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- Publisher
- Pubmed Central
- Copyright
- Copyright © 2003, The Rockefeller University Press
- ISSN
- 0022-1007
- eISSN
- 1540-9538
- DOI
- 10.1084/jem.20021686
- Publisher site
- See Article on Publisher Site
Abstract
Members of the Cbl family of molecular adaptors play key roles in regulating tyrosine kinase- dependent signaling in a variety of cellular systems. Here we provide evidence that in B cells Cbl-b functions as a negative regulator of B cell antigen receptor (BCR) signaling during the normal course of a response. In B cells from Cbl-b–deficient mice cross-linking the BCRs re- sulted in sustained phosphorylation of Ig, Syk, and phospholipase C (PLC)-2, leading to prolonged Ca mobilization, and increases in extracellular signal–regulated kinase (ERK) and c-Jun NH -terminal protein kinase (JNK) phosphorylation and surface expression of the activa- tion marker, CD69. Image analysis following BCR cross-linking showed sustained polarization of the BCRs into large signaling-active caps associated with phosphorylated Syk in Cbl-b–defi- cient B cells in contrast to the BCRs in Cbl-b–expressing B cells that rapidly proceeded to form small, condensed, signaling inactive caps. Significantly, prolonged phosphorylation of Syk correlated with reduced ubiquitination of Syk indicating that Cbl-b negatively regulates BCR signaling by targeting Syk for ubiquitination. Key words: B cells • antigen receptor • ubiquitination • tyrosine kinase • capping Introduction B lymphocyte responses are initiated by the binding of 9), and chicken DT40 B cells lacking Syk are unresponsive multivalent antigens to the B cell antigen receptors to BCR stimulation (10). Given the central role of Syk in (BCRs),* an event that triggers signaling cascades resulting B cell activation, regulation of Syk is likely to be essential in the transcription of a variety of genes associated with B to set the appropriate signaling thresholds for both the initi- cell activation (1). The BCR is composed of antigen bind- ation and dampening of immune responses. However, the two known negative regulators of BCR signaling, namely ing chains, the Ig molecules, and a noncovalently associated signal-transduction complex, Ig/Ig, containing in its SHIP and SHP-1, do not appear to target Syk. SHIP is a lipid phosphatase that influences the levels of PIP-3 (11– cytoplasmic domain immunoreceptor tyrosine-based acti- vation motifs (ITAMs; reference 2). Cross-linking the 13) and SHP-1, a protein phosphatase, regulates BCR sig- naling by dephosphorylating Lyn (14). BCR results in the phosphorylation of the ITAMs by the Src family kinase, Lyn followed by recruitment and activa- Recent studies have provided evidence that two mem- bers of the Cbl family of molecular adaptors, c-Cbl and tion of the nonreceptor protein tyrosine kinase, Syk (3–5). Recruitment of Syk by the phosphorylated BCR is a key Cbl-b, function to regulate signaling in immune cells downstream of the immune receptors (15, 16). Members of event in the assembly of the BCR signalosome composed of the adaptor protein BLNK and key downstream signal- the Cbl family share several highly conserved features in- ing components including phospholipase C (PLC)-2, cluding: a tyrosine kinase binding (TKB) domain composed Vav, and Bruton’s tyrosine kinase (Btk) (6, 7). Syk defi- of a four-helix bundle, a Ca -binding EF hand motif and ciencies in mice result in aberrant B cell development (8, an unusual SH2 domain; a RING finger domain that in- teracts with ubiquitin conjugating enzymes allowing Cbl Address correspondence to Susan K. Pierce, NIAID/NIH/Twinbrook II, proteins to function as ubiquitin ligases; a proline rich region 12441 Parklawn Dr., Rm. 200B, MSC 8180, Rockville, MD 20852. involved in SH3-domain interactions; multiple tyrosine res- Phone: 301-496-9589; Fax: 301-402-0259; E-mail: [email protected] idues that when phosphorylated allow interactions with *Abbreviations used in this paper: BCR, B cell antigen receptor; Btk, SH2 domains and luicine zippers (16). Current evidence Bruton’s tyrosine kinase; RT, room temperature; TKB, tyrosine kinase binding; Ub, ubiquitin. indicates that Cbl proteins regulate the activity of protein 1511 The Journal of Experimental Medicine • Volume 197, Number 11, June 2, 2003 1511–1524 http://www.jem.org/cgi/doi/10.1084/jem.20021686 The Journal of Experimental Medicine tyrosine kinases in part through ubiquitination and degra- Technology; and the phosphotyrosine specific PY20 mAb from Transduction Lab. AlexaFluor 488-conjugated to streptavidin, dation of their targets (17–19). Evidence for the role of phalloidin and goat Abs specific for rabbit IgG were purchased c-Cbl and Cbl-b in lymphocyte cell signaling was provided from Molecular Probe. The WASP-GBD-GFP fusion protein by the phenotypes of c-Cbl– and Cbl-b–deficient mice, the used for the measurement of active CDC42 (25) was a kind gift most dramatic of which were in T cell compartments (20– of Dr. Michael Rosen (University of Texas Southwestern Medi- 23). These phenotypes provided evidence that c-Cbl and cal Center, Dallas, TX). Rabbit Abs specific for Ig was pre- Cbl-b have distinct functions in T cells. Loss of c-Cbl re- pared as described (26). sulted in severe defects in developing thymocytes but only B Cell Activation Assay. B cells were purified from red cell mild defects in mature peripheral T cells. Thymocytes from depleted spleen cells by complement-mediated cytotoxicity after c-Cbl mice showed enhanced activation of ZAP-70 af- incubation with a Thy-1–specific mAb. The resulting cells were ter TCR engagement that was uncoupled from the need 90% B220 . The cells were incubated with a rat mAb specific for mouse IgM (1 g/10 cells) on ice for 30 min, washed and in- for CD4 costimulation and Lck activation. Consequently, cubated with F(ab) goat Abs specific for rat IgG Fc at 37 C. positive selection of CD4 thymocytes was enhanced in 2 / Alternatively, cells were incubated with F(ab) goat Abs specific c-Cbl mice (20, 21). In contrast, thymocyte development for mouse IgM at 37 C for the indicated times. was normal in Cbl-b–deficient mice but the peripheral T Immunoprecipitation, Immunoblotting, and Lyn Kinase Assays. cells were hyperreactive. Activation of mature peripheral T Immunoprecipitation and immunoblotting of cell lysates were as cells from Cbl-b mice was independent of the engage- described previously (27). For immunoprecipitation of Syk, Ig, ment of the coreceptor CD28 and consequently Cbl-b 7 BLNK, phospho-Btk, Vav1, and PLC-2, 5 10 cells per time mice were highly susceptible to autoimmunity (22, 23). point were lysed in the Tris or MES-buffered saline containing The effect of Cbl deficiencies in B cells has not been as rig- 1% NP-40, sodium orthovanadate, and protease inhibitors. For orously explored. B cells in c-Cbl mice appeared in immunoprecipitation of Cbl-b and Lyn, 2 10 cells were lysed in the RIPA buffer containing 1% NP-40, 0.5% sodium deoxy- normal numbers although in some mice an increase in the cholate, 0.1% SDS, sodium orthovanadate, and protease inhibi- number of immature IgM IgD cells was noted (20), hint- / tors. Lyn kinase activity was measured in Lyn immunoprecipitates ing at a developmental defect. B cells from Cbl-b mice by the transfer P into a peptide substrate using the SignaTECT showed enhanced proliferative responses to BCR cross- protein tyrosine kinase assay system according to the manufac- linking and to CD40 stimulation analogous to the T cell turer’s protocol (Promega). Briefly, the Lyn immunoprecipitates phenotype in these mice (23). Recent evidence indicates were incubated with kinase buffer including biotin-conjugated that Cbl-b plays a positive role in signaling in immature Src kinase–specific peptide substrates and P -ATP for 20 min at DT40 B cells (24). Here we provide evidence that in ma- C. The reaction was stopped and the reaction mixtures dotted ture splenic B cells Cbl-b is a negative regulator of BCR- on to membranes to which streptavidin was coupled. The mem- mediated B cell activation in part through its ubiquitination branes were washed and counted using a -scintillation counter. of Syk. For immunoblotting of unfractionated lysates, cells were lysed in the RIPA buffer and 20 g of protein per time point was ana- lyzed. The immunoblot bands were quantified by densitometry Materials and Methods and expressed as a ratio of phosphotyrosine containing protein to Mice. Cbl-b mice on a mixed genetic background be- total protein. / 2 tween 129 and C57BL/6 were as described (22). Cbl-b mice Measurement of Intracellular Ca Mobilization. For detection 2 7 on a mixed genetic background between 129 and C57BL/6 were of cytosolic Ca , 10 splenocytes were incubated with 0.75 M obtained from Taconic. For some analyses, Cbl-b and Cbl- Indo-1 acetoxy-methyl ester (Indo-1; Molecular Probe) in 1% b littermates on the same mixed genetic background were HBSS-FBS containing 0.01% F-127 (Molecular Probe) for 45 used. min at 30 C. Cells were subsequently stained with PE-conjugated Reagents and Abs. The mouse IgM-specific rat IgG2a mAb B220 specific mAb to detect B220 cells in flow cytometry. The (R6–60.2) used for BCR cross-linking was purchased from BD primary IgM-specific Ab was added followed by the secondary Biosciences. F(ab) goat Abs specific for mouse Ig or for Ab and cytosolic Ca fluxes were recorded in real time using a IgM G, biotin-conjugated F(ab) goat Abs specific for rat Ig, FACS vantage (Becton Dickinson) and analyzed by the FlowJo HRP-conjugated goat Abs specific for mouse Ig, Rhodamine software program (True Star, Inc.). Red X (RRX)-conjugated Fab goat Abs specific for mouse Ig, Determination of F-actin Polymerization and CDC42 Activation. PE-conjugated F(ab) donkey Abs specific for rabbit IgG, F-actin was detected by phalloidin staining quantified by flow FITC-conjugated F(ab) goat Abs specific for mouse IgG, and cytometry. GTP-bound CDC42 was detected using WASP- Texas-Red–conjugated streptavidin were purchased from Jack- GBD-GFP fusion protein as described previously (25). Cells son ImmunoResearch Laboratories. Latrunculin B, Piceatannol were fixed, permeabilized with 0.05% saponin and stained with and PP2 were purchased from Cal Biochem Co. Cytochalasin D rabbit Abs specific for GFP followed by FITC-labeled goat Abs was purchased from Sigma-Aldrich. The following Abs were specific for rabbit Ig and analyzed by flow cytometry (FAC- purchased: rabbit Abs specific for Syk, Cbl-b, Vav1, BLNK, Scan™; Becton Dickinson). Lyn, PLC-2, ubiquitin, phospho-ERK, ERK or JNK, goat Abs Detection of Phospho-Btk. The level of intracellular phospho- specific for Btk, and a mouse mAb specific for BLNK (2B11) Btk was determined by flow cytometry using phospho-Btk–spe- from Santa Cruz Biotechnology, Inc.; rabbit Abs specific for cific antibodies. Splenic B cells (10 cells/ml) were incubated in phospho-JNK from Biosource International; rabbit Abs specific HBSS for 1 h at 37 C and stimulated by adding 20 g/ml F(ab) for phospho-Syk (tyrosine 519/520), phospho-Btk (tyrosine goat Abs specific for mouse Ig for the indicated time periods. 223), phospho-Akt (serine 473), and Akt from Cell Signaling After washing twice with PBS containing 0.2% sodium azide, 1512 Cbl-b Negatively Regulates BCR Signaling The Journal of Experimental Medicine cells were fixed, permeabilized, blocked with 10% normal mouse phospho-Syk or Syk, the LSM5 imaging examiner software pro- and donkey sera on ice, stained with rabbit Abs specific for phos- gram was used. To compare the intensity of phospho-Syk colo- / / pho-Btk (Y223)-or as a control nonspecific rabbit Ig detected us- calized with IgM between Cbl-b and Cbl-b splenic B ing PE-conjugated F(ab) donkey Abs specific for rabbit IgG. cells, the images from five randomly chosen fields each contain- After acquisition by FACScan™ (Becton Dickinson), the data ing at least 400 cells were acquired as grayscale with linear con- were analyzed by the FlowJo software program. The percentage trast. The mean fluorescence intensity (MFI) of phospho-Syk of the phospho-Btk–positive cells relative to control Ab stained colocalized with IgM was calculated based on the colocalization cells was calculated after gating on live cells by the forward- and scatter diagrams by multiplying both IgM and phospho-Syk posi- side-scatter. tive, matched pixel numbers by the MFI of the matched pixels Immunofluorescence Microscopy. To quantify the number of and then dividing by the number of cells in a chosen field. cells showing patch, Cap I or Cap II BCR structures, purified splenic B cells were treated with a rat mAb specific for mouse IgM at 4 C for 30 min then transferred to poly-l-lysine treated Results coverslips at 4 C. Biotin-conjugated goat antibodies specific for Prolonged Phosphorylation of Ig and Syk after BCR Cross- rat IgG were added to further cross-link the BCR and the cells linking in Cbl-b B Cells. B cells from Cbl-b–deficient warmed to 37 C for the times indicated. At the end of each time point the cells were fixed with 3.7% paraformaldehyde, mice were shown previously to hyper-proliferate in re- quenched with 50 M NH Cl, blocked with PBS containing 1% sponse to BCR cross-linking suggesting a role for Cbl-b in BSA (PBS-BSA) for 30 min at room temperature (RT) and the negative regulation of B cell signaling. One of the earli- stained with AlexaFluor 488-conjugated streptavidin in the dark est events in BCR signaling is the tyrosine phosphorylation for 30 min at RT. The coverslips were mounted in Prolong An- of the Ig/Ig complex by the Src-family kinase Lyn (3). tifade (Molecular Probe) and examined by fluorescence micros- The phosphorylation of Ig results in the recruitment of copy (Olympus IX70 microscope, 100 W mercury lamp, a Syk to Ig via its SH2 domains and the subsequent phos- cooled CCD camera, 60 1.4 oil objectives). The cells having a phorylation of Syk (4). The subsequent Syk-mediated BCR patch, Cap I, or Cap II morphology were counted at each phosphorylation of the B cell adaptor protein BLNK links time point. the BCR to both PLC-2 and Btk. To investigate the mo- To image Ig and phosphotyrosine in patch-, Cap I–, Cap II– positive cells, splenic B cells were incubated with a rat mAb spe- lecular mechanisms underlying the hyper-responsiveness of cific for IgM at 4 C, washed, allowed to settle on coverslips on B cells from Cbl-b mice, we began by monitoring the ice, and further cross-linked with biotin-conjugated goat Abs tyrosine phosphorylation of Cbl-b, Lyn, Ig, and Syk after / / specific for rat IgG for 5 min for patch and Cap I and for 30 min BCR cross-linking. B cells from Cbl-b and Cbl-b for Cap II at 37 C. Fixed cells were incubated with Texas Red– mice were incubated with F(ab) IgM-specific Abs to conjugated Streptavidin washed, permeabilized with 0.05% sapo- cross-link the BCR for increasing lengths of time at 37 nin in PBS-BSA, blocked with 50 g/ml of purified normal goat and at the end of each time point the phosphorylation state IgG for 30 min at RT, and incubated with PY20 mAb specific of Cbl-b, Lyn, Ig, and Syk was determined by immuno- for phosphotyrosine for 1 h at RT. The secondary FITC-conju- precipitation of each protein followed by immunoblots gated goat F(ab) anti–mouse, Fc -specific Ab, was added for 30 probing with a phosphotyrosine-specific mAb. min at RT. For imaging of IgM and phospho-Syk, and IgM and In B cells from Cbl-b mice, Cbl-b was not detectably Syk, splenic B cells were incubated with Fab goat Abs specific for mouse Ig conjugated with Rhodamine Red-X (RRX) for 15 phosphorylated in resting cells, but became phosphorylated min at RT, washed, and allowed to settle onto an poly-l-lysine immediately upon cross-linking (Fig. 1 A). Phosphorylation coated 8-well glass-bottom chamber slide (Labtek/Nunc) on ice of Cbl-b increased after 2 min at 37 C then decreased ap- for 30 min. Cells were stimulated with 50 g/ml of F(ab) goat proaching unactivated levels by 30 min. In control experi- Abs specific for mouse IgM G at 37 C for the indicated times ments, Cbl-b was not detected in Cbl-b B cells (Fig. 1 and fixed. After quenching, cells were permeabilized with 0.05% A). The phosphorylation of Cbl-b following BCR cross- saponin in PBS-Gelatin (PBS containing 1% fish skin gelatin), linking suggests that it plays a direct role in BCR signaling. blocked with 10% normal mouse sera and 50 g/ml of purified / / In both Cbl-b and Cbl-b B cells Lyn showed a normal goat IgG for 30 min at RT. Cells were stained with rabbit high level of constitutive phosphorylation that increased Abs specific for Syk or phospho-Syk (Y519/520) detected using slightly upon BCR cross-linking and remained phosphory- goat Abs specific for rabbit IgG conjugated with AlexaFluor 488. lated for the 30-min time course (Fig. 1 B). The level of For two-color confocal and differential interference contrast (DIC) images, a confocal laser scanning microscope (Zeiss Axio- Lyn phosphorylation, assessed by the ratio of the densities vert 200M LSM 510 META; Carl Zeiss Microimaging, Inc.) fit- of the Lyn bands in immunoblots to the densities of the ted with a 1.4 oil planapochromat 63 objective was used. Im- phospho-Lyn bands, was slightly higher (10%) in B cells ages were acquired with configuration of Ex488/Em 505–530BP / / from Cbl-b as compared with Cbl-b mice. Other- for AlexaFluor 488 or FITC and Ex543/Em 560LP for Texas wise, the absence of Cbl-b did not appear to have a signifi- Red Dye or RRX and under the scan control of fixed pixel den- cant effect on Lyn phosphorylation. Direct measurements sity at 512 512 pixels, 8 bit (phosphotyrosine) or 12 bit (phos- of the kinase activity of Lyn in vitro showed no significant pho-Syk and Syk) pixel depths, linear contrast of grayscale, 7.8 differences in Lyn activity in Lyn immunoprecipitates from ms scan time, and pinhole size of 45 m (phosphotyrosine and cell lysates prepared from B cells from Cbl-b as com- IgM) or 66 m (phospho-Syk/Syk and IgM). No significant sig- pared with Cbl-b mice after BCR cross-linking (Fig. 1 nal saturation was noted in any of the images used for analysis. To quantify the colocalization between IgM and phosphotyrosine, C). Resting Cbl-b B cells may have a slightly higher 1513 Sohn et al. The Journal of Experimental Medicine The phosphorylation of Syk was similarly prolonged in Cbl-b B cells (Fig. 2 B). BCR cross-linking resulted in slightly higher phosphorylation of Syk immediately follow- ing cross-linking in Cbl-b B cells (a ratio of 1.1) as compared with Cbl-b B cells (a ratio of 0.9). Syk re- mained phosphorylated for a longer period of time in Cbl- / / b B cells compared with Cbl-b B cells (a ratio of 0.8 versus 0.4 measured at 10 min). The phosphorylation of Syk on Y519/520 within its activation loop is essential for Syk’s activity (17, 28). Immunoblots of Syk immunopre- cipitates probed with polyclonal Abs specific for Syk phos- phorylated on Y519/520 showed approximately twofold more Y519/520 phospho-Syk in Cbl-b as compared with Cbl-b B cells 2 min after BCR cross-linking (Fig. 2 C). Significantly, phosphorylated Syk remained associated with Ig for longer periods of time in Cbl-b B cells as compared with Cbl-b B cells (Fig. 2 A, right panel). Syk was immunoprecipitated with Ig in resting cells, however, the Ig-associated Syk was not phosphorylated. Immediately upon BCR cross-linking the Syk associated with Ig was phosphorylated. The amount of phospho-Syk associated with Ig showed a significant decrease in Cbl- b B cells 10 min after BCR cross-linking relative to that in Cbl-b B cells (a ratio of 0.2 versus 0.9). The association of phospho-Syk with the BCR was im- aged in B cells following BCR cross-linking. B cells were incubated with RRX-conjugated Fab goat Abs specific for Ig, washed and allowed to settle on coverslips before Figure 1. Cbl-b is phosphorylated following BCR cross-linking but cross-linking the BCR by addition of goat Abs specific for does not affect Lyn activity. Purified splenic B cells from Cbl-b and Cbl-b mice were incubated with F(ab) goat Abs specific for mouse mouse IgM. Cells were incubated at 37 C for 0 to 30 min, Ig at 37 C for the indicated times. The cells were washed, lysed, and the fixed, permeabilized, and stained for phospho-Syk using lysate subjected to immunoprecipitation using Abs specific for Cbl-b (A) phospho-Syk (Y519/520)-specific rabbit Abs detected us- or Lyn (B) and the immunoprecipitate analyzed by SDS-PAGE and im- ing AlexaFluor 488-conjugated goat Abs specific for rabbit munoblotting probing for phosphotyrosines using the phosphotyrosine- IgG. The cells were imaged by laser scanning confocal mi- specific mAb PY20, stripped, and reprobed for either Cbl-b (A) or Lyn (B). The results shown are representative of three independent experi- croscopy and representative merged images acquired 2 min ments. The Lyn kinase activity was measured in Lyn immunoprecipitates after BCR cross-linking are shown (Fig. 2 D, top). In un- / / of lysates of B cells from Cbl-b and Cbl-b mice at various times af- treated resting cells no phospho-Syk was detected in either ter cross-linking the BCR (C). The results from three experiments were / / Cbl-b or Cbl-b B cells. 2 min after BCR cross-link- averaged and the results expressed relative to the Lyn activity in resting Cbl-b B cells. ing the images appeared to show a larger number of cells with cap structures in which the BCR and phospho-Syk / / were colocalized in Cbl-b as compared with Cbl-b level of Lyn activity as compared with Cbl-b B cells re- B cells. To quantify the amount of BCR and phospho-Syk flecting the slightly higher level of Lyn phosphorylation in that were colocalized, the images were analyzed pixel by Cbl-b B cells. pixel and the FI of the IgM RRX and phospho-Syk Cross-linking the BCR resulted in the tyrosine phos- (Y519/520) AlexaFluor 488 within each pixel plotted in a phorylation of Ig in B cells from both Cbl-b and Cbl- histogram (Fig. 2 D, bottom). Pixels that lie along the diag- b mice detected immediately upon cross-linking (Fig. 2 onal contain equal amounts of colocalized IgM and phos- A, left panel). Quantitation of the bands corresponding to pho-Syk (Y519/520). For those above the diagonal the in- Ig and phosphorylated Ig showed slightly greater phos- tensity of the phospho-Syk (Y519/520) is less than that of phorylation of Ig in Cbl-b B cells (a ratio of tyrosine- the BCR and for pixels below the diagonal the phospho- phosphorylated Ig to total Ig of 1) as compared with Syk (Y519/520) intensity is greater than that of the BCR. Cbl-b B cells (a ratio of 0.75). Significantly, the phos- The histograms indicate less colocalization of phospho-Syk phorylation of Ig was prolonged in B cells lacking Cbl-b with the BCR at the 2 min time point in Cbl-b B cells / / (Fig. 2 A). In Cbl-b B cells Ig phosphorylation re- as compared with Cbl-b B cells (Fig. 2 D, bottom). For turned to nearly unstimulated levels 10 min following each time point, the MFI of the phospho-Syk (Y519/520) BCR cross-linking (a ratio of 0.2). In contrast, a large por- colocalized with the BCR was quantified (Fig. 2 E). The tion of Ig in Cbl-b B cells remained phosphorylated at degree of colocalization of phospho-Syk and the BCR was 10 min (a ratio of 0.7). similar immediately after the addition of the BCR cross- 1514 Cbl-b Negatively Regulates BCR Signaling The Journal of Experimental Medicine Figure 2. Phosphorylation of Ig and Syk and associa- tion of phospho-Syk with the BCR is prolonged in acti- vated Cbl-b B cells. (A) Purified splenic B cells from / / Cbl-b and Cbl-b mice were treated to cross-link the BCR by incubation with a rat mAb specific for mouse IgM on ice for 30 min followed by incubation with F(ab) goat Abs specific for rat IgG at 37 C for the times indi- cated. At the end of each time point the cells were lysed, and the lysates sub- jected to immunoprecipitation using Ig-specific Abs. The Ig immunopre- cipitates were analyzed by SDS-PAGE and immunoblotting probing for phosphotyrosine-containing proteins, stripped, and reprobed for either Ig (left panel) or Syk (right panel) using specific Abs. (B) B cells from Cbl-b and Cbl-b mice were treated as in panel A to cross-link the BCR and at the end of each time point cells were lysed and Syk immunoprecipated from the lysates. The Syk immunoprecipitates were analyzed by SDS-PAGE and immunoblotting probing first with a phosphotyrosine-specific mAb, stripped, / / and reprobed for Syk. (C) Cbl-b and Cbl-b B cells were treated in panel A to cross-link the BCR and incubated for 2 min at 37 C and lysed. Syk was immunoprecipitated from the lysates and the immunoprecipitates an- alyzed by SDS-PAGE and immunoblotting. The blots were first probed using phospho-Syk (Y519/520) specific Abs, stripped and reprobed with Syk-spe- / / cific Abs. (D) B cells from Cbl-b and Cbl-b mice were imaged by laser scanning confocal microscopy to determine the colocalization of the BCR and phospho-Syk (Y519/520) as detailed in Materials and Methods. Briefly, B cells were incubated with RRX-conjugated Fab goat Abs specific for mouse Ig, washed, allowed to settle onto coverslips, and activated by the addition of goat Abs specific for mouse IgM for 0 to 30 min at 37 C. The cells were fixed, permeabilized, and stained with rabbit Abs specific for phospho-Syk (Y519/520) detected using AlexaFluor 488-conjugated goat Abs specific for rabbit Ig. The cells were imaged by confocal laser scanning microscopy using a Zeiss Axiovert 200M LSM 510 META. A representative field is shown 2 min after BCR cross-linking (top) and the RRX and AlexaFluor 488 intensi- ties of each pixel are plotted (bottom). (E) The average MFI of the AlexaFluor 488 colocalized with RRX for five fields of cells at each time point is given. linking Abs (the 0 min time point) in Cbl-b and Cbl- ity that Cbl-b influences Syk through ubiquitination, Syk / / b B cells. 2 min after BCR cross-linking the amount of was immunoprecipitated from lysates of Cbl-b and Cbl- phospho-Syk (Y519/520) associated with the BCR in- b B cells treated to cross-link the BCR for varying creased significantly in Cbl-b B cells and then decreased lengths of time and the immunoprecipitates analyzed by with time. In contrast, in Cbl-b B cells the amount of immunoblot probing for ubiquitin, Syk and phosphoty- phospho-Syk (Y519/520) colocalized with the BCR had rosine. The results from the 2-min time point are shown / / already decreased 2 min after BCR cross-linking. These re- (Fig. 3). In resting B from both Cbl-b and Cbl-b a sults taken together with those above indicate that Syk is a portion of Syk is tyrosine phosphorylated. A high molecu- target of Cbl-b activity after BCR cross-linking resulting in lar weight band reacting with both Syk- and phosphoty- the rapid turnover of active phospho-Syk (Y519/520) asso- rosine-specific antibodies appears to be ubiquitinated and ciated with Ig. may represent a product of constitutive turnover. Cross- Increased Syk Ubiquitination in Cbl-b B Cells. The linking the BCR resulted in increased tyrosine phosphory- members of the Cbl family have been shown to function as lation of Syk and heterogeneous and poly-ubiquitination of E3 ubiquitin ligases and to target protein tyrosine kinase Syk in Cbl-b B cells. In contrast, although Syk is phos- substrates for degradation (17). To investigate the possibil- phorylated the ubiquitination of Syk was significantly less 1515 Sohn et al. The Journal of Experimental Medicine curred immediately following BCR cross-linking and de- creased rapidly thereafter. In contrast, BLNK remained sig- nificantly phosphorylated 10 min after BCR cross-linking in Cbl-b B cells. Thus, Cbl-b influenced the duration of the phosphorylation of BLNK. Cbl-b also affects the phosphorylation state of Btk (Fig. 4 / / B). B cells from Cbl-b and Cbl-b mice were treated with IgM-specific antibodies to cross-link the BCR and at various times afterward the cells were permeabilized, stained with Abs specific for phospho-Btk (Y223) and ana- lyzed by flow cytometry. The levels of phosphorylated Btk increased immediately after BCR cross-linking in B cells / / from both Cbl-b and Cbl-b mice. However, Btk phosphorylation reached higher levels in Cbl-b B cells as compared with Cbl-b B cells 2–10 min following BCR cross-linking. Figure 3. The activation-induced ubiquitination of Syk is reduced in To further characterize the phospho-Btk (Y223) in / / / Cbl-b B cells. Splenic B cells from Cbl-b and Cbl-b mice were terms of its association with PLC-2 and Syk, cells were treated as in Fig 2, A–C, to cross-link the BCR and warmed to 37 C for lysed at various times after BCR cross-linking and the ly- increasing lengths of time. The cells were lysed, and Syk was immuno- precipitated and analyzed by SDS-PAGE and immunoblotting. Duplicate sates subjected to immunoprecipitation using phospho-Btk immunoblots were probed for either ubiquitin (anti-Ub) or phosphoty- (Y223)-specific Abs. The immunoprecipitates were immu- rosine (Anti-PTyr). The phosphotyrosine blot was stripped and reprobed noblotted probing for phosphotyrosine, stripped and re- for Syk (anti-Syk). The results for the 2-min time point are shown at probed for Btk, PLC-2, and Syk using specific antibodies which point ubiquitination of Syk was maximal. The result shown is rep- resentative of three independent experiments. (Fig. 4 C). The phospho-Btk (Y223) immunoprecipitates contained Btk, only weakly detected in immunoblot, and phospho-PLC-2 and phospho-Syk. Significantly, the in Cbl-b B cells and appeared to involve primarily the phospho-Btk/phospho-PLC-2/phospho-Syk complex re- most highly ubiquitinated forms of Syk detected in resting mains assembled for longer periods of time in Cbl-b as cells. In controls, there was no detectable ubiquitination of compared with Cbl-b B cells (Fig. 4 C). / / Ig in Cbl-b or Cbl-b B cells at any time following The direct effect of Cbl-b on PLC-2 phosphorylation BCR cross-linking (unpublished data). However, it is pos- was measured. In resting B cells from both Cbl-b and sible that in addition to Syk proteins coimmunoprecipitated Cbl-b mice there was no significant phosphorylation of with Syk are ubiquitinated. Ubiquitination of Syk was PLC-2 (Fig. 5, top). Upon BCR cross-linking PLC-2 maximal at two min following BCR cross-linking (unpub- was phosphorylated in Cbl-b mice and by 15 min fol- lished data) at a time at which the levels of phospho-Syk lowing BCR cross-linking the amount of phosphorylated (Y519/520) began decreasing in Cbl-b B cells (Fig. 2, PLC-2 had decreased significantly. In Cbl-b B cells, D and E) suggesting that Cbl-b targets phospho-Syk for PLC-2 was phosphorylated upon BCR cross-linking but ubiquitination resulting in its subsequent degradation. in contrast to the PLC-2 in Cbl-b B cells, PLC-2 Prolonged Phosphorylation of BLNK, Btk and PLC-2 and was strongly phosphorylated at 15 min and a decrease in 2 / Ca Fluxes in Cbl-b B Cells. A key downstream ef- phosphorylation was not detected until 30 min after BCR fector of BCR signaling is PLC-2 that cleaves PIP2 (4, 5) cross-linking. Consistent with this observation Ca fluxes releasing IP3 and DAG resulting in release of intracellular after BCR cross-linking in Cbl-b B cells were more 2 / Ca stores. PLC-2 is phosphorylated by Syk and Btk persistent as compared with those in Cbl-b B cells (Fig. which are activated by phosphorylation by Lyn (6). BLNK 5, bottom). Taken together these results indicate that in the plays a key role in Ca signaling by functioning as a scaf- absence of Cbl-b, complexes containing phosphorylated fold for the assembly of complexes of PLC-2 and Btk Syk, BLNK, Btk and PLC-2 persist leading to prolonged after Syk-mediated phosphorylation. Recent evidence in- Ca fluxes. dicates that normal Ca signaling requires BLNK-depen- The Association of Vav-1 with Syk Is Prolonged in Cbl-b dent PLC-2–BLNK interactions and BLNK-independent B Cells. Vav, a guanine-nucleotide exchange factor or Btk activation as well as the assembly of BLNK-PLC-2– GEF for the Rho/Rac family of GTPases, plays a central Btk complexes (29). To determine if a Cbl-b deficiency role in integrating signaling from the BCR. Vav activity is influenced these events the levels of phosphorylation of regulated, in part, by phosphorylation by Syk (30). Evi- BLNK, Btk, and PLC-2 and Ca fluxes were measured dence for a role for Cbl-b in regulating the function of Vav / / / in Cbl-b or Cbl-b and Cbl-b B cells after BCR in lymphocytes was provided by the observation that the cross-linking. phosphorylation of Vav-1 was increased in Cbl-b T In Cbl-b B cells the phosphorylation of BLNK was cells as compared with Cbl-b T cells (22, 23). To inves- prolonged as compared with Cbl-b B cells (Fig. 4 A). In tigate the effect of a Cbl-b deficiency on Vav-1 phosphory- Cbl-b B cells maximal BLNK phosphorylation oc- lation, Vav was immunoprecipitated from Cbl-b and 1516 Cbl-b Negatively Regulates BCR Signaling The Journal of Experimental Medicine Figure 4. The phosphoryla- tion of BLNK and Btk is influ- enced by Cbl-b. (A) B cells from / / Cbl-b and Cbl-b mice were treated as in Fig. 1 to cross- link the BCR at 37 C for the times indicated. The cells were lysed and BLNK was immuno- precipitated from the lysates. The BLNK immunoprecipitates were analyzed by SDS-PAGE and im- munoblotting probing with a phosphotyrosine-specific mAb or with a BLNK-specific mAb. (B) B / / and Cbl-b cells from Cbl-b mice were treated as in Fig. 1 to cross-link to BCR at 37 C for 0 to 30 min, fixed, permeabilized, and stained with rabbit phospho- Btk (Y223)-specific Abs detected using PE-conjugated Abs specific for rabbit Ig. The cells were ana- lyzed by flow cytometry and the percent phospho-Btk positive cells relative to the negative control is given for each time point. (C) B / / and Cbl-b cells from Cbl-b mice were treated as in B to cross- link the BCR, lysed and the ly- sates immunoprecipitated using the phospho-Btk (Y223)-specific Abs and the immunoprecipitates analyzed by immunoblot probing first using a phosphotyrosine-spe- cific mAb, stripped, and re- probed using antibodies specific for PLC-2, Btk, or Syk. Cbl-b B cells before and at various times after BCR lated upon BCR cross-linking (Fig. 4) and associates with cross-linking and the immunoprecipitates analyzed by im- phospho-Syk (Fig. 4) and based on its MW, Btk is a good munoblot probing for phosphotyrosine. In resting B cells candidate for this 77 kD phosphoprotein. / / from both Cbl-b and Cbl-b mice low levels of Syk has also been shown to play an important role in acti- phosphorylated Vav-1 were detected (Fig. 6 A). BCR vating PI3K through phosphorylation (31). Little differences / / cross-linking resulted in the rapid phosphorylation of Vav-1 were detected in Cbl-b versus Cbl-B B cells in the / / in both Cbl-b and Cbl-b B cells which persisted phosphorylation of PI3K (unpublished data) or Akt (Fig. 6 for the 30-min course of the experiment. Densitometry B), a downstream effector of PI3K. Thus, Cbl-b deficiencies analyses of the intensities of the bands in the Vav and phos- do not appear to significantly affect the phosphorylation of photyrosine immunoblots in Fig. 6 A indicated a small in- proteins in the PI3-K pathway downstream of Syk. crease in the level of phosphorylation of Vav at the 2 and 5 Prolonged Phosphorylation of ERK and JNK in Cbl-b B / / min time point in Cbl-b versus Cbl-b B cells, how- Cells. The MAP kinases, ERK and JNK, that regulate ever, this increase was not completely reproducible. Signif- nuclear transcription events are also major downstream tar- icantly, the association of Vav with Syk was prolonged in gets of BCR signaling cascades leading to B cell activation / / Cbl-b B cells as compared with Cbl-b B cells. Prob- (4). To determine if these pathways were influenced by ing immunoblots of Vav-1 immunoprecipitates for Syk Cbl-b, the level of tyrosine phosphorylation of ERK 1 and showed larger amounts of Syk associated with Vav at early 2 and JNK 1 and 2 were monitored at various times after time points after BCR cross-linking that persisted for BCR cross-linking. As compared with Cbl-b B cells, / / longer in Cbl-b as compared with Cbl-b B cells ERK was more strongly phosphorylated throughout a 20 (Fig. 6 A). Probing the immunoblot for phosphotyrosine- min time course in Cbl-b B cells (Fig. 7 A). Similarly, containing proteins indicated that the Syk associated with both forms of JNK were more strongly phosphorylated in / / Vav was phosphorylated (Fig. 6 A). In addition to phos- Cbl-b as compared to Cbl-b B cells (Fig. 7 A). phorylated Syk, a phosphoprotein migrating at 77 kD was Thus, the signaling pathways involving ERK and JNK ap- also more strongly associated with Vav-1 in Cbl-b as pear to be regulated by Cbl-b. Consistent with the in- / / / compared with Cbl-b B cells. The identity of this pro- creased activation of ERK in Cbl-b B cells, Cbl-b B tein is not known, however, given that Btk is phosphory- cells showed greater expression of the early activation 1517 Sohn et al. The Journal of Experimental Medicine 2 Figure 5. PLC-2 phosphorylation and downstream Ca fluxes are / / prolonged in activated Cbl-b B cells. (Top) B cells from Cbl-b and Cbl-b mice were treated to cross-link the BCR as in Fig. 1, lysed, and the lysates subjected to immunoprecipitation using PLC-2–specific anti- bodies. The immunoprecipitates were analyzed by immunoblot for phos- Figure 6. The association of phosphorylated Syk with Vav1 is pro- / / photyrosine-containing proteins, stripped, and reprobed for PLC-2. A longed in Cbl-b B cells. Purified splenic B cells from Cbl-b and representative result of three independent experiments is shown. (Bot- Cbl-b mice were treated to cross-link the BCR as in Fig. 1. (A) The tom) B cells were incubated with Indo-1 for 45 min at 30 C and stained cells were lysed and the lysates subjected to immunoprecipitation using with a PE-conjugated B220-specific mAb to allow gating on the B cells. Vav1-specific Abs. The immunoprecipitates were analyzed by immuno- The cells were treated at 37 C with a rat mAb specific for IgM for 30 s blotting probing for phosphotyrosine using the mAb PY20. The blots followed by F(ab) goat antibodies specific for rat IgG to cross-link the 2 were stripped and probed for Vav1 or Syk. (B) Alternatively, the unfrac- BCR. The cells were monitored for 512s by flow cytometry for bound tionated lysates were analyzed by immunoblot probing for phospho-Akt (violet) and unbound (blue) Indo-1. The arrow indicates the time of addi- (S473) stripped and reprobed for Akt. tion of the secondary antibody. permeabilized and incubated with the phosphotyrosine- marker CD69 that is partially dependent on ERK activa- specific mAb, PY20, detected using FITC-conjugated F(ab) goat Abs specific for mouse IgG. To characterize tion (32). Maximal expression of CD69 was induced by treating the cells with 10 g/ml IgM-specific antibody to the patched and capped structures with regard to Syk and / / phospho-Syk colocalization, B cells were incubated with cross-link the BCR in both Cbl-b and Cbl-b B cells but maximal CD69 expression was over twofold greater in RRX-conjugated Fab goat Abs specific for Ig, washed, and the BCR cross-linked by the addition of goat Abs spe- Cbl-b B cells (Fig. 7 B). The Formation of Signaling Active BCR Cap Structures Is cific for mouse IgM. The cells were warmed to 37 C for varying lengths of time, fixed, permeabilized, and stained Prolonged in Cbl-b B Cells. After cross-linking, the BCR clusters to form patches that subsequently move to for Syk and phospho-Syk using rabbit Abs specific for Syk or phospho-Syk (Y519/520) detected using AlexaFluor one pole of the B cell to form a cap that is ultimately con- densed and internalized (33). Recent evidence suggests that 488-conjugated Abs specific rabbit Ig. The cells were im- aged by confocal laser scanning microscopy and the merged the cap is a region of active signaling and that Syk promotes and stabilizes cap formation in B cells (34). As described images of the BCR and either phosphotyrosine, phospho- Syk or Syk are shown (Fig. 8, a–c). The images were ana- above (Fig. 2, D and E) after BCR cross-linking the BCR remained colocalized with phospho-Syk for a longer period lyzed pixel by pixel as described above (Fig. 2 D) and the / / data displayed in histograms. Similar structures were ob- of time in Cbl-b as compared with Cbl-b B cells. / / The cross-linking-induced capping of the BCR and colo- served in B cells from Cbl-b and Cbl-b mice and shown are the images of Cbl-b B cells. Cross-linking calization of the BCR with tyrosine phosphorylated pro- teins, specifically with Syk and phospho-Syk, was further the BCR resulted immediately in BCR patches (Fig. 8, a–c, top) that with time at 37 C formed large caps at one pole characterized. B cells were incubated at 4 C with IgM-spe- cific rat mAbs followed by biotin-conjugated goat Abs spe- of the cell (Cap I structures; Fig. 8, a–c, middle) that pro- ceeded to form tight, condensed Cap II structures (Fig. 8, cific for rat Ig, washed, and warmed to 37 C for increasing lengths of time. The cells were fixed, and incubated with a–c, bottom). The BCR in the patches and large Cap I structures were nearly completely coincident with phos- Texas Red–conjugated streptavidin to detect the BCR. To detect phosphotyrosine-containing proteins, the cells were photyrosine-containing proteins as indicated in the merged 1518 Cbl-b Negatively Regulates BCR Signaling The Journal of Experimental Medicine Figure 7. ERK and JNK phosphorylation are prolonged and downstream CD69 expres- sion increased in Cbl-b B cells. (A) Purified splenic B cells were treated to cross-link the BCR as in Fig. 1. The cells were lysed and equal amounts of whole-cell lysates subjected to immunoblotting using Abs spe- cific for phospho-ERK (anti- PERK) and phospho-JNK (anti-PJNK). The blots were stripped and reprobed for ERK1 and 2 and JNK1 and 2. (B) After BCR cross-linking the cells were incubated for 20 h at which time the surface expression of CD69 was quantified by flow cytometry. The results are the mean of duplicate cultures of one of three representative experiments. image showing colocalization of the red and green fluores- The number of patch, Cap I and Cap II structures / / cent dyes yielding yellow (Fig. 8 a, top, middle). In addi- formed by B cells from Cbl-b and Cbl-b B cells after tion, the analyses of the distribution of the fluorescence in- BCR cross-linking were counted and compared (Fig. 8 d). / / tensities of the green and red dyes showed that most pixels Similar numbers of B cells from Cbl-b and Cbl-b lie along the diagonal of the histogram indicating colocal- mice formed patches with nearly all B cells showing a ization of IgM and phosphotyrosine-containing proteins patched morphology immediately after BCR cross-linking (Fig. 8 a, top, middle). Thus, both the patched and Cap I which decreased with time at 37 C (Fig. 8 d, top). Signifi- structures appear signaling active. In contrast, the Cap II cantly, Cbl-b B cells formed BCR Cap I structures BCR structures showed no staining for phosphotyrosine more rapidly as compared with Cbl-b B cells such that indicating that these BCR structures are signaling inactive after 2 min at 37 C the BCR was in Cap I structures in / / (Fig. 8 a, bottom). The analyses of the red and green fluo- 62% of Cbl-b B cells as compared with 32% of Cbl-b rescence intensities showed little colocalization of red and B cells (Fig. 8 d, middle). In several experiments the time green dye with most pixels lying significantly above the di- course of polarization varied by 2–3 min but the acceler- / / agonal (Fig. 8 a, bottom). The analysis of cells stained with ated rate of polarization in Cbl-b versus Cbl-b was Syk-specific Abs showed near complete colocalization of observed in each experiment. The presence of BCR in Syk with the BCR in patched and Cap I structures yielding Cap I structures was sustained in B cells over the course of / / yellow in the image (Fig. 8 c, top, middle) and quantifica- the experiment in Cbl-b as compared with Cbl-b B tion of the image showed a near complete colocalization of cells (50% versus 32%) in agreement with the observation the BCR and Syk. However, not all of the Syk associated above (Fig. 2, D and E) that the BCR remained colocalized with the BCR in the patched and Cap I structures was ac- with phospho-Syk for longer in Cbl-b as compared / / tive phospho-Syk (Y519/520). The images of cells stained with Cbl-b B cells. In addition, more Cbl-b B cells to detect phospho-Syk (Y519/520) and the BCR show progressed to form signaling inactive Cap II structures as distinct red areas indicating that the BCR was not com- compared with Cbl-b B cells (Fig. 8 d, bottom) sug- pletely colocalized with phospho-Syk (Y519/520). The gesting that Cbl-b by negatively regulating the BCR sig- quantification of the image showed significant proportion naling promoted the condensing of the BCR into signaling of the pixels lie above the diagonal indicating regions that inactive Cap II structures. contain the BCR but not phospho-Syk (Y519/520). The The capping of the BCR has been shown to be depen- Cap II structures showed virtually no staining with either dent on the association of the BCR with the actin cyto- the Syk-specific Abs (Fig. 8 c, bottom panel) or with the skeleton (35). The difference observed in the rate of BCR phospho-Syk (Y519/520)-specific Abs (Fig. 8 b, bottom). caps was reflected in the rate of activation of CDC42 and The condensed Cap II structures are often associated with of F-actin polymerization (Fig. 9). CDC42 is a member of intracellular vesicles containing the BCR as shown in Fig. the Rac family of GTPases that when activated binds to 8 c, bottom panel. Although the Cap II structures showed WASP which promotes actin cytoskeleton association. The no staining for Syk the vesicles containing internalized level of activated CDC42 in B cells from Cbl-b and BCR stained for Syk. The quantitation of the MFI of the Cbl-b mice at various times after BCR cross-linking image showed two clear BCR areas one of which contains was determined by permeabilizing the cells and incubating Syk and one of which did not. This observation suggests with the recombinant fusion protein WASP-GBD-GFP. that the internalized BCR associated with Syk enters the The GFP signal of the recombinant protein was amplified cell from active Syk-containing Cap I structures and not by incubation with GFP-specific Abs detected using FITC- from the signaling inactive Cap II structures. The BCR in conjugated secondary Abs. Cbl-b B cells showed an im- the Cap II structure may also be internalized but degraded mediate increase in active CDC42 which decreased by 2 too rapidly to be imaged. min and remained at an elevated level for the course of the 1519 Sohn et al. The Journal of Experimental Medicine / / Figure 8. Signaling active BCR caps are prolonged in Cbl-b B cells. (a) Splenic B cells from Cbl-b mice were incubated with a rat mAb specific for IgM at 4 C, washed and incubated with biotin-labeled goat antibodies specific for rat IgG and warmed to 37 C for increasing lengths of time. At the end of each time point the cells were fixed and incubated with Texas Red–conjugated streptavidin to detect the BCR. The cells were permeabilized and incubated with the phosphotyrosine-specific mAb PY20 detected using FITC-labeled F(ab) goat Abs specific for mouse IgG (b and c). Splenic B cells from Cbl-b mice were incubated with RRX-conjugated Fab goat Abs specific for Ig for 15 min at 25 C and the BCR was cross-linked by the addition of goat Abs specific for mouse IgM. The cells were incubated at 37 C for increasing lengths of time, permeabilized, and incubated with either rabbit phospho-Syk (Y519/520)-specific Abs (b) or with rabbit Syk-specific Abs (c) each detected using AlexaFluor 488-labeled goat Abs specific for rabbit IgG. In each case, the cells were examined by confocal laser scanning microscopy and shown are the merged images of the optimal single planes at a magnification of 63. Shown are typical images of patch, Cap I and Cap II structures taken for phosphotyrosine images at 5 min for patch and Cap I structures and 30 min for Cap II structures and for Syk and phospho-Syk images at 0 min for patch, 2 min for Cap I, and 30 min for Cap II structures. The images were analyzed by the LSM 5 software program to quantify the colocalization of red and green fluorescence and the data plotted in histo- / / grams. (d) Purified splenic B cells from Cbl-b and Cbl-b mice were treated as in panel a to cross-link the BCR and cells were incubated at 37 for increasing lengths of time. At the end of each time point the cells were fixed with 3.7% paraformaldehyde, stained with Alexa 488-conjugated strepta- vidin, and examined by fluorescence microscopy. The mean percentage ( SD) of B cells exhibiting the patch, Cap I and Cap II structures with time after warming to 37 C is given. At least 500 cells at random were counted per time point. experiment (Fig. 9 A). In contrast Cbl-b B cells showed tochalasin D (CytD; reference 36), inhibitors of the actin no immediate rapid increase in active CDC42 but rather cytoskeleton; PP2, an inhibitor of Src family kinases (37); active CDC42 increased slowly in Cbl-b B cells reach- and piceatannol (Pic), an inhibitor of Syk (38) that has also ing levels comparable to those in Cbl-b B cells by 2 been reported to inhibit additional protein tyrosine kinases min. Actin polymerization was measured after BCR cross- (39). As shown in Fig. 9 C, 2 min after BCR cross-linking linking in permeabilized and fixed cells using Alexa 488- the BCR were patched on the majority of untreated cells. conjugated phalloidin. Cbl-b B cells showed slightly ac- Patching at 2 min after BCR cross-linking was not affected celerated actin polymerization as compared with Cbl-b by any inhibitor indicating that patching was not depen- B cells (Fig. 9 B). Consistent with these observations a dent on Src or Syk signaling nor on an intact cytoskeleton. small but significantly larger proportion of the BCR was This is despite the observation that phospho-Syk (Y519/ found associated with the actin cytoskeleton in a high-den- 520) colocalized with the BCR in patches (Fig. 8 b). 10 sity pellet from lysates of Cbl-b as compared with Cbl- min following BCR cross-linking the BCR were observed b B cells (unpublished data). in Cap I (60%) and Cap II structures (25%). Each inhibitor To directly assess the requirements for signaling and the significantly blocked the formation of Cap I structures and actin cytoskeleton in the formation of patch, Cap I and the BCR in treated cells remained patched. Thus, the for- Cap II structures, Cbl-b B cells were treated with the mation of Cap I structures required both Src and Syk sig- following inhibitors: latrunculin (LN; reference 36) or Cy- naling and an intact actin cytoskeleton. Significantly, in B 1520 Cbl-b Negatively Regulates BCR Signaling The Journal of Experimental Medicine Figure 9. CDC42 activation and actin polymer- ization are enhanced in Cbl-b–deficient B cells. (A) / / Purified splenic B cells from Cbl-b and Cbl-b mice were treated to cross-link the BCR as in Fig. 2, A–C, incubated at 37 C for the times indicated, fixed, permeabilized, and incubated with the WASP-GBD-GFP fusion protein followed by in- cubation with FITC-conjugated-rabbit antibodies specific for GFP and then FITC-conjugated goat antibodies specific for rabbit Ig. The cells were ana- lyzed by flow cytometry and the results presented as the percent increase in mean fluorescence of acti- vated B cells relative to the mean fluorescence of resting B cells. (B) B cells were treated as in panel A and permeabilized cells were stained with Alexa 488–conjugated phalloidin and analyzed by flow cytometry. Shown is the percent increase of the mean fluorescence of stimulated B cells relative to the mean fluorescence intensity of resting cells at each time point. (C) Splenic B cells from Cbl-b mice were pretreated with the following inhibitors: piceatannol (100 M for 1 h at 37 C) a Syk inhibi- tor(38); PP2 (100 M for 1 h at 37 C) a Src-family kinase inhibitor (reference 37); and Cytochalasin D (10 M for 1 h at 0 C; reference 36) or Latrunculin (10 M for 30 min at 37 C; reference 36) inhibi- tors of the actin cytoskeleton. The pretreated cells were incubated with a rat mAb specific for IgM, washed and incubated for 2 or 10 min at 37 C with biotin-labeled F(ab) goat antibodies specific for rat IgG, fixed and stained with Alexa 488–labeled streptavidin to visualize the BCR. The cells were ex- amined by fluorescence microscopy and the number of cells showing a patch, Cap I or Cap II morphology were scored. cells treated with PP2 the number of cells with Cap I struc- contrast, Cbl-b does not appear to effect PI3K activity or tures decreased and the number of cells with a Cap II mor- PI3K-downstream effectors but does influence ERK activ- phology increased indicating that when Src-dependent sig- ity. SHP-1, a tyrosine phosphatase, was recently shown to naling is blocked the BCR progresses to signaling inactive down-regulate the activation of Lyn and Lyn-induced ty- Cap II structures. rosine phosphorylation of the CD19 receptor in B cells re- sulting in reduced B cell activation (14). As shown here, Cbl-b does not appear to affect Lyn activity. Thus, these Discussion three negative regulators of BCR signaling appear to target Here we provide evidence that in B cells Cbl-b func- different elements of the BCR signaling pathways leading to down modulation of B cell responses. It will be of inter- tions to negatively regulate BCR signaling by targeting Syk for ubiquitination. In Cbl-b–deficient B cells Syk fails to be est to understand the mechanisms which trigger the activity of these regulators and how their activities are coordinated. ubiquitinated following BCR cross-linking in contrast to B cells from wild-type mice in which case BCR cross-linking Lymphocytes express both c-Cbl and Cbl-b. In T cells Cbl-b and c-Cbl appear to function at different stages of leads to rapid ubiquitination of Syk. The results presented suggest that Cbl-b ubiquitinates active phosphorylated Syk development and to target different substrates for ubiquit- ination. c-Cbl has been reported to target Syk for ubiquit- and thus functions to dampen BCR signaling after signaling is initiated and thus plays a role in the normal down modu- ination in human Ramos B cells upon BCR cross-linking and in this way to function as a negative regulator of B lation of BCR signaling. Two additional negative regula- tors of BCR signaling have been described recently, cell activation (17). Thus, Cbl-b and c-Cbl may have somewhat redundant functions in regulating mature B cell namely SHIP and SHP-1. However, the targets of their regulation appear distinct from that described here for Cbl-b. activation through the ubiquitination of Syk. However, recent studies suggest that Cbl-b and c-Cbl may function SHIP, a 5 inositol phosphatase, was first described to block BCR signaling when recruited to the low affinity Fc differently and regulate different targets in developing ver- sus mature B cells. In immature DT40 chicken B cells ev- receptor, FcRIIb, after the coligation of the BCR and FcRIIb by the binding of immune complexes (40). SHIP idence was provided that c-Cbl negatively regulates BCR signaling through its affect on the essential adaptor protein dephosphorylates PIP(3,4,5)P3, the phospholipid product of PI-3K activity, to PI(3,4)P2. Recently, SHIP-deficient BLNK resulting in a block in the recruitment of PLC-2 to BLNK and PLC-2 phosphorylation (41). In contrast, B cells were shown to have elevated levels of PI(3,4,5)P3 (11) increased recruitment of Btk to the plasma membrane Cbl-b was shown to positively regulate Btk-mediated ac- tivation of PLC-2 in immature DT40 B cells (24). Evi- (12) and enhanced Ca signaling (13). However, ERK ac- tivation appeared unaffected in SHIP-deficient cells (13). In dence was also provided that Cbl-b functioned similarly in 1521 Sohn et al. The Journal of Experimental Medicine the immature mouse WEHI B cells. In control experi- Cbl-b also appeared to influence the formation of stable, ments, PLC-2 was shown to be phosphorylated to simi- signaling active BCR caps after BCR cross-linking. In T / / lar levels in mature Cbl-b and Cbl-b B cells 1 min cells the polarization of the TCR after ligation and the for- after BCR cross-linking consistent with the results pre- mation of the supramolecular activation complex and the sented here. Taken together these findings suggest that immunological synapse appear to be crucial prerequisites Cbl-b and c-Cbl may provide overlapping functions in for T cell activation (44). Evidence has been provided that the regulation of Syk in mature B cells but have distinct Cbl-b influences this process by negatively regulating the targets in immature B cells resulting in both positive and coupling of the TCR to Vav-1 and downstream CDC42 negative regulation. and WASP, leading to actin cytoskeleton-dependent TCR The molecular mechanism by which Cbl-b facilitates the clustering (45). In B cells, the BCR has been shown to po- ubiquitination of Syk is not known. Ubiquitination is initi- larize after cross-linking into structures that concentrate ated by the linkage of ubiquitin (Ub) to an Ub-activating Syk, Vav, Btk, and Rac and thus appear analogous to the T enzyme, E1. Ub is then transferred by E1 to an Ub con- cell immunological synapses (46). Here we show that in B jugating enzyme, E2, that subsequently conjugates the cells from Cbl-b–deficient mice as compared with wild- COOH terminus of Ub to a lysine on the target protein type B cells the BCR remains in a signaling active Cap I through a isopeptide bond. The specificity of ubiquitina- structure for longer periods of time and less BCR is found tion is largely determined by Ub protein ligases, E3s, which condensed in signaling inactive Cap II structures. Al- interact with both E2s, through RING finger domains, and though the functional significance of the Cap II structures the E2 substrates and thus facilitate the conjugation of Ub is not known, it is possible that they represent preendocy- to the target protein (42). Members of the Cbl family are totic structures involved in the removal of signaling inac- E3 ligases that bind to the kinases targeted for ubiquitina- tive BCR from the surface. However, although Cap II tion through their TKB domains (16). The ubiquitination structures often appear near intracellular vesicles that con- of Syk by c-Cbl minimally requires the TKB and RING tain internalized BCR we show here that the intracellular finger domain of c-Cbl. The TKB domain of c-Cbl binds BCR is associated with Syk while the Cap II BCR is not. to tyrosine 317(Y317) in the linker region between the Earlier studies showed that internalization of the BCR is SH2 and the catalytic domains of mouse Syk when it is dependent on BCR signaling although the exact nature of phosphorylated following BCR cross-linking. A mutant the signals required is not known. The studies presented form of Syk that lacks the Y317 showed an enhanced abil- here suggest that BCRs may be internalized from active ity to interact with Ig suggesting that the association of Cap I structures as a part of normal down modulation of c-Cbl with Syk blocks its ability to interact with Ig and to the response. As receptors are internalized and signaling couple the BCR to downstream signaling pathways (43). dampened the BCRs remaining on the surface may con- Based on these observations a model can be proposed for dense into Cap II structures which are then internalized the regulation of BCR signaling by Cbl-b in which BCR and degraded. cross-linking results in the phosphorylation of Ig, the re- In summary, the studies presented here provide evidence cruitment of Syk to Ig through the SH2 domains of Syk that Cbl-b negatively regulates Syk through ubiquitination. and phosphorylation of Syk at multiple tyrosines including Thus, Cbl-b has the potential to block BCR signaling at an Y317. Cbl-b would bind to Syk Y317 through its TKB early step effectively uncoupling the BCR from many domain resulting in both the modulation of Syk/Ig associa- downstream signaling pathways. Consequently, it will be of tion and the ubiquitination of Syk by an E2 conjugating significant interest to determine the factors that induce and enzyme bound to the RING finger domain of Cbl-b. The regulate Cbl-b activity. ubiquitinated Syk would be subsequently degraded. Con- The authors are grateful to Dr. Michael Rosen (University of Texas sistent with this model we observe that in Cbl-b–deficient Southwestern Medical Center) for graciously providing the WASP- B cells Syk is not ubiquitinated and the phosphorylation of GBD-GFP fusion protein for analysis of CDC42 activation and to Ig and association of Ig with phosphorylated Syk is pro- Dr. Tian Jin for his invaluable guidance in obtaining the confocal longed. These events correlated with the prolonged associ- images shown here. ation of phosphorylated Syk with Vav, the prolonged Submitted: 24 September 2002 phosphorylation of BLNK, PLC-2, ERK, and JNK, in- 2 Revised: 2 April 2003 creased and prolonged Ca fluxes, and increases in the ex- Accepted: 7 April 2003 pression of the activation marker CD69. Also consistent with a model in which Cbl-b targets Syk for degradation was the observation that the activation of Lyn was not sig- nificantly affected in Cbl-b-deficient B cells. Thus, by tar- References geting Syk for degradation and modulating its association 1. Reth, M., and J. Wienands. 1997. Initiation and processing of with Ig, Cbl-b regulates several downstream signaling signals from the B cell antigen receptor. Annu. Rev. Immunol. pathways of Syk. It is of interest that the PI3K pathway was 15:453–479. apparently not influenced in Cbl-b–deficient B cells. It re- 2. Cambier, J.C. 1995. Antigen and Fc rceptor signaling. 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The Journal of Experimental Medicine – Pubmed Central
Published: Jun 2, 2003