 MAPK antibody was used as control for the equivalent loading of whole cell lysates. with PMA (lane 5) but impaired in the mutant cells stimulated with anti-IgM antibodies (lane 6). This suggests that the failure of NF-␬B to translocate to the nucleus in BCR-stimulated BLNKϪ/Ϫ B cells is due to a specific impairment in the degradation of I␬B factors. Taken together, the data indicate that BLNK is required for BCR-induced NF-␬B activation in B cells and this occurs via a mechanism that involves the degradation of the I␬B subunits and the subsequent nuclear translocation of the NF-␬B/Rel transcription factors. The Activation of Bruton’s Tyrosine Kinase Is Normal, but That of PLC-␥2 Is Impaired in BCR-stimulated BLNKϪ/Ϫ B Cells—Recently Btk has been shown to be essential for the BCR-induced activation of NF-␬B in B cells (37, 38), and this involves a similar mechanism that requires the degradation of I␬B subunits. BLNK has been shown to associate with Btk (9, 10). Hence, it is possible that the inactivation of BLNK may affect the expression and/or activation of Btk, which in turn leads to the impairment in NF-␬B activation in BLNKϪ/Ϫ B Role of BLNK in Cell Cycle Entry and NF-␬B Activation FIG. 9. Expression and activation of Btk and PLC-␥2 in antiIgM-stimulated BLNK؊/؊ B cells. Btk (A) and PLC-␥2 (B) were immunoprecipitated from whole cell lysates of wild-type and BLNKϪ/Ϫ B cells that were treated with anti-IgM antibodies for various times and probed with anti-phosphotyrosine (pY) and the immunoprecipitating antibodies in Western blot (WB) analyses. The figures shown are representative of five separate experiments. cells. We therefore immunoprecipitated Btk from lysates of non-treated or anti-IgM-stimulated wild-type and mutant B cells to explore this possibility. As shown in Fig. 9A (lower panel), Btk is expressed at equivalent levels in both the wildtype and BLNKϪ/Ϫ B cells that were either non-treated or stimulated with anti-IgM antibodies. In addition, Btk is also activated with the same kinetics in both the wild-type and mutant B cells, as revealed by the anti-phosphotyrosine antibody that reveals the phosphorylation and activation status of Btk (Fig. 9A, upper panel). Thus, the inability of BLNKϪ/Ϫ B cells to activate NF-␬B in response to BCR stimulation is not due to a defect in the expression or activation of Btk per se. While the current work was in progress, Petro and Khan (39) showed that the enzyme PLC-␥2 that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5trisphosphate and diacylglycerol is also essential for the BCRinduced activation of NF-␬B. We thus immunoprecipitated PLC-␥2 from BLNKϪ/Ϫ B cells that were stimulated with antiIgM antibodies for various times to determine if the enzyme is activated normally in these cells. As shown in Fig. 9B, PLC-␥2 is fully activated within min, and its phosphorylation persisted for at least 10 after BCR engagement in wild-type B cells. In contrast, PLC-␥2 remains non-phosphorylated and therefore not activated after BCR stimulation for the same time duration examined in BLNKϪ/Ϫ B cells. Thus, the defect in NF-␬B activation in BLNKϪ/Ϫ B cells can be correlated to a defect in PLC-␥2 activation in response to BCR engagement. DISCUSSION BLNKϪ/Ϫ B cells not proliferate upon BCR stimulation (Fig. 1). Our data presented here indicate that this defect is due to the failure of mutant B cells to enter the cell cycle in response to this stimulus. In contrast to wild-type B cells, BLNKϪ/Ϫ B cells fail to incorporate BrdUrd and are arrested prior to the S phase of the cell cycle (Fig. 2). Indeed, this specific impairment is correlated at the molecular level to the inability of anti-IgM activated BLNKϪ/Ϫ B cells to express the cell cycle regulatory proteins such as cyclin D2 and cdk4 (Fig. 3) that are necessary for the progression of cell cycle beyond the G0/G1 phase (29). Anti-IgM treatment of BLNKϪ/Ϫ B cells also fails to induce the expression of the cell survival protein Bcl-xL (Fig. 4) that has been postulated to be critical for the viability of proliferating B cells (11, 13, 30). Thus, BLNKϪ/Ϫ B cells may have a compound defect in not being able to sustain cell survival for the progression of the cell cycle after anti-IgM stimulation. Taken together, the data strongly suggest that BLNKϪ/Ϫ B 20061 cells fail to enter and abort the cell cycle upon BCR engagement. The defect in cell cycle progression and cell proliferation may explain why, at the physiological level, BLNKϪ/Ϫ mice failed to mount an effective humoral immune response to T cell-independent antigen that involved extensive BCR cross-linking (18, 19). Antigen-specific B cells are rare and undergo clonal expansion when activated. It is likely that antigen-activated B cells are not clonally expanded in BLNKϪ/Ϫ mutant mice due to their inability to undergo the cell cycle and to their higher propensity to undergo apoptosis. Interestingly, BLNKϪ/Ϫ mice are able to mount a normal T cell-dependent immune response (18, 19). This would suggest that T cell help in the form of co-stimulation or secreted cytokines might overcome the cell cycle defect associated with the BCR signaling resulting from a BLNK deficiency. Of the major signaling pathways that are known to regulate cell proliferation and/or survival such as those of Akt (34), MAPKs (33) and NF-␬B (35, 36), we show here that only the latter is impaired in BCR-activated BLNKϪ/Ϫ B cells (see Figs. and 8). The finding that Bcl-xL, which is regulated by NF-␬B (40), is not induced in BLNKϪ/Ϫ B cells in response to BCR stimulation is consistent with this signaling defect. Previous analyses of BLNKϪ/Ϫ mice (18 –21) and our current biochemical study of BLNKϪ/Ϫ B cells suggest that the defects resulting from a BLNK deficiency mirror more closely to those resulting from a lack of Btk as in xid mice and BtkϪ/Ϫ B cells (23, 24). BLNKϪ/Ϫ and xid B cells are both unable to upregulate the cell cycle regulatory proteins and enter the cell cycle upon BCR engagement (12), and they both show a propensity to undergo a higher rate of spontaneous apoptosis in culture (11, 12). In addition, BCR-stimulated BtkϪ/Ϫ B cells also fail to activate the NF-␬B signaling pathway (37, 38). We demonstrate here that the expression and activation of Btk is normal in BLNKϪ/Ϫ B cells (Fig. 9). Hence, the phenotypes of BLNKϪ/Ϫ mice and cells are not likely due to defective expression and activation of Btk per se. Recently, PLC-␥2 was also shown to be essential for the activation of NF-␬B in response to BCR engagement (39). Our demonstration that this enzyme is not activated in BCR-stimulated BLNKϪ/Ϫ B cells suggests that this is the likely defect that impairs NF-␬B activation in these mutant cells. Taken together, a detailed model for the activation of NF-␬B in response to BCR stimulation is now emerging (see Fig. 10). Engagement of the BCR activates the immediate downstream tyrosine kinases Syk and Btk (2). It is known that both Syk and Btk are required for the full phosphorylation and activation of PLC-␥2 (41). BLNK is the adaptor molecule that couples Syk to PLC-␥2 (4). Since BLNK has also been shown separately to associate with Btk (9, 10), it is likely to couple Btk to PLC-␥2 as well, although this has not been directly proven. Our data of normal Btk but impaired PLC-␥2 activation in BCR-stimulated BLNKϪ/Ϫ B cells are consistent with this model of tri-molecular interaction. Thus, BLNK emerges as the key adaptor molecule that couples Syk and Btk, either in concert or sequentially to activate PLC-␥2, which in turn activates NF-␬B that regulates genes involved in proliferation and survival such as bcl-x. These signaling molecules Syk, Btk, BLNK, and PLC-␥2 together form a “signalosome” (42). Inactivation of BLNK (this report) or Btk or PLC-␥2 will disrupt this signalosome and lead to a common NF-␬B signaling defect. This may provide an explanation for the similar B cell defects found in BLNKϪ/Ϫ (18 –21), BtkϪ/Ϫ (23, 24), and PLC-␥2Ϫ/Ϫ (43, 44) mice. By extrapolation, one would then expect that PLC-␥2Ϫ/Ϫ B cells would also not up-regulate cell cycle regulatory and cell survival molecules upon BCR engagement, and this awaits further 20062 Role of BLNK in Cell Cycle Entry and NF-␬B Activation FIG. 10. A model for the BCR-induced activation of NF-␬B. Engagement of the BCR activates Syk and Btk (1). Syk phosphorylates BLNK (2). BLNK couples Syk and Btk to PLC-␥2 (3), and this results in its activation and to the eventual activation of NF-␬B that regulates genes involved in cell survival and proliferation such as bcl-x. confirmation. Finally, it is worthy to note that B cells deficient in the various components of NF-␬B also exhibit proliferation defects (35, 36, 45, 46), again consistent with the model that we presented here. Our current analyses indicate that BLNKϪ/Ϫ B cells are able to proliferate upon LPS stimulation. This contrasts with previous reports that have stated otherwise (19, 21). The discrepancy in the response to LPS may be due to the relative sensitivity of the various assays used to examine cellular proliferation. It may be that the MTT assay used in our current study is more easily saturated compared with the thymidine incorporation assay used by others (19, 21) and thus failed to measure the reduction in the LPS-induced proliferation of BLNKϪ/Ϫ B cells compared with the wild-type B cells. However, it is noted that, in one of the previous published reports (19), BLNKϪ/Ϫ B cells did respond to some extent to LPS stimulation and thus did not contradict our current data qualitatively. Indeed, the various experiments outlined in this paper that examine the incorporation of BrdUrd (Fig. 2), the induction of cell cycle regulatory proteins (Fig. 3), and cell survival protein, Bcl-xL (Fig. 4), are all consistent with the fact that BLNKϪ/Ϫ B cells are able to respond to LPS stimulation and proliferate. Finally, examination of LPS-stimulated BLNKϪ/Ϫ B cells (Fig. 6) indicates that they are bigger in size (as reflected by the forward scatter profile) compared with anti-IgM-stimulated or non-treated BLNKϪ/Ϫ B cells, again consistent with the notion that BLNKϪ/Ϫ B cells are activated by LPS. LPS signals through CD14 and the Toll-like receptors (47), and, although it activates NF-␬B (48), it appears to so via a pathway that does not require BLNK. Incidentally, it was reported that PLC-␥2Ϫ/Ϫ B cells, which have defects similar to those of BLNKϪ/Ϫ B cells, also proliferate normally in response to LPS stimulation (43). Much of the recent work in BCR signaling had been done using the chicken DT40 B cell line. It was shown in this system that the BCR-induced activation of ERK and JNK was impaired in the absence of Syk and Btk (49). Given that BLNK interacts with both Syk and Btk (4, 9, 10), it would be reasonable to assume that some of the MAPKs might also be affected by a BLNK deficiency. Indeed, it was shown recently that the BCR-induced activation of ERK, JNK, and p38 MAPK was also perturbed in the DT40 cell line lacking BLNK (8). However, our data presented in this paper seem to contradict the latter observation. We show here that the BCR-induced activation of all three classes of MAPKs remain intact in mouse primary B cells lacking BLNK (see Fig. 7). A likely explanation in the discrepancy is the cellular context of the systems used, namely the DT40 chicken B cell line used by others (4, –10) versus the mouse primary B lymphocytes used in this report. Such a discrepancy has indeed been observed in several instances previously. First of all, it was shown that the BCR-induced Ca2ϩ response was reduced in human B cell lines and mouse primary B lymphocytes lacking Btk (50) but totally abolished in the DT40 chicken B cells (41). Similarly, DT40 chicken B cells lacking BLNK not flux Ca2ϩ in response to BCR crosslinking (8), whereas mouse B cells lacking BLNK although the response was again reduced (19). Thus, there may exist real signaling differences in mouse primary B lymphocytes and the chicken DT40 cell line. Alternatively, it remains possible that the DT40 chicken B cell line is in a different state of maturation compared with the splenic B cells that we used in this paper. It is interesting to note that mouse primary B cells lacking BLNK (19) or Btk (50) still retain the ability to flux Ca2ϩ to some extent, although the activation of PLC-␥2 is impaired in these mutant cells. On the same note, mouse primary B cells lacking PLC-␥2 (43) also retain the ability to flux Ca2ϩ in response to BCR engagement, although the magnitude is again much reduced. Finally, it has been suggested that BCR specificity and hence signaling may play a role in the development of CD5ϩ B cells (51). Mice deficient in BLNK (18 –21), Btk (23, 24), or PLC-␥2 (43, 44), which are molecules involved in BCR signaling, all lack CD5ϩ B cells. Recently, it was shown that cyclin D2 expression is also essential for CD5ϩ B cell development (52). CD5ϩ B cells are thought to undergo self-renewal, a process that is most likely induced through the recognition of an antigen by their BCR (51). Our current data and those previously published (37, 38) indicate that BLNK and Btk are part of a signalosome that transduces the BCR signal that leads to the expression of cyclin D2 and the entry into cell cycle. Thus, in the absence of BLNK or Btk, the BCR signal that leads to the expression of cell cycle regulatory molecules is impaired and this will result in the subsequent defect in BCR-induced cellular proliferation that may affect the self-renewal of a population of CD5ϩ B cells. Acknowledgment—We thank the Institute of Molecular and Cell Biology In Vivo Model Unit for the care and maintenance of mice. 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Today 14, 84 –91 52. Solvason, N., Wu, W., Parry, D., Mahony, D., Lam, E., Glassford, J., Klaus, G., Sicinski, P., Weinberg, R., Liu, Y., Howard, M., and Lees, E. (2000) Int. Immunol. 12, 631– 638 THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 277, No. 34, Issue of August 23, pp. 30707–30715, 2002 Printed in U.S.A. Peritoneal CD5؉ B-1 Cells Have Signaling Properties Similar to Tolerant B Cells* Received for publication, March 14, 2002, and in revised form, June 12, 2002 Published, JBC Papers in Press, June 17, 2002, DOI 10.1074/jbc.M202460200 Siew-Cheng Wong‡, Weng-Keong Chew‡, Joy En-Lin Tan‡, Alirio J. Melendez§, Florence Francis‡, and Kong-Peng Lam‡¶ From the ‡Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore and the §Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore CD5؉ B (or B-1) cells are the normal precursors of B cell chronic lymphocytic leukemia. They differ from conventional B (B-2) cells with respect to their phenotype and mitogenic responses and are often secretors of the natural polyreactive antibodies in the serum. The origin of B-1 cells remains controversial, and the relationship between B-1 cells and autoreactive B cells is unclear. Here, we compare the signaling pathways that are activated by the engagement of the B cell antigen receptor (BCR) in B-1 and B-2 cells. Stimulation of the BCR leads to the induced activation of the three major classes of mitogen-activated protein kinases (MAPKs), ERK, JNK, and p38 MAPK, as well as the Akt kinase and the transcription factors nuclear factor of activated T cells (NF-AT) and NF-␬B in B-2 cells. In contrast, B-1 cells have constitutive activation of ERK and NF-AT but exhibit delayed JNK and lack p38 MAPK and NF-␬B induction upon BCR cross-linking. The lack of NF-␬B activation in B-1 cells may be due to a lack of Akt activation in these cells. Furthermore, our study using specific inhibitors reveals that the extended survival of B-1 cells in culture is not due to the constitutive activation of ERK; nor is it due to Akt signaling or Bcl-xL upregulation, since these are not induced in B-1 cells. The current findings of altered MAPK and NF-AT activation and lack of NF-␬B induction in B-1 cells indicate that these cells have signaling properties similar to tolerant B cells that are chronically exposed to self-antigens. Indeed, BCR stimulation of B-1 cells does not lead to their full activation as indicated by their lack of maximal up-regulation of specific markers such as CD25, CD69, and CD86. CD5ϩ B (or B-1) cells are a unique subset of B cells that are distinguishable from the conventional B or B-2 cells in terms of their phenotype, anatomical localization, and self-renewal properties (1). For example, B-1 cells are found in the pleural and peritoneal cavities and express a high level of IgM and low levels of IgD and the pan B-cell marker B220 on their cell surfaces. In addition, they express an intermediate level of the T-cell marker CD5. On the other hand, B-2 cells predominate in the spleen and lymph nodes and express intermediate levels of IgM and IgD and a high level of B220, and they lack CD5 * This work is supported by grants from the Biomedical Research Council of the Agency for Science, Technology, and Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed. Tel.: 65-6874-3784; Fax: 65-6779-1117; E-mail: mcblamkp@imcb.nus.edu.sg. This paper is available on line at http://www.jbc.org expression on their cell surfaces. The origin of B-1 cells is controversial, and it remains to be determined whether they are derived from a separate B cell lineage (2) or represent a state of differentiation or activation of normal B lymphocytes (3). However, B-1 cells are known to secrete natural polyreactive antibodies found in the serum and often have specificities directed toward self-antigens such as phosphatidylcholine (4), single-stranded DNA (5), ribonucleoprotein (6), and the cell surface Thy-1 antigen (7). In addition, B-1 cells frequently give rise to B-cell chronic lymphocytic leukemia (B-CLL).1 B-1 cells also differ from B-2 cells in their functional responses to external stimuli. For example, engagement of the B cell antigen receptor (BCR) leads to the proliferation of B-2 cells, but such entry into the cell cycle is blocked in B-1 cells (8, 9). This difference in physiological response suggests that B-1 cells may have signaling properties that are different from B-2 cells. The BCRs on both B-1 and B-2 cells are composed similarly of the Ig heavy and light chains in complex with the signaling subunits Ig␣ and Ig␤ (10). BCR signaling is known to activate numerous signal transduction pathways, and the induction of a particular pathway may depend on the state of differentiation of the B lymphocyte and may lead to distinct cellular outcomes (11). Signaling differences in response to BCR engagement have been documented between immature and mature B cells, and these differences may lead to cell death in the former but activation in the latter (12, 13). Naïve B cells that have yet to encounter antigens and tolerant B cells that are chronically exposed to self-antigens also differ in their BCR signaling events, in particular the activation of the mitogen-activated protein kinases (MAPKs) and the transcription factors NF-␬B and NF-AT (14). The MAPKs are serine/threonine protein kinases, and they couple receptor signaling to cellular responses such as proliferation, differentiation, and cell death (15). The three major classes of MAPKs are the extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and the p38 MAPK (16). ERK has been implicated in cell growth and proliferation (17), whereas JNK and p38 MAPK appear to be involved in stress response and apoptosis (18, 19). Cross-linking of the BCR activates all three classes of MAPKs in naïve B cells, but only ERK is activated in tolerant B cells (14), whereas the activation of p38 MAPK is not known. The transcription factor NF-␬B regulates genes involved in survival and prolif1 The abbreviations used are: B-CLL, B-cell chronic lymphocytic leukemia; Ab, antibody; BCR, B cell antigen receptor; ERK, extracellular signal-regulated kinase; IP3, inositol 1,4,5-triphosphate; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; NF-AT, nuclear factor of activated T cells; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PMA, phorbol 12-myristate 13-acetate; PLC, phospholipase C; FACS, fluorescence-activated cell sorting; Btk, Bruton’s tyrosine kinase. 30707 30708 BCR Signaling in B-1 and B-2 Cells eration (20), whereas NF-AT seems to regulate genes involved in cellular homeostasis and differentiation (21). BCR signaling induces the activation of both NF-␬B and NF-AT in naïve B cells, whereas the activation of NF-AT is constitutive but that of NF-␬B is blocked in tolerant B cells (14). Since differences in BCR signaling have been documented between immature and mature B cells and between naïve and tolerant B cells, it is assumed that B-1 and B-2 cells may also differ in their induction of the various signaling pathways. Indeed, it is known that the activation of the transcription factor STAT-3 is constitutive in B-1 cells but only induced in B-2 cells (22). Therefore, in this report, we systematically examine whether the various common signaling pathways that are induced by BCR engagement in B-2 cells, namely those of phospholipase C (PLC)-␥2, MAPKs, Akt, NF-␬B, and NF-AT, are also differentially activated in B-1 cells. The study of the signaling pathways activated by BCR engagement on B-1 cells may shed light to the origins of this subset of B lymphocytes. EXPERIMENTAL PROCEDURES Mice—The VH12f (23) and wild-type BALB/c mice were maintained in our animal facility and used to isolate B-1 and B-2 cells, respectively. All mice were used between and months of age and in accordance with institutional guidelines. Flow Cytometry—Peritoneal cavity and splenic B cells were stained with fluorochrome-conjugated antibodies (Abs) for 15 on ice. After washing in phosphate-buffered saline containing 3% fetal calf serum and 0.01% NaN3, the cells were analyzed on a FACScan (Becton Dickinson) using Cell Quest Software. The following Abs used in the FACS analyses were obtained from PharMingen (San Diego, CA): antiIgM (R6 – 60.2), anti-IgD, anti-B220 (RA3– 6B2), anti-CD5, anti-CD23, anti-CD25, anti-CD69, and anti-CD86 (B7.2). The anti-VH12 (5C5) Ab was obtained previously from Dr. G. Haughton (University of North Carolina, Chapel Hill, NC). Purification and Treatment of Cells—To obtain a pure population of B-1 cells, peritoneal cavity washout of VH12f mice was seeded onto a tissue culture dish for 2–3 h to remove adherent macrophages. B-2 cells were isolated from splenocytes of wild-type mice by MACS using negative selection with anti-CD43 microbeads (Miltenyl Biotech). The purity of B-1 and B-2 cells obtained is Ͼ85% as assessed by FACS analysis using anti-IgM and anti-B220 Abs. Purified cells were cultured in complete RPMI 1640 medium with serum except in the JNK and p38 MAPK experiments, where the serum supplement was omitted. For the NF-␬B experiment, the cells were cultured in OPTI-MEM௡ I reduced serum medium (Invitrogen). For the I␬B␣ assays, cells were treated with 50 ␮M cycloheximide (Sigma) before and during the various stimulations. B cells (1–5 ϫ 106) were left untreated or stimulated with 10 –50 ␮g/ml goat anti-mouse IgM F(abЈ)2 fragment (Jackson Immunoresearch), a combination of 0.1 ␮g/ml PMA and ␮g/ml ionomycin (Sigma), or ␮g/ml lipopolysaccharide (LPS) (Sigma) for various times at 37 °C prior to the conduct of the various assays. Treatment of cells with the MEK1 inhibitor U0126 (Cell Signaling Technology, Beverly, MA), PD98059, or the inactive analog SB202474 (Calbiochem) was performed at concentrations ranging from to 100 ␮M. Proliferation Assay—Purified B cells (5 ϫ 105) that were either nontreated or stimulated with various amounts of anti-IgM F(abЈ)2 antibody or LPS were cultured for 42 h in a 96-well flat-bottomed plate at 37 °C in the presence of 7% CO2. Cells were subsequently pulsed with ␮Ci of [3H]thymidine (Amersham Biosciences) and harvested h later with a Skatronas cell harvester (Skatronas Instruments Inc.). The incorporation of radioactivity was measured by a Wallac LKB 1219 Rackbeta liquid scintillation counter (PerkinElmer Life Sciences). Preparation of Nuclear Extracts—Cells were lysed on ice in hypotonic buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 ␮g/ml aprotinin, and 2.5 ␮g/ml leupeptin) for min. After the addition of 0.2% Nonidet P-40, the cell lysate was passed through a 26-gauge needle to ensure the complete lysis of cells and centrifuged at 13,000 rpm for at °C. The nuclear pellet was washed twice in the hypotonic buffer; resuspended in a high salt buffer that contains 20 mM Hepes, pH 7.9, 0.4 M NaCl, mM EDTA, 0.02% Nonidet P-40, mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 ␮g/ml aprotinin, and 2.5 ␮g/ml leupeptin; and incubated on a Spiramix roller mixer for 30 at °C. The nuclear fraction was subsequently cleared of insoluble material by centrifugation at 13,000 rpm for at °C before desalting and concentrating with a microcon-3 column (Millipore Corp.). The nuclear extracts were stored at Ϫ80 °C prior to use, and the protein content was measured using a Bio-Rad DC protein assay (Bio-Rad). Electrophoretic Mobility Shift Assays—For the NF-␬B gel shift assay, 10 ␮g of the nuclear extracts was incubated with a [␣-32P]dATP-labeled probe that contains the sequence 5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј and ␮g of poly(dI⅐dC) in buffer A (12 mM Hepes, pH 7.9, mM Tris-HCl, pH 7.9, 60 mM KCl, 30 mM NaCl, mM MgCl2, mM dithiothreitol, and 12.5% glycerol). For the NF-AT gel shift assay, 10 ␮g of nuclear extracts was incubated with ␮g of poly(dI⅐dC) and a labeled probe that contains the sequence 5Ј-CGCCCAAAGAAGAAAATTTGTTTCATA-3Ј in gel shift buffer B (21.5 mM Hepes, pH 7.9, 84 mM NaCl, mM EDTA, 1.2 mM dithiothreitol, 0.1% glycerol, and 300 ␮g/ml bovine serum albumin). The reaction mixture was incubated for 20 at room temperature prior to electrophoresis in a 5% nondenaturing PAGE. Immunoprecipitations and Western Blot Analyses—Cells (106 to 107) were lysed on ice for 15 in a buffer that contains 1% (v/v) Nonidet P-40, 10 mM Tris-HCl, pH 8, 150 mM NaCl, mM EDTA, 0.2 mM Na3VO4, mM phenylmethylsulfonyl fluoride, and 10 ␮g/ml aprotinin, and the debris was removed by centrifugation at 13,000 rpm for 12 at °C. For immunoprecipitations, cell lysates were sequentially incubated with 2–2.5 ␮g of appropriate antibodies and protein A/G PLUSagarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoprecipitates or lysates were electrophoresed in a 7–10% SDSPAGE, electroblotted onto polyvinylidene difluoride membrane, and probed with Abs that recognize specific proteins. Protein bands were visualized using horseradish peroxidase-coupled Abs and the enhanced chemiluminescence detection system (Amersham Biosciences). The following Abs were used: anti-phosphotyrosine (PY20); anti-PY20-agarose and anti-Bcl-xL (Transduction Laboratories, San Diego, CA); antiBruton’s tyrosine kinase (anti-Btk) (PharMingen); anti-phospho-ERK, anti-ERK2, anti-JNK1, anti-TFIID, anti-I␬B␣, anti-PLC-␥2, and antitubulin (Santa Cruz Biotechnology); anti-phospho-JNK, anti-phosphop38, anti-p38 MAPK, anti-Akt, and anti-phospho-AktS473 (Cell Signaling Technology, Beverly, MA); and anti-phospho-AktT308 (Upstate Biotechnology, Inc., Lake Placid, NY). In Vitro Kinase Assay for ERK—ERK was immunoprecipitated from lysates of nontreated or anti-IgM-stimulated cells using anti-ERK2 Ab and protein A/G PLUS-agarose beads. After washing in 1% Nonidet P-40 buffer, the immunoprecipitate was resuspended in a mixture that contains the myelin basic protein substrate as detailed in the MAPK assay kit (Upstate Biotechnology). D-myo-Inositol 1,4,5-Trisphosphate Assay for PLC␥ Activity—5 ϫ 10 purified B cells that were either nontreated or stimulated with anti-IgM were lysed, and the generation of D-myo-inositol 1,4,5-trisphosphate (IP3) was measured as previously described (24, 25), using the BIOTRAK TRK 1000 kit (Amersham Biosciences). Briefly, unlabeled IP3 generated by the cells was used to compete with a fixed, known amount of [3H]IP3 for binding to a limited number of IP3 receptors present in the bovine adrenal glands homogenate provided by the kit. The bound IP3 is separated from the free IP3 by centrifugation, which pellets the IP3-receptor complexes. The free IP3 in the supernatant was discarded by decantation. Measurement of the amount of radioactivity bound to the receptor enables one to estimate the amount of unlabelled IP3 in the sample as determined by interpolation from a standard curve. RESULTS VH12-expressing B-1 Cells Are Found in Normal Mice—B-1 cells represent a minor B cell subset and typically constitute 1–3% of the B cells found in the spleen, although they are enriched in the peritoneum of mice (26). As shown in Fig. 1A, comparison of the B cells found in the spleen and peritoneal cavity of normal mice revealed that peritoneal B-1 cells differ from the splenic conventional B (or B-2) cells with respect to their phenotypes. B-1 cells express a high level of IgM and low levels of B220 and IgD compared with B-2 cells. In addition, they are CD5ϩ and CD23Ϫ. Thus, B-1 and B-2 cells are distinct B cell subsets. Because of the paucity of B-1 cells in normal mice, there is a need to obtain an enriched source of these cells in order to study BCR signaling in B-1 cells. We have previously generated a strain of immunoglobulin knock-in mice, designated VH12f (23), that carry a VH12 Ig heavy chain transgene. The VH12 heavy chain is derived from an antibody that recognizes phosphatidylcholine (27), an anti- BCR Signaling in B-1 and B-2 Cells FIG. 1. Identification and purification of VH12-expressing B-1 cells. A, FACS analysis depicting the phenotypic differences between B-2 and B-1 cells found in the spleen and peritoneal cavity (PerC) of normal mice, respectively. The phenotype of transgenic VH12-expressing B-1 cells is also shown. Cells were stained with anti-IgM and anti-IgD or anti-B220 or anti-CD5 antibodies. The numbers indicate percentage of total B cells. B, FACS analysis showing the presence of VH12-expressing B cells in the peritoneal cavity of wild-type and VH12f mice. The numbers indicate percentage of total B cells present. C, FACS analysis showing the purity of the B-1 and B-2 cell populations isolated from the peritoneal cavity of VH12f and spleen of wild-type mice, respectively. The numbers indicate percentage of total B cells present. 30709 genic specificity that is enriched in B-1 cells. Not surprisingly, as shown in Fig. 1A, VH12f mice develop predominantly B-1 cells that are IgMhighB220lowCD5ϩIgDϪ (23). In addition, as shown in Fig. 1B, close to 90% of the B cells in the peritoneal cavity of VH12f mice express the knock-in VH12 heavy chain as identified by FACS staining with the idiotypic anti-VH12 Ab. As such, these mice would provide an ideal source of B-1 cells for biochemical analyses. To ensure the relevance of using the transgenic VH12-expressing B-1 cells, we show that VH12-expressing B cells are naturally occurring and formed a sizable fraction (close to 2%) of the normal B-1 cell repertoire in the peritoneal cavity of wild-type mice. Next, we showed that VH12-expressing B-1 cells can be isolated with great purity from the peritoneal cavity of VH12f mice (Fig. 1C) using a procedure that does not lead to the stimulation of the BCR. Similarly, conventional B-2 cells can also be purified from the spleens of wild-type mice for comparative study. Thus, the use of transgenic VH12-expressing B-1 cells from the peritoneal cavity of VH12f mice (23) would greatly facilitate the study of the biochemical properties of B-1 cells. Transgenic VH12-expressing B-1 Cells Do Not Proliferate in Response to BCR Engagement but Exhibit Extended Survival in Vitro—To further ensure that the transgenic VH12-expressing B-1 cells behave like normal B-1 cells found in wild-type mice, we examined their response to BCR stimulation. In all experiments described in this paper, BCR signaling is induced using anti-IgM F(abЈ)2 antibodies. It is known that B-1 cells, unlike B-2 cells, not proliferate when their BCRs are cross-linked (28). Indeed, transgenic VH12-expressing B-1 cells did not respond to anti-IgM stimulation regardless of the dosage given (Fig. 2A). This was in contrast to the dose-dependent proliferation of B-2 cells. However, as control, we showed that VH12expressing B-1 cells were not completely refractory to stimulation, since they did proliferate in response to LPS treatment, albeit to a much lesser extent compared with B-2 cells (Fig. 2B). Another property of B-1 cells is their extended survival in culture compared with B-2 cells (29). In contrast to B-2 cells that undergo apoptosis rapidly within a day in culture, VH12expressing B-1 cells can survive for extended period of time in vitro without dying, as shown in Fig. 2C. Taken together, the data suggest that the transgenic VH12-expressing B-1 cells behave like normal B-1 cells and further support the notion that they would serve as a suitable model system to study the properties of this unique subset of B cells. Henceforth, VH12expressing B-1 cells will be designated simply as B-1 cells. Lack of BCR-induced NF-␬B Activation in B-1 Cells—Given the physiological differences seen in B-1 and B-2 cells, in particular in their different proliferative response to BCR crosslinking, we proceeded to examine if BCR stimulation would induce different signaling events in these two B cell subsets. BCR signaling is known to activate the transcription factor NF-␬B that regulates genes involved in cell proliferation and survival (30). The predominant form of NF-␬B in B cells is the p50-c-Rel heterodimer (31), and in particular, c-Rel is shown to be essential for B cell proliferation after BCR engagement (31–33). As expected, treatment of B-2 cells with anti-IgM or a combination of PMA and ionomycin led to the activation of NF-␬B in these cells, as evidenced by the increased binding of nuclear NF-␬B proteins to an oligoprobe that contained the NF-␬B consensus binding site (Fig. 3A, right). In contrast, there was no significant induction of NF-␬B above the background level in BCR-stimulated B-1 cells, and as control, the treatment of B-1 cells with PMA and ionomycin did result in the activation of this transcription factor (Fig. 3A, left). To confirm that NF-␬B was indeed not activated in BCR- 30710 BCR Signaling in B-1 and B-2 Cells FIG. 2. B-1 cells not proliferate in response to anti-IgM stimulation but exhibit extended survival in culture. Purified B-1 and B-2 cells were stimulated for 48 h with increasing concentrations of goat anti-mouse IgM F(abЈ)2 fragment (A) or LPS (B). Cell proliferation was quantified by H3 incorporation. C, purified B-1 and B-2 cells were cultured for various numbers of days, and the number of remaining live cells was quantified by trypan blue exclusion. Results shown are representative of three independent experiments. FIG. 3. Lack of NF-␬B activation in B-1 cells due to the absence of induced degradation of I␬B proteins. A, nuclear extracts from nontreated (U), anti-IgM (Ig), or PMA/ionomycin (P/I)-stimulated B-1 and B-2 cells were examined for NF-␬B binding activity in an electrophoretic mobility shift assay. B, Western blot analysis of Bcl-xL expression in B-1 and B-2 cells that were nontreated (U) or stimulated with anti-IgM (Ig) or LPS for 48 h. The anti-tubulin blot serves as a control for the loading of whole cell lysates. C, Western blot analysis of I␬B␣ degradation in nontreated (U) or PMA/ionomycin (P/I)- or anti-IgM (Ig)-stimulated B-1 and B-2 cells. The anti-c-Rel blot was used as a control for the loading of whole cell lysates. D, Western blot analysis of c-Rel translocation into the nuclei of B-1 and B-2 cells that were either nontreated (U) or stimulated with anti-IgM (Ig) or PMA/ionomycin (P/I). The blot was first probed with anti-c-Rel Ab and subsequently reprobed with anti-TFIID Ab to control for the amount and integrity of the nuclear extracts used. stimulated B-1 cells, we examined the induction of one of its target genes, bcl-x (34, 35). As shown in Fig. 3B, the expression of Bcl-xL was up-regulated in B-2 cells in response to either anti-IgM or LPS treatment. However, treatment of B-1 cells BCR Signaling in B-1 and B-2 Cells with anti-IgM antibodies did not lead to the expression of Bcl-xL, consistent with a lack of NF-␬B activation in these cells. As control, B-1 cells did express Bcl-xL after LPS treatment. Taken together, the data indicate that NF-␬B is not activated in B-1 cells after BCR stimulation. To determine the reason for the lack of NF-␬B activation in B-1 cells, we next examined the molecular events leading to its activation. In nonstimulated cells, NF-␬B/Rel factors are sequestered in the cytoplasm by the inhibitory (I)-␬B family of proteins (36). Upon specific stimulation, the I␬B kinases are activated, and this leads to the serine/threonine phosphorylation and subsequent degradation of I␬B proteins and the release of NF-␬B/Rel proteins for translocation into the nucleus to effect gene transcription (37, 38). As shown in Fig. 3C, treatment of B-2 cells with anti-IgM antibodies or a combination of PMA and ionomycin led to the degradation of the I␬B proteins as indicated by the loss of I␬B␣ subunit in Western blot analyses of whole cell lysates. Concomitantly, there was an increase in c-Rel translocation into the nucleus of anti-IgM- or PMA/ionomycin-stimulated B-2 cells (Fig. 3D). In contrast, I␬B␣ proteins were not degraded in IgM-stimulated B-1 cells (Fig. 3C), and there was a lack of c-Rel translocation into the nucleus of these cells (Fig. 3D). Again, as control, I␬B␣ could be degraded, and nuclear translocation of c-Rel was effected in PMA/ionomycin treated B-1 cells. Thus, in B-1 cells, I␬B proteins specifically not degrade in response to BCR signaling, and this results in a lack of NF-␬B activation in these cells. Intact Btk but Reduced PLC-␥2 and Lack of Protein Kinase B/Akt Activation in IgM-stimulated B-1 Cells—Two signaling pathways have been linked to NF-␬B activation (39 – 41). In B cells, the Btk-PLC-␥2 pathway has been shown to be essential for NF-␬B activation in B cells (42). Hence, it is possible that the expression or activation of these signaling molecules may be altered in B-1 compared with B-2 cells. Thus, we first examined the activation of Btk in nontreated and anti-IgM-stimulated B-1 and B-2 cells. Western blot analysis of tyrosinephosphorylated Btk indicated that Btk was activated with the same kinetics in both anti-IgM-stimulated B-1 and B-2 cells (Fig. 4A). Next, we examined the activation of the downstream PLC-␥2 in nontreated and anti-IgM-stimulated B-1 and B-2 cells. As shown in Fig. 4B, PLC-␥2 was expressed equivalently in both B-1 and B-2 cells and could be activated by anti-IgM treatment. However, in B-1 cells, the PLC-␥2 activation appeared to tail off faster at the 30 s time point. Since the intact phosphorylation of PLC-␥2 in B-1 cells seems inconsistent with the lack of NF-␬B activation, we directly examined the enzymatic activity of PLC-␥2. PLC-␥2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) into diacylglycerol and IP3. Hence, its activity can be assayed by measuring the production of IP3. As shown in Fig. 4C, the generation of IP3 was observed to be reduced in anti-IgM-stimulated B-1 cells as compared with that of B-2 cells. Hence, the reduced activity of PLC-␥2 in B-1 cells may impact upon NF-␬B activation after BCR stimulation. Another signaling pathway that is activated by the crosslinking of the BCR on B-2 cells is that of the serine/threonine kinase, Akt, or protein kinase B (43, 44). Akt is an important signaling molecule that has also been implicated in the activation of NF-␬B (39, 45– 47) and in cell survival (48, 49) in many different biological systems. We therefore also examined the activation status of Akt in B-1 cells. The activity of Akt is dependent on phosphorylation, and Akt can be phosphorylated on two potential sites: Thr308 and Ser473. Western blot analyses using specific antibodies that recognized either of the phosphorylated residues of Akt indicated that both sites were phosphorylated in BCR-stimulated 30711 FIG. 4. Intact Btk, reduced PLC-␥2, and lack of Akt activation in anti-IgM-stimulated B-1 cells. A, normal activation of Btk in B-1 and B-2 cells. Whole cell lysates from B-1 and B-2 cells that were treated with 20 ␮g/ml anti-IgM Abs for various times were immunoprecipitated with anti-phosphotyrosine (PY20)-agarose and probed with anti-Btk Ab. The numbers below the blot indicate the -fold difference in phosphorylation with respect to the unstimulated sample. B, reduced phosphorylation of PLC-␥2 in B-1 cells. Cell lysates from B-1 and B-2 cells treated as in A were immunoprecipitated with anti-PLC-␥2, probed with anti-PY20, and later reprobed with the immunoprecipitating Ab for loading control. Numbers below the blot indicate the -fold difference in phosphorylation with respect to the unstimulated sample. C, kinetics of IP3 generation from nontreated and anti-IgM stimulated B-1 and B-2 cells. Results shown are representative of two separate experiments. D, absence of Akt activation in B-1 cells. Whole cell lysates from B-1 and B-2 cells treated as in A were immunoprecipitated with anti-Akt and probed with anti-pAktS473 (upper panel) or anti-pAktT308 (lower panel) Abs. The blots were reprobed with the immunoprecipitating Ab to check for equal loading of cell lysates. 30712 BCR Signaling in B-1 and B-2 Cells B-2 cells (Fig. 4D). In contrast, neither Thr308 nor Ser473 was phosphorylated in B-1 cells, regardless of the duration of BCR stimulation. Thus, Akt is not activated in IgM-stimulated B-1 cells. This, together with the reduced PLC␥2 activity, could explain the lack of NF-␬B activation in B-1 cells upon BCR engagement. Differential Induction of Mitogen-activated Protein Kinases in B-1 and B-2 Cells—BCR engagement is also known to activate the MAPK signaling pathways that have been shown to regulate cell growth, differentiation, and death in various biological systems (15). The three major classes of MAPKs are the ERK, JNK, and p38 MAPK. B cells at different stages of differentiation may activate different MAPKs when triggered via their BCRs (50); for example, tolerant B cells have constitutive ERK but failed to induce JNK activation (14). To determine the pattern of MAPK activation in B-1 cells, we stimulated these cells with anti-IgM antibodies for various times, and the activation of the different classes of MAPKs was examined using phosphorylation state-specific antibodies. Anti-IgM-treated B-2 cells were used as controls, since all classes of MAPKs could be activated in these cells following stimulation. As shown in Fig. 5A, Western blot analysis using anti-phospho-ERK antibody indicated that ex vivo B-1 cells had a basal level of constitutive ERK activation compared with B-2 cells. This basal level of ERK activation in B-1 cells could be further up-regulated as indicated by the increased amount of phosphorylated ERK that was detected after anti-IgM stimulation (Fig. 5B). An in vitro kinase assay using myelin basic protein as a substrate indicates that this basal level of phospho-ERK in B-1 cells was indeed active (Fig. 5C). In contrast, although ERK was activated in B-2 cells within and sustained for at least 30 after anti-IgM stimulation, there was clearly a lack of basal ERK activity in these cells as shown by a lack of anti-phospho-ERK antibody staining or ERK activity in phosphorylating the myelin basic protein substrate (Fig. 5, A and C). Furthermore, BCR stimulation seemed to induce a greater level of phosphorylation and hence activation of ERK in B-1 cells compared with B-2 cells (Fig. 5B). Examination of JNK activation also revealed a difference in the induction of this MAPK in B-1 and B-2 cells. Whereas JNK activation occurred within and was sustained for at least 10 after anti-IgM treatment of B-2 cells, the kinetics of JNK activation was very much delayed to the 10-min time point in B-1 cells (Fig. 5D). Finally, analysis of p38 MAPK activation using anti-phospho-p38 MAPK antibody indicated that there was hardly any induction of this kinase above the basal level in anti-IgMstimulated B-1 cells (Fig. 5E) compared with similarly treated B-2 cells, where the activation of p38 MAPK was noticeable within and maintained for at least 10 after BCR stimulation. Taken together, the above data indicate that B-1 cells have constitutive basal ERK activity and delayed JNK and lack p38 MAPK activation compared with B-2 cells, where all of these MAPKs are inducibly activated after BCR engagement. Extended Survival of B-1 Cells in Culture Is Not Due to Constitutive ERK Activation—The basal level of constitutive ERK activation in B-1 cells is of potential interest. Ex vivo B-1 cells possessed a considerable amount of the phosphorylated form of ERK compared with ex vivo B-2 cells (Fig. 5A). Constitutive ERK activation has been implicated in the maintenance of cell survival in many biological systems (51). One of the distinguishing features of B-1 cells as shown in Fig. 2C is their extended survival in culture compared with B-2 cells, which undergo cell death rapidly ex vivo in the absence of stimulation FIG. 5. Differential activation of ERK, JNK, and p38 MAPK in B-1 and B-2 cells. A, ex vivo B-1 but not B-2 cells have constitutive ERK activation. Whole cell lysates of ex vivo B-1 and B-2 cells were probed with anti-phospho-ERK (p-ERK) and anti-ERK Abs in Western blot analysis. B, ERK activation can be further up-regulated by antiIgM stimulation of B-1 cells. Cells were treated with 10 ␮g/ml of anti-IgM Abs for various times, and Western blot analysis was performed as in A. C, in vitro kinase assay for ERK activity. ERK2 was immunoprecipitated from the lysates of nontreated and anti-IgM-stimulated B-1 and B-2 cells and used to phosphorylate myelin basic protein substrate. D, JNK activation is delayed in B-1 cells. B-1 and B-2 cells were treated with 10 ␮g/ml anti-IgM Abs for various times, and whole cell lysates were probed with anti-pJNK and anti-JNK Abs in Western blot analysis. E, lack of p38 activation in B-1 cells. B-1 and B-2 cells were treated with 10 ␮g/ml anti-IgM Abs for various times, and whole cell lysates were probed with anti-phospho-p38 MAPK and anti-p38 MAPK Abs in Western blot analysis. (52). Interestingly, the phosphorylated and hence activated form of ERK could be detected not only in ex vivo B-1 cells but also in B-1 cells that were in culture for up to 10 days without any BCR stimulation (Fig. 6A). We thus determined whether BCR Signaling in B-1 and B-2 Cells 30713 FIG. 7. Constitutive activation of NF-AT in B-1 cells. Nuclear extracts from nontreated (U) or anti-IgM (Ig)- or PMA/ionomycin (P/ I)-stimulated B-1 and B-2 cells were examined for NF-AT activity in an electrophoretic mobility shift assay. FIG. 6. Constitutive ERK activation is not responsible for the extended survival of B-1 cells in culture. A, detection of activated ERK in nonstimulated B-1 cells that were in culture for various numbers of days. B, titration of PD98059 and U0126 for the inhibition of ERK activation. B-1 cells were cultured overnight with different concentrations (shown in ␮M) of U0126 or PD98059 or the inactive analog SB202474 (50 ␮M) and assayed for the presence of phospho-ERK in Western blot analysis. C, viability of B-1 cells after 24 h of culture either in the absence (U) or the continuous presence of a 50 ␮M concentration of the inhibitor PD98059 (PD) or 10 ␮M of U0126. the constitutive activation of ERK plays a role in B-1 cell survival in culture by incubating B-1 cells in the continuous presence of the inhibitor PD98059 or U0126 that acts on the upstream kinase MEK1, which phosphorylates ERK. The ad- dition of the compound PD98059 but not its inactive analog SB202474 at a concentration of 50 ␮M was sufficient to completely abrogate the phosphorylation of ERK and yet remained nontoxic to the cells (Fig. 6B, upper panel). Similarly, the addition of ␮M U0126 was sufficient to inhibit ERK activation (Fig. 6B, lower panel). However, the number of viable B-1 cells remained unchanged after 24 h (Fig. 6C) or 48 h (data not shown) of culture in the presence of either of the inhibitors. Thus, the constitutive activation of ERK appears not to be responsible for the extended survival of B-1 cells in culture. Constitutive Activation of NF-AT in B-1 Cells—The lack of NF-␬B activation and the differential induction of MAPKs in B-1 cells are reminiscent of that of tolerant B cells that are chronically exposed to self-antigens (14). This raises the interesting possibility that B-1 cells may have signaling properties similar to those found in tolerant B cells. Another signaling pathway that is differentially induced in tolerant and naïve B-2 cells is that of NF-AT, which is constitutively active in the former but inducible in the latter (14). In addition, it was reported that human B-CLL cells, which are CD5ϩ, have constitutive NF-AT activation (53). Since B-1 cells frequently give rise to B-CLL (54, 55) and have BCR specificities directed toward self-antigens, we examined the pattern of NF-AT activation in B-1 cells. Indeed, as shown in Fig. 7, ex vivo B-1 cells had significant levels of constitutive NF-AT activation, as evidenced by the enhanced binding of an oligoprobe that contained the NF-AT consensus site. Furthermore, the level of NF-AT activation in B-1 cells could be further up-regulated by antiIgM or PMA/ionomycin treatment. In comparison, significant levels of NF-AT activation were only observed in B-2 cells after anti-IgM or PMA/ionomycin stimulation. Thus, normal B-1 cells, like tolerant B cells (14) and B-CLL cells (53), exhibit constitutive NF-AT activation. B-1 Cells Do Not Fully Up-regulate Their Activation Markers upon BCR Stimulation—Thus far, the biochemical analysis of BCR signaling suggests that B-1 cells resemble tolerant B cells in the induction of MAPKs, NF-AT, and NF-␬B and raises the interesting possibility that B-1 cells may be anergic B cells. Indeed, unlike B-2 cells, B-1 cells also resemble anergic B cells in that they both not enter the cell cycle upon BCR engagement (8, 9, 11, 14). To determine whether other parameters of activation are also altered in B-1 cells, we examined the upregulation of activation markers on these cells after BCR stimulation. As shown in Fig. 8, anti-IgM or LPS stimulation of B-2 cells and LPS stimulation of B-1 cells led to high level expression of CD25 (IL-2R␣), the early activation marker CD69, and the co-stimulatory molecule CD86 (B7.2) on these cells. In contrast, anti-IgM stimulation of B-1 cells only led to a partial increase in cell surface expression of these activation molecules that was significantly lower than the levels induced by LPS stimulation of B-1 cells or by anti-IgM and LPS stimulation of B-2 cells. Hence, B-1 cells not fully up-regulate their activation markers upon BCR engagement. 30714 BCR Signaling in B-1 and B-2 Cells TABLE I Differences in BCR-Induced signaling pathways in B-1 and B-2 cells a Signaling pathways Naïve B-2 B-1 Tolerant Ba NF-␬B NF-AT MAPKs ERK JNK p38 MAPK Akt Induced Induced Not activated Constitutive Not activated Constitutive Induced Induced Induced Induced Constitutive Delayed Not activated Not activated Constitutive Not activated NDb ND Data on signaling events in tolerant B cells were obtained from Ref. 14. b FIG. 8. Anti-IgM stimulation does not fully activate B-1 cells. B-1 and B-2 cells were left untreated (U) or stimulated overnight with 10 ␮g/ml anti-IgM Ab (Ig) or ␮g/ml LPS and examined for the upregulation of CD25 (IL-2R␣), CD69 and CD86 (B7.2) in FACS analyses. DISCUSSION Studies presented here indicate that B-1 and B-2 cells have differential induction of multiple signaling pathways. Specifically, B-1 cells have constitutive ERK and NF-AT signaling, reduced PLC-␥2 activation, and delayed JNK activation, and they lack p38 MAPK, Akt and NF-␬B induction upon BCR engagement. In contrast, all of these signaling pathways are activated by BCR cross-linking in B-2 cells. The lack of NF-␬B activity in the nucleus of BCR-stimulated B-1 cells had previously been documented (56). However, in this report, we explore further the reason for the lack of NF-␬B induction in BCR-stimulated B-1 cells and show that this is due to a lack of induced degradation of the I␬B proteins in the cytoplasm. Two major signaling pathways are known to lead to NF-␬B activation, namely that of the PLC-␥2 and the Akt pathways. Whereas the activation of the nonreceptor tyrosine kinase Btk is normal in both BCR-stimulated B-1 and B-2 cells, the activity of PLC-␥2 as assessed by PI(4,5)P2 hydrolysis was observed to be reduced in B-1 cells in response to the cross-linking of the BCR. It is not clear at present if the lower amount of IP3 generated in B-1 cells is due to a reduction in PLC-␥2 activation, its localization, or a shortage of its substrate PI(4,5)P2. The reduction in PLC-␥2 activity and the impaired Akt activation in B-1 cells may explain the lack of NF-␬B induction in these cells. In turn, the lack of NF-␬B activity in BCR-stimulated B-1 cells may be the reason why these cells not proliferate upon BCR stimulation, since NF-␬B is known to induce the expression of cyclin D1 and Bcl-xL, both of which are required for cells to enter the cell cycle (57–59). Another interesting feature of B-1 cells is their ability to survive for extended periods of time in culture in contrast to normal primary B cells, which undergo rapid cell death ex vivo in the absence of stimuli. The extended survival of B-1 cells is not due to NF-␬B or Akt signaling, since these two pathways, ND, not determined. which have been implicated in cellular survival, are neither constitutively activated nor induced by BCR engagement in these cells. The MAPKs regulate cell growth, proliferation, differentiation, and cell death in various biological systems (15–19), and it is currently not known what aspects of B-1 cell physiology are regulated by these kinases. Our data indicate that the enhanced survival of ex vivo B-1 cells is also not due to the constitutive activation of ERK, since inhibiting the activation of this kinase did not lead to more pronounced cell death. Since both JNK and p38 MAPK have been implicated in apoptosis (18, 19), future experiments involving the enforced expression of these kinases are required to examine whether the failure to induce or sustain p38 MAPK and JNK activation in B-1 cell may explain their extended survival in vitro (52). One interesting finding in this report is the constitutive activation of the transcription factor NF-AT in B-1 cells. NFAT activation is regulated by Ca2ϩ flux, which in turn can be regulated by PLC-␥2 activation through its hydrolysis of PI(4,5)P2 into IP3 and diacylglycerol. However, we are at present unable to correlate the constitute activation of NFAT in B-1 cells with the inducible manner of PLC-␥2 activation. Perhaps some intermediate product downstream of PLC-␥2 is altered in B-1 cells that leads to this phenomenon. It was reported previously that human B-CLL cells, which were often CD5ϩ, had constitutive activation of NF-AT (53). Recently, the enhancer region of the CD5 gene was found to contain multiple NF-AT binding sites (60). Taken together, the data temptingly suggest that one of the characteristic features of the B-1 cell phenotype, namely the cell surface expression of CD5, may be due to the constitutive activation of this transcription factor. Indeed, consistent with this view, treatment of VH12 transgenic mice that develop predominantly B-1 cells with cyclosporin A, an immunosuppressive drug that interferes with calcium signaling and hence NF-AT activation, prevented the generation of CD5ϩ B cells (61). The origins of B-1 cells have been controversial and debated for years (2, 3). The pattern of MAPK, NF-AT, and NF-␬B signaling in B-1 cells described in this study closely resembles that of tolerant B cells that are chronically exposed to selfantigens (Table I). B-1 cells also behave like tolerant B cells in that they not proliferate upon BCR engagement (8, 9). In addition, we show here that BCR-stimulated B-1 cells are not fully activated in terms of the expression of specific activation markers. These findings and the fact that B-1 cells often have autoreactive specificities temptingly suggest that B-1 cells may be a special class of anergic or tolerant B cells. They differ from the classical anergic B cells only in that B-1 cells persist in vivo and can survive for extended periods of time ex vivo. In support of the argument that B-1 cells may be a special class of anergic B cells, we noted that B-1 cells are part of a normal B cell repertoire and often have specificities directed toward self-antigens such as DNA (5), ribonucleoprotein (6), and Thy-1 (7). The acquisition of CD5 expression on B-1 cells BCR Signaling in B-1 and B-2 Cells may be a consequence of the continuous recognition of selfantigen by their BCR, which constitutively activates the transcription factor NF-AT that may regulate CD5 gene expression (60). Consistent with this observation, anti-Thy-1 B cells are CD5ϩ only in the presence of Thy-1 (7). In the absence of Thy-1, anti-Thy-1 B cells resemble normal B-2 cells. Furthermore, transgenic B cells chronically exposed to the specific soluble antigen hen egg lysozyme express low levels of cell surface CD5 and become unresponsive to BCR stimulation. However, upon the removal of CD5, these cells become hyperresponsive (62). The expression of CD5, an inhibitory molecule that dampens BCR signaling (63), may allow for the persistence of B-1 cells with polyreactive specificities and, at the same time, prevent a full-blown autoimmunity. Finally, the hypothesis that B-1 cells are anergic B cells would argue against the lineage origin of B-1 cells and support the activation-induced model for their generation. Indeed, altering the strength of BCR signaling could alter the phenotype of B-1 cells. VH12-expressing B-1 cells that are normally CD5ϩ become B-2-like in their phenotype in the absence of Btk (64) and BLNK (65), which are the signaling molecules directly in the BCR signal transduction pathway. 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Immunol. 12, 397– 404 [...]... the B cell receptor As the BCR has no intrinsic protein tyrosine kinases (PTKs) activity, it utilizes several distinct families of PTKs and protein tyrosine phosphatases (PTPases) Signaling events upon engagement of the BCR involves the activation of an array of intracellular PTKs and PTPases (Figure 1 -3) 17 CHAPTER 1 INTRODUCTION 1. 4.2 .1 Protein tyrosine kinases There are three distinct families of. .. 14 1 Figure 5.5 Subcellular localization of DIP in resting and activated cells 14 4 Figure 5.6 Interaction of DIP with specific domains of Dok- 3 14 8 Figure 5.7 Interaction of DIP with Dok- 1 15 2 Figure 5.8 DIP causes apoptosis of mammalian cells via a caspase 3dependent mechanism 15 7 Figure 5.9 Inhibition of DIP- mediated apoptosis by overexpression of Dok- 1 and Dok- 3 16 0... associated with the BCR to form a heterodimer 16 CHAPTER 1 INTRODUCTION regions, with the variable regions of IgH and IgL chains forming the antigen-binding site As the cytoplasmic region of the IgH chain of mIg is short and in the case of IgM, consisting of only three amino acids (lysine, valine, and lysine), it is unlikely that the IgH chain is capable of mediating signaling from BCR It has been...LIST OF FIGURES Figure 4.6 Inhibiting FcγRIIB signaling prevents Dok- 3 localization to lipid rafts 12 7 Figure 5 .1 A schematic view of Dok- 3 protein domains 13 3 Figure 5.2 Sequence analysis of DIP 13 7 Figure 5 .3 Interaction of DIP and Dok- 3 through overexpression studies 13 8 Figure 5.4 Expression of DIP in different tissues and at various stages of B and T cell development... FcγRIIB inhibitory pathway To further examine the role of Dok- 3 in cellular signaling, we have screened for interacting partners of Dok- 3 and identified a novel protein which we termed Dok- 3interatcing protein (DIP) DIP was found to be ubiquitously expressed and possesses an ICE-like protease (caspase) p20 domain Over-expression of DIP in SH-SY5Y XIV SUMMARY cells induces in apoptosis in a caspase 3- dependent... treatment of caspase inhibitors z-VAD and DEVD DIP- mediated cell death can also be partially overcome by the expression of the anti-apoptotic protein Bcl-xL Interestingly, DIP is also able to interact with Dok- 3 homologue, Dok- 1 and both Dok- 3 and Dok- 1 are able to inhibit DIP- mediated cell death in a dose-dependent manner The discovery of DIP therefore links Dok- 3 to possible roles in B cell elimination... abrogated of their normal B cell function and these fail to proliferate upon mitogen stimulation (Pike et al., 19 82) The BCRs on anergic B cells remain capable of 10 CHAPTER 1 INTRODUCTION binding antigens, but BCR signaling is down regulated implying the importance of BCR signaling in the induction of B cell anergy (Cyster and Goodnow, 19 95; Cyster et al., 19 96; Healy et al., 19 97; Inaoki et al., 19 97;... downstream role for Btk and Syk kinases in a signaling pathway which is initiated by the Src kinases 1. 4.2 .1. 1 Activation of src protein tyrosine kinases Upon BCR ligation, Src PTKs are the first to become phosphorylated The activated Src PTKs subsequently phosphorylate the tyrosine residues within the ITAMs of Igα and Igβ However, the mechanism underlying the activation of Src PTKs is unclear In vitro,... signaling pathways (Satterthwaite and Witte, 19 96) The combinational acitivty of these various PTKs determine the quality and quantity of BCR signaling Time course studies implicate temporal activation of these proteins Src family kinases are activated first (5 -10 seconds) This is followed by activation of Btk (2-5 minutes) and then Syk family of kinases (10 -60 minutes) (Saouaf et al., 19 94) This indicates... that Dok- 3 may act in an inhibitory manner through the FcγRIIB signaling pathway Dok- 3 was found to localize to lipid rafts only after treatment of whole intact Ig (which triggers the BCR together with FcγRIIB, but not with F(ab’)2 fragment Ig (which triggers the BCR alone) SHIP and FcγRIIB were also found in the lipid rafts with Dok- 3 upon whole Ig treatment, suggesting that Dok- 3 may play a role in the . CHARACTERIZATION OF A DOK- 3- INTERACTING PROTEIN, DIP 13 0 5 .1 INTRODUCTION 13 1 5.2 CLONING OF FULL LENGTH DOK- 3 CDNA AND IDENTIFICATION OF A DOK- 3- INTERACTING PROTEIN, DIP 13 2 5 .3 INTERACTION OF DOK- 3. Adaptors involved in positive regulation of BCR signaling 39 1. 6.2 .1. 1 Bam32 39 1. 6.2 .1. 2 BCAP 40 1. 6.2 .1 .3 BLNK 41 1. 6.2.2 Adaptors involved in negative regulation of BCR signaling 43 1. 6.2.2 .1. RAFTS IN BCR SIGNALING 34 1. 6 REGULATION OF BCR SIGNALING BY ADAPTOR PROTEINS 37 1. 6 .1 Domains and motifs found in adaptor proteins 37 1. 6.2 Adaptor proteins in BCR signaling 38 1. 6.2 .1 Adaptors