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International Immunology Advance Access originally published online on February 17, 2008
International Immunology 2008 20(4):485-497; doi:10.1093/intimm/dxn010
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Dual role of Cbl links critical events in BCR endocytosis

Michele Jacob, Leslie Todd, Maame F. Sampson and Ellen Puré

Wistar Institute and Ludwig Institute for Cancer Research, 3601 Spruce Street, Philadelphia, PA 19104-4268, USA

Correspondence to: M. Jacob; E-mail: mjacob{at}wistar.org


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl–/– cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.

Keywords: capping, Crk, cytoskeleton, Rac


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Endocytosis can attenuate receptor-mediated signaling in various cell types. It is also essential for antigen presentation by B lymphocytes, although the mechanisms of B cell receptor (BCR) endocytosis have not been fully investigated. Antigens bound to BCR must be internalized in order to be proteolytically processed and shuttled back to the surface of B cells for presentation to cognate T cells [reviewed in (1–3)]. Depending on the conditions in which BCR ligation occurs, proximal activation of members of the Src and Syk families of protein tyrosine kinases (PTKs) then induce different effector molecules that regulate intermediate and late events to drive specific biological responses (proliferation, apoptosis or differentiation). Among the intermediate events regulated by tyrosine kinases, rearrangement of the cytoskeleton is of particular interest because it is fundamental to panoply of cellular processes. Its importance is underscored by the fact that de-regulation of actin dynamics has been repeatedly linked to cell transformation [reviewed in (4–7)]. Similarly, de-regulation of receptor endocytosis can lead to transformation. Nevertheless, the mechanisms connecting these two essential processes are still not fully understood.

Cbl has emerged as an important regulator of receptor-induced signaling in numerous cell types. Initially described as an adaptor protein, Cbl was shown to interact with various components of signaling cascades through multiple protein interaction domains (8–17). The ring finger domain was then shown to confer E3 ubiquitin ligase activity on Cbl (18–21) and attention has shifted toward the role of Cbl in receptor ubiquitylation. In fact, over-expression of Cbl and ubiquitin was shown to enhance degradation of various growth factor receptors (18, 20–24). In the case of BCR, different groups have reported an effect of Cbl on ligand-induced signaling (14, 25–27). Dragone et al. (28) studied the effect of Cbl on BCR levels. However, they only investigated ligand-independent regulation of BCR levels in the context of B cell development. The role of Cbl in ligand-induced BCR endocytosis is thus still unknown.

Although it is known that Cbl can associate with members of the Crk family and that CrkII can regulate cytoskeletal dynamics, the physiological relevance of Cbl–Crk complexes is still unclear. We hypothesized that Cbl promotes endocytosis not only by ubiquitylating receptors but also by linking receptor engagement and ubiquitylation to the re-organization of F-actin. Using the DT40 B cell line as a model system, we found that Cbl and its ring finger are required for BCR to undergo efficient ligand-induced endocytosis. Furthermore, we report the first evidence that Cbl is critical for the regulation of CrkII by tyrosine phosphorylation, the activation of Rac downstream of CrkII and Rac-mediated remodeling of cortical actin leading to BCR capping. Our data suggest that BCR-induced formation of Cbl–CrkII complexes leads to the activation of Rac which, in turn, mediates actin rearrangement. Interestingly, the requirement for Cbl to maintain the constitutive tyrosine phosphorylation of CrkII may contribute to poise the cytoskeleton to promptly respond to receptor engagement. We thus conclude that Cbl promotes BCR endocytosis through both its ubiquitin ligase and protein interaction domains: the N-terminal ubiquitin ligase domain mediates ubiquitylation of BCR [or BCR-associated molecules (26, 29)] while a C-terminal protein interaction motif mediates Cbl–CrkII complex formation, which regulates Rac-dependent actin remodeling leading to BCR capping.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cells and reagents
DT40 cells were maintained as previously described (30, 31), including the DT40Cbl–/– line which we purchased from the Riken Cell Bank (Tsukuba, Japan) (32). Monoclonal M4 antibody was purified by ammonium sulfate precipitation from serum-free hybridoma culture supernatant generated in Integra CL-350 flasks (Integra Biosciences, Ijamsville, MD, USA). Anti-Cbl (C15), anti-CrkII (C18) and anti-phosphotyrosine (PY99) antibodies, as well as protein A- and protein A/G-agarose conjugates, were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal anti-Cbl and anti-phosphotyrosine (4G10) antibodies were from Transduction Laboratories (Lexington, KY, USA) and Upstate Biotechnology (Charlottesville, VA, USA), respectively. FITC- and Texas red-labeled goat F(ab')2 anti-mouse Ig antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). FITC-labeled phalloidin was from Molecular Probes (Eugene, OR, USA) and 125I-labeled protein A was from PerkinElmer (Boston, MA, USA). Cytochalasin D and puromycin were from Sigma (St Louis, MO, USA). Latrunculin A and hygromycin B were from EMD Biosciences (San Diego, CA, USA) and Roche (Indianapolis, IN, USA), respectively. Chromerge was from Fisher Scientific (Pittsburgh, PA, USA).

cDNAs and transfections
The cDNAs coding for hemagglutinin (HA)-tagged wild-type (wt) human Cbl and 70ZCbl were kindly provided by L. Samelson (National Institutes of Health). We generated the cDNA coding for chicken Cbl by PCR. Reverse transcription was performed using 5'-GCTATTGTTACAGGTAAGAGGGAAAGCC-3' as a gene-specific primer. Cbl cDNA was then amplified with the forward primer 5'-AATCAGCGTGCGGCGTG-3' and the same reverse gene-specific primer in a typical three-step cycling program. The HA-tagged cDNAs were sub-cloned in the pApuroII or pMJ103 vector. Chicken Cbl cDNA was cloned in TOPO-XL (Invitrogen), prior to sub-cloning in pApuroII or pMJ103 vector. Resulting constructs were used to transfect DT40 cell lines, as previously described (30). Empty vectors were used as controls. Stable transfectants were selected based on drug resistance (puromycin or hygromycin B), immunoblotting and Southern blotting. At least two unrelated clones of each type were used in our experiments.

Immunoblotting and immunoprecipitation
DT40 cells were left untreated or stimulated at 37°C with 10 µg ml–1 M4 antibody for the indicated time before being lysed in PBS, pH 7.2, containing 1% NP40, 0.1% deoxycholate, 1 mM Na3VO4, 10 mM NaF, 10 mM sodium pyrophosphate, 0.4 mM EDTA and protease inhibitors. After post-nuclear extracts (15 min, 4°C, 13 000 x g) were assayed for protein concentration, immunoprecipitation was done at 4°C for 3–16 h, followed by 3 h with protein A- or protein A/G-agarose conjugates. Samples were then washed, reduced in Laemmli buffer, resolved by SDS–PAGE and transferred onto polyvinylidene difluoride membrane. Incubation with primary antibodies, secondary antibodies and 125I-protein A was done in blocking buffer (30 mM Tris–HCl, pH 7.6, 75 mM NaCl and 3% BSA), with washes (30 mM Tris–HCl, pH 7.6, 75 mM NaCl and 0.05% Tween 20) in-between steps. Tyrosine phosphorylation of total post-nuclear extracts was routinely assessed as a control for the phenotype of all cell lines.

Internalization assay by flow cytometry
Cells (2 x 107 ml–1) were washed with RPMI, resuspended and chilled on ice for 15 min. An aliquot was retained at this stage as a control for auto-fluorescence. The remaining cells were incubated with 10 µg ml–1 M4 antibody (30 min, 4°C). Cells were then washed to remove excess/unbound antibody, aliquoted and incubated at 37°C for different times prior to fixation in PBS containing 3.7% formaldehyde and 0.1% NaN3 (20 min, 4°C). Fixed cells were washed, incubated with FITC-labeled F(ab')2 goat anti-mouse Ig antibody (30 min, 4°C), washed to remove excess antibody, re-suspended in PBS and analyzed by flow cytometry using a FACSscan cytometer (Becton Dickinson, San Jose, CA, USA). Maximum fluorescence was determined at t = 0, i.e. prior to incubating the cells at 37°C to allow endocytosis. Since saturating amounts of M4 and secondary antibodies were used, a shift to lower fluorescence levels over time (compared with t = 0) indicated lower levels of BCR on the cell surface due to endocytosis. After we established the signal from auto-fluorescence (negative control) and maximum BCR labeling, the percentage of BCR internalization was calculated as follows: percentage of cells in the negative fluorescence range at a given time – percentage of cells in the negative fluorescence range at t = 0. Alternatively, mean fluorescence intensity (MFI) was used to determine the extent of BCR endocytosis. The MFI of cells prior to internalization was used to compare BCR expression in the various cell lines. Values from three independent experiments were used for the calculations. The level of BCR expression on parental cells was used as reference (100%).

Internalization assay by fluorescence microscopy
Pre-chilled DT40 cells were incubated with M4 antibody (30 min, 4°C), washed to remove excess antibodies, incubated with Texas red-labeled F(ab')2 goat anti-mouse Ig antibodies (30 min, 4°C) and washed again to remove excess antibodies before being aliquoted, incubated at 37°C for the indicated times and fixed as described above. Cells were attached to Chromerge-washed poly-L-lysine-coated glass slides (Carlson Scientific, Peotone, IL, USA) for 1 h [room temperature (RT)] in a moist chamber. Unattached cells were removed by gentle washing with PBS. Attached cells were permeabilized using 0.05% Triton X-100 in PBS–2% FCS (15 min, RT), washed with PBS and blocked in PBS–2% FCS (15 min, RT). F-actin was detected using FITC-labeled phalloidin (30 min, RT). Slides were washed, briefly air-dried and mounted with Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL, USA). Observation and analysis were done on a Leica microscope (Leica Microsystems Inc., Bannockburn, IL, USA) with IPLab 3.6 software (Scanalytics, Fairfax, VA, USA).

Rac and Ras activation assays
Rac and Ras activation was measured using affinity precipitation kits from Upstate Biotechnology. The procedure was modified as follows: 8.5–10 x 106 cells were washed with RPMI, chilled on ice (15 min) and stimulated with 10 µg ml–1 M4 (37°C) for the indicated times. The reaction was terminated by pelleting (13 000 x g, 10 s) and lysing the cells (5 min, 4°C) in lysis buffer supplemented with phosphatase and protease inhibitors. To pull down Rac, an aliquot of post-nuclear extract (2 min, 4°C, 13 000 x g) was nutated with 4 µl PAK1-binding domain agarose conjugate (30 min, 4°C). To pull down Ras, the lysate was mixed with 20 µl Raf1-binding domain agarose conjugate. Affinity complexes were then pelleted (10 s, 13 000 x g), washed with lysis buffer, reduced in Leammli buffer and processed as described above. The level of Rac and Ras activation was determined by the amount of GTP-loaded protein that was detected with anti-Rac or anti-Ras antibodies, respectively, and was normalized to the amount of total proteins. Total Rac protein was determined by immunoblotting total post-nuclear extracts with anti-Rac antibody.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Over-expression of Cbl enhances ligand-induced BCR endocytosis
The role of Cbl in endocytosis of growth factor receptors is well established. The effect of Cbl on TCR endocytosis has also been investigated by different groups (33–35). By comparison, much less is known about the role of Cbl in endocytosis of BCR. Based on reports showing that BCR-mediated activation of the PTKs Syk and Lyn results in tyrosine phosphorylation of multiple protein species, including a 120-kD species (31, 36–38), as well as pharmacologic evidence previously reported by our laboratory that activation of PTKs is required for ligand-induced BCR endocytosis (37), we used immunochemistry and identified the 120-kD species as Cbl (Fig. 1A). Furthermore, we found that the extent of tyrosine phosphorylation of Cbl in various DT40 mutant cell lines correlated with the extent of BCR internalization in these cell lines (data not shown). We therefore hypothesized that Cbl plays a role in ligand-induced BCR endocytosis, similar to growth factor receptors. To test this hypothesis, we stably transfected DT40 cells with HA-tagged cDNA coding for wt Cbl or 70ZCbl [an oncogenic mutant that has a 17-amino acid deletion within the ring finger and lacks E3 ubiquitin ligase activity (9, 18)]. We then compared the extent of BCR internalization in two or more independent clones of each type of transfectant to that of parental DT40 cells, as well as DT40 cells transfected with empty vector.


Figure 1
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  		Fig. 1. Cbl facilitates BCR endocytosis in DT40 cells. (A) Ligand-induced tyrosine phosphorylation of Cbl. Cbl was immunoprecipitated from resting (–) or stimulated (+) DT40 cells using polyclonal anti-Cbl antibodies and immunoblotted with monoclonal anti-Cbl versus anti-phosphotyrosine (pY) antibodies (top 2 panels). The bottom panel shows an anti-pY blot of post-nuclear extracts from the same samples (n > 3). (B) Over-expression of Cbl but not 70ZCbl accelerates BCR endocytosis. The internalization assay was performed as described in Methods. BCR internalization is indicated by a decrease in fluorescence intensity (FL1-H) over time. The results are representative of >10 separate experiments. (C) Quantitation of the data from B (see the Methods for details). (D) Over-expression of wt or 70ZCbl in DT40 cells. Post-nuclear extracts were immunoblotted with anti-Cbl or anti-HA antibodies (n = 3). (E) Tyrosine phosphorylation of wt Cbl and 70ZCbl. Immunoprecipitation (IP) and immunoblotting (IB) were done as in (A) (n = 3); endo: endogenous and exo: exogenous.

 
We found that the rate of BCR endocytosis was higher in DT40 cells over-expressing wt Cbl, compared with parental cells, and that the magnitude of Cbl's effect correlated with its level of over-expression (Fig. 1B–D). The enhancing effect of Cbl was remarkable at early time points but not significant at later time points (Fig. 1B and C). This indicates that over-expression of Cbl affects the kinetics but not the maximum level of BCR internalization. Alternatively, it could mean that cells over-expressing Cbl reach equilibrium between endocytosis and recycling to the cell surface faster than parental cells. In contrast, over-expression of 70ZCbl did not enhance BCR internalization (Fig. 1B and C). Furthermore, the rate of endocytosis of 70ZCbl over-expressing cells was comparable to that of parental cells, suggesting that 70ZCbl did not act as a dominant negative (DN) to inhibit the function of endogenous Cbl in BCR endocytosis.

The inability of 70ZCbl to enhance BCR endocytosis does not appear to be due to its level of expression being lower than that of exogenous wt Cbl (Fig. 1D). As discussed below, we found similar amounts of exogenous wt and 70ZCbl associated with CrkII, despite this difference in expression levels. This thus suggests that the lower level of 70ZCbl proteins was not limiting and that the lack of effect of 70ZCbl on BCR internalization is due to the enzymatically inactive ring finger in 70ZCbl. Similarly, the inability of 70ZCbl to enhance BCR internalization was not due to reduced tyrosine phosphorylation of the protein compared with exogenous wt Cbl (Fig. 1E). In fact, after normalizing for protein levels, it appears that BCR ligation induced hyper-phosphorylation of 70ZCbl, compared with wt Cbl, which was also observed in fibroblasts (38). In interpreting these data, it is important to keep in mind that the anti-Cbl antibodies used in this study were generated using peptides from the C-terminal region of human Cbl, which is not as well conserved among species as the N-terminal region. Consequently, these antibodies exhibit greater reactivity toward exogenous human Cbl, which partly accounts for the apparent extent of over-expression of the transgene product compared with the endogenous avian protein (Fig. 1D).

Interestingly, BCR expression was slightly reduced in DT40 cells over-expressing wt Cbl (DT40.Cbl #1: 86 ± 6% and DT40.Cbl #2: 70 ± 3%) but not 70ZCbl (105 ± 9%) compared with parental cells. This is likely due to a higher level of basal turnover of the BCR mediated by an increase in Cbl-associated ubiquitin ligase activity in these cells. Consistent with this conclusion, Dragone et al. (28) recently showed that over-expression of Cbl and SLAP down-regulates BCR expression in unstimulated B cells. Furthermore, we detected notable levels of tyrosine phosphorylated Cbl in unstimulated DT40.Cbl and DT40.70ZCbl cells (Fig. 1E). Therefore, our results indicate that the enhancing effect of Cbl on BCR endocytosis requires the ring finger and its inherent E3 ubiquitin ligase activity.

BCR endocytosis is delayed in Cbl–/– cells
We next investigated the impact of deleting Cbl. Importantly, BCR expression was comparable in the Cbl–/– (98 ± 7%) and parental cells. However, BCR internalization was markedly delayed in DT40Cbl–/– cells compared with parental cells (Fig. 2A) and the effect of Cbl deficiency was most obvious at early time points. At first glance, our data may seem contradictory to those by Kitaura et al. (39) who showed ablation of BCR endocytosis in Cbl-deficient murine B cells. However, they used Cbl–/–Cbl-b–/– (double knock-out) B cells and, perhaps more important, their time points were limited to 10 and 20 min. Our results thus extend their observations by showing that in the absence of Cbl, BCR endocytosis is delayed but not ablated as previously reported. Furthermore, the endocytic phenotype of Cbl–/– cells was reversible by forced expression of Cbl (Fig. 2B). By 20 min, BCR endocytosis was already substantially restored in DT40Cbl–/– cells expressing wt Cbl. By 30 min, restoration was complete. In contrast, 70ZCbl did not reconstitute the endocytic phenotype (even as late as 40 min), despite a level of expression comparable to wt Cbl (Fig. 2C). These data confirm that regulation of BCR endocytosis by Cbl is dependent on its ubiquitin ligase activity. Interestingly, the avian Cbl protein seemed more efficient at reconstituting the endocytic phenotype of Cbl–/– cells than the human counterpart. After only 10 min at 37°C, the percentage of BCR internalization of Cbl–/–.chkCbl cells was approximately three times higher than that of untransfected cells (Fig. 2D). By comparison, the enhancing effect of human Cbl was not detectable until 20 min (Fig. 2B).


Figure 2
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  		Fig. 2. BCR internalization is delayed in DT40Cbl–/– cells. (A) The percentage of BCR internalization was determined as described in the Methods. The results are representative of four separate experiments. The absence of Cbl in DT40Cbl–/– cells was confirmed by immunoprecipitation and immunoblotting as described in Fig. 1(A) (n = 3). (B) Reconstitution of DT40Cbl–/– cells with wt Cbl but not 70ZCbl restores the endocytic phenotype. Transfections with HA-tagged human cDNAs and internalization assays were done as described in the Methods. (C) Levels of exogenous wt Cbl versus 70ZCbl proteins in DT40 cell lines. (D) Cbl-deficient DT40 cells were reconstituted with chicken Cbl and assayed for BCR endocytosis as above. The results are representative of three separate experiments.

 
Cbl down-regulates BCR-induced signaling
If Cbl down-regulates receptor-induced signaling by ubiquitylating components of BCR complexes, which would mark them for endocytosis and degradation or recycling, one would expect to observe accumulation or hyper-induction of signaling proteins in Cbl-deficient cells. Consistent with this logic, we detected hyper-phosphorylation of a number of protein species in DT40Cbl–/– cells, even when comparing the extent of tyrosine phosphorylation at a time point that we determined to be optimal for parental DT40 cells (Fig. 3). The effect appears to be largely a quantitative rather than qualitative one as the species detected in Cbl-deficient cells could all be detected in parental cells after a longer exposure of the blots (data not shown). Importantly, reconstitution of DT40Cbl–/– cells with wt Cbl reversed the hyper-phosphorylation phenotype to that of parental DT40 cells, establishing a specific role for Cbl in BCR-induced signaling leading to BCR internalization (Fig. 3). These data thus confirm that Cbl contributes to the timely down-regulation of ligand-induced signaling by promoting BCR endocytosis.


Figure 3
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  		Fig. 3. DT40Cbl–/– cells exhibit protein hyper-phosphorylation. The tyrosine phosphorylation pattern was obtained by immunoblotting (IB) post-nuclear extracts from resting (–) and BCR-induced (+; 3 min, 37°C) cells with anti-phosphotyrosine (pY) antibodies (n > 4).

 
Cbl regulates F-actin dynamics underlying BCR endocytosis
To demonstrate that the more rapid removal of BCR from the surface of Cbl-expressing cells was due to endocytosis, as opposed to shedding, we used fluorescence microscopy to follow ligand-induced redistribution of BCR in DT40 and DT40Cbl–/– cells. Since Cbl has been shown to also modulate cytoskeletal events leading to cell adhesion, spreading and motility in other cell types (40–44), we hypothesized that Cbl regulates receptor internalization not only by ubiquitylating receptors but also by linking it to cytoskeletal remodeling as well. Accordingly, we investigated whether Cbl regulates the cytoskeletal dynamics during ligand-induced BCR endocytosis by dual staining of wt and Cbl knock-out cells for BCR and F-actin. These experiments not only confirmed that BCR were endocytosed at different rates in Cbl–/– and parental cells but also revealed that ligand-induced BCR capping was markedly impaired in the absence of Cbl expression. Moreover, co-localization of BCR patches and caps with F-actin was altered in Cbl-deficient cells compared with wt cells.

Specifically, wt DT40 cells exhibited significant BCR capping early after BCR cross-linking (Fig. 4A and B). Some BCR capping was even apparent in cells kept at 4°C (t = 0). Co-localization of BCR caps with F-actin was observed early after BCR cross-linking at 37°C. Consistent with our flow cytometry data (Figs 1B and C and 2 A, B and DGo), BCR endocytosis in these cells was obvious by 30 min when BCR was detected intracellularly and no longer co-localized with F-actin (Fig. 4D). This suggests that the re-organization and co-localization of F-actin with BCR is important in the early steps of endocytosis and that, once internalized, ligand-receptor complexes dissociate from cortical F-actin. Also consistent with our observations by flow cytometry, over-expression of wt Cbl in parental cells accelerated BCR capping and co-localization with F-actin (data not shown).


Figure 4
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  		Fig. 4. Cbl regulates F-actin re-organization and BCR capping. The internalization assay was performed as described in the Methods (n = 3). BCR were detected with a combination of mouse anti-chicken IgM and Texas red-labeled F(ab')2 goat anti-mouse whereas F-actin was detected with FITC-phalloidin. Co-localization of BCR with F-actin is indicated by the yellow areas. Brightness and color balance were adjusted to enhance contrast and visual quality. Portions of different fields may have been grouped to keep the image size to a minimum while being representative of a much larger field.

 
To confirm that actin polymerization is essential for BCR endocytosis in DT40 B cells, as it is in other B cell lines and primary B cells, we compared the level of BCR internalization in DT40 cells that were pre-treated (1.5 h, 37°C) with inhibitors of actin polymerization, latrunculin and cytochalasin, or vehicle control. We found BCR internalization to be dramatically reduced after pre-treatment with latrunculin (73 ± 6% inhibition) or cytochalasin (99 ± 1% inhibition). Importantly, the effects were reversed by washing out the drugs and thus not due to cytoxicity (n = 3).

Contrary to our observations in wt cells, BCR capping was consistently delayed in DT40Cbl–/– cells (Fig. 4E–H). BCR caps did not appear as compact as in wt cells at any time point, as if BCR patches never fully coalesced. BCR patches and loose caps also remained on the surface of Cbl–/– cells for a prolonged period, before being internalized at 45 and 60 min (data not shown). Moreover, co-localization of BCR caps with F-actin was rarely observed and the re-organization of actin into cortical F-actin was severely impaired in Cbl–/– cells (Fig. 4E–H) compared with parental cells (Fig. 4A–D). These data thus provide direct evidence that Cbl promotes BCR endocytosis, as opposed to shedding. More importantly, these data indicate that Cbl affects F-actin remodeling leading to BCR capping, providing the first evidence that Cbl regulates both receptor ubiquitylation and cytoskeletal re-organization in the context of receptor endocytosis.

Rac activation depends on Cbl
Rac is a small GTPase that can mediate actin re-organization and regulate endocytosis (45–47). Moreover, Yu et al. (48) showed that TCR capping is defective in Rac-deficient primary T cells. To further delineate the mechanisms by which Cbl affects BCR-mediated actin re-organization leading to BCR capping and endocytosis, we tested whether Cbl affects Rac activation downstream of BCR-mediated signaling. We compared the levels of GTP-loaded/active Rac in DT40 and DT40Cbl–/– cells. We found that there is a significant level of active Rac in unstimulated wt DT40 cells (Fig. 5A and B). Following BCR cross-linking, Rac activity transiently increased, peaking at 2.5 min (Fig. 5A and B). This suggests that Rac activation is important in the early steps of receptor signaling and/or endocytosis, which is consistent with the kinetics of BCR–actin co-localization in wt DT40 cells (Fig. 4A–D). In marked contrast, Rac activation was essentially abolished in DT40Cbl–/– cells (Fig. 5A and B), although similar levels of Rac protein were detected in Cbl-deficient and wt cells (data not shown). Interestingly, over-expression of exogenous wt Cbl did not cause further activation of Rac (Fig. 5A and B), suggesting that Rac and/or additional molecules involved in its activation by Cbl were limiting or subject to control by mechanisms that limit maximal Rac activation.


Figure 5
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  		Fig. 5. Activation of Rac is Cbl-dependent. (A) DT40 and DT40Cbl–/– cells were left untreated or stimulated for the indicated times. GTP-loaded Rac was affinity purified, immunoblotted and quantitated based on their integrated optic density (n = 3). (B) Quantitation of the data from (A). (C) DT40 cells over-expressing wt Cbl or 70ZCbl were treated and processed as in (A) to determine their levels of Rac activation. (D) Quantitation of the data from (C). (E) The levels of Ras activation in resting versus stimulated DT40 and DT40Cbl–/– cells were evaluated as described in the Methods. (F) Quantitation of the data from (E).

 
Although exogenous 70ZCbl did not reduce endocytosis mediated by endogenous Cbl and thus did not act as a DN in this process (Fig. 1B and C), it appeared to act as a DN regulator of Rac, preventing much of the transient increase in activity observed in parental and Cbl over-expressing cells. This effect of 70ZCbl is particularly evident at later time points. For example, the level of GTP–Rac at 30 min in DT40.70ZCbl cells was only half that measured in DT40.Cbl cells (Fig. 5C and D) and parental cells (Fig. 5A and B). These results thus suggest that the effect of Cbl on BCR-induced cytoskeletal remodeling requires a functional ring finger and its inherent ubiquitin ligase activity. Alternatively, it could be that hyper-phosphorylated 70ZCbl (Fig. 1E) sequesters Rac or upstream molecules involved in its ligand-dependent activation, rendering them unavailable to optimally activate Rac. In this case, the effect of 70ZCbl would not be a direct consequence of the mutated ring finger.

To determine whether the effect of Cbl on Rac was selective, compared with other small G-proteins, we measured active Ras levels in parental and Cbl-deficient cells. Considering that Ras activation did not change appreciably in parental cells and was not ablated in Cbl–/– cells (Fig. 5E and F), our data demonstrate that Cbl is essential to activate Rac but not Ras. Our data may in fact suggest that Cbl is involved in sustaining Ras activation over time, as evidenced by the lower level of active Ras in Cbl-deficient cells stimulated for 10 min compared with parental cells. In any case, these data clearly show that Rac activation depends on Cbl and further suggest that Cbl affects both components of receptor endocytosis: receptor ubiquitylation and cytoskeletal re-organization.

Cbl inducibly associates with CrkII in B cells
Cbl has been shown to associate with CrkII in other cell types but the physiological relevance of Cbl–CrkII complexes is still unclear (reviewed in (49)]. We tested the hypothesis that BCR-mediated signaling leading to endocytosis induces a physical interaction between Cbl and CrkII in co-immunoprecipitation studies. We found that Cbl indeed associates with CrkII in DT40 cells (Fig. 6). The association is transient and depends on ligand-induced signaling. While no Cbl protein was detected in CrkII immunoprecipitates from unstimulated cells (Fig. 6A), complex formation peaked between 2.5 (data not shown) and 5 min of stimulation, and then decreased at 20 min. Dissociation of the complexes over time might be due to dephosphorylation of CrkII and/or Cbl. However, we have determined in other studies (M. Jacob, L. A. Todd, R. S. Majumdar, Y. Li, K. -I. Yamamoto and E. Puré, submitted for publication) that the association of Cbl and CrkII does not require tyrosine phosphorylation of CrkII. Therefore, Cbl transiently associates with CrkII in a ligand-dependent fashion and dephosphorylation of Cbl over time likely leads to dissociation of the complexes.


Figure 6
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  		Fig. 6. BCR ligation induces the formation of Cbl-CrkII complexes. (A) DT40 and DT40Cbl–/– cells were left untreated (–) or stimulated (+) for the indicated times. Anti-CrkII immunoprecipitates were immunoblotted with anti-phosphotyrosine (pY), anti-CrkII or anti-Cbl antibodies (n = 3). Association between Cbl and CrkII was also observed after stimulating for 3 min (data not shown). (B) The ring finger of Cbl is not required for its association with CrkII. Immunoprecipitation (IP) with anti-CrkII or an isotype-matched control antibody and immunoblotting were done as in (A), using parental DT40 cells and DT40 cells over-expressing wt Cbl or 70ZCbl (n = 3). (C) Crk-binding sites in Cbl (Y700–Y774) are critical to BCR endocytosis. Cbl–/– cells were transfected with wt versus Y2FCbl and BCR endocytosis was determined as in Fig. 1. (D) Y700 and Y774 are important for the inducible association of Cbl and CrkII. Cbl–/– cells were reconstituted with wt Cbl or 70ZCbl and co-precipitation studies were done as in (A). Hyper, hyper-phosphorylated; hypo, hypo-phosphorylated and Y2FCbl, Y700F/Y774FCbl. (E) Tyrosine phosphorylation of CrkII is restored after reconstitution of Cbl–/– cells with wt Cbl.

 
Importantly, complex formation between Cbl and CrkII was observed in anti-CrkII immune complexes isolated from DT40 and DT40Cbl–/– cells transfected with wt Cbl (Fig. 6B and C, left panels). In contrast, neither Crk nor Cbl was detected in immune precipitates obtained with an isotype-matched control antibody, even in cells over-expressing wt Cbl or 70ZCbl (Fig. 6B, right panels). The association did not require the ubiquitin ligase activity of Cbl since the extent of association with CrkII was comparable in cells over-expressing wt Cbl or 70ZCbl (Fig. 6B, left panels). Furthermore, transfection of DT40Cbl–/– cells with the Y700F/Y774FCbl (Y2F) mutant demonstrated that these two residues are critical for the BCR-induced association of Cbl and CrkII (Fig. 6C), similar to what was reported for Cbl and CrkL in fibroblasts (50).

Interestingly, our investigations revealed that transfection of DT40Cbl–/– cells with the Y700F/Y774FCbl (Y2F) mutant did not reconstitute BCR endocytosis compared with parental DT40 cells or Cbl–/– transfected with wt Cbl (Fig. 6D). Considering that the ring finger is intact in the Y2F mutant, these results demonstrate that the association between Crk and Cbl is as important to BCR endocytosis as the ring finger itself. Based on these data, as well as other data from our laboratory (Jacob et al., submitted for publication), it appears that tyrosine phosphorylation of Cbl is required for the formation of Cbl–CrkII complexes and that tyrosine phosphorylation of CrkII is required for activation of Rac. We therefore suggest that Cbl–CrkII complexes couple Cbl-mediated ubiquitylation of engaged receptors and Rac-dependent capping of engaged receptors, the cooperation of which ensures efficient endocytosis.

Cbl regulates the tyrosine phosphorylation of CrkII
Our investigation of the Cbl–CrkII association revealed that the constitutive tyrosine phosphorylation of CrkII observed in parental cells is practically abolished in Cbl–/– cells (Fig. 6A, panels 2 and 4). This effect was specific to CrkII since anti-phosphotyrosine immunoblotting of post-nuclear extracts from stimulated Cbl–/– cells showed most protein species to be hyper-phosphorylated compared with wt cells (Fig. 3). Furthermore, the effect was reversed after reconstitution of Cbl-deficient cells with wt Cbl (Fig. 6E).

Since endogenous Cbl-b—which was shown to be expressed in DT40 cells (51)—apparently cannot compensate for the absence of Cbl in sustaining tyrosine phosphorylation of CrkII, it thus appears that Cbl but not Cbl-b plays an unexpected role in maintaining the basal tyrosine phosphorylation of CrkII. Cbl could act by either positively regulating the PTKs or negatively regulating the tyrosine phosphatases responsible for CrkII phosphorylation and dephosphorylation, respectively. In any case, the Cbl-dependent basal phosphorylation of CrkII may contribute to the cytoskeleton being poised to mediate prompt and efficient receptor signaling and endocytosis, following receptor engagement. Interestingly, over-expression of wt or 70ZCbl in DT40 cells accelerated the dephosphorylation of CrkII (Fig. 6B, panels 2 and 4 on the left), implicating Cbl in the regulation of CrkII by tyrosine phosphorylation for the first time (as far as we are aware). This function of Cbl is independent of the ring finger, as is the association of Cbl and CrkII (see above).


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Our data demonstrate that, although not essential for ligand-induced BCR endocytosis, Cbl strongly enhances the efficiency at which it occurs. They also establish that the ring finger-associated ubiquitin ligase activity is critical for Cbl to accelerate BCR endocytosis. Reduction rather than ablation of BCR endocytosis in Cbl–/– cells raises two possibilities: (i) BCR endocytosis occurs through a single mechanism that is enhanced by Cbl but not absolutely dependent on it and (ii) there are two pathways for BCR endocytosis, one being fast and Cbl-dependent while the other is slower and Cbl-independent. In either case, it is possible that Cbl-b partially compensated for the lack of Cbl expression. Cbl-b is indeed known to have partially overlapping functions with Cbl and to be expressed in the DT40 cell line (51). Our data clearly showed that Cbl-b cannot compensate for the absence of Cbl in mediating CrkII phosphorylation and Rac activation. However, it may have ubiquitylated components of the proximal BCR-signaling complex to signal endocytosis, albeit less efficiently than Cbl. It will be interesting to investigate this possibility by generating DT40Cbl–/–Cbl-b–/– cells. It will also be important to identify the targets of ubiquitylation in B cells. Although there is some evidence that Syk is a target of Cbl- and Cbl-b-mediated ubiquitylation in Ramos (29) and primary splenic B cells (26), respectively, the overall role of ubiquitylation of various components of the BCR-signaling complex still remains to be determined. Furthermore, future studies should investigate whether BCR and/or its ligand traffic (or are processed) differently in the presence versus absence of Cbl. This could have a major impact not only on the outcome of antigen binding to BCR in particular but also on the outcome of ligand binding to receptors in general.

We showed that, through an as yet unknown mechanism, Cbl is required for the regulation of CrkII by phosphorylation. Interestingly, the regulatory role of Cbl on the tyrosine phosphorylation of CrkII was not rescued by Cbl-b, suggesting a highly specific and physiologically important interaction between Cbl and CrkII. Given that Cbl is not a kinase, that CrkII has been shown to be a major in vivo substrate for the PTK Abl in DT40 B cells (M. Jacob, L. A. Todd, R. S. Majumdar, Y. Li, K. -I. Yamamoto and E. Puré, submitted for publication) and that Miyoshi-Akiyama et al. (52) recently reported that Cbl activates Abl by engaging Abl's SH3 domain, future studies will focus on determining whether Cbl regulates phosphorylation of CrkII through activation of Abl or, otherwise, inactivation of a tyrosine phosphatase. This mechanism implies the participation of one of Cbl's Pro-rich motifs but, as far as we know, it remains to be identified.

We also showed that the absence of Cbl has an effect on early BCR-mediated signaling. Indeed, tyrosine phosphorylation of signaling proteins is known to be one of the earliest events to occur following BCR ligation. The accumulation of tyrosine-phosphorylated species in DT40Cbl–/– cells compared with parental cells (or cells transfected with wt Cbl) thus indicates that Cbl regulates early BCR-mediated signaling. Cbl plays an essential role in the activation of Rac (a known effector of CrkII), as well as ligand-induced remodeling of F-actin leading to BCR capping. Our data are consistent with reports on a role for CrkII in Rac activation in other cell types (46–48). They are also consistent with a recent report on a role for Cbl in Rac activation leading to membrane ruffling and migration of fibroblasts (53). On the other hand, they seem to contradict the conclusions reached by Phee et al. (54). In their studies, Phee et al. used a DN Rac mutant and reported that Rac is essential for ligand-induced endocytosis of BCR. However, Debreceni et al. (55) demonstrated that the use of such DN mutants can generate misleading results because they act by sequestering upstream guanine nucleotide exchange factors that interact with multiple small GTPases. It is thus possible that over-expression of DN Rac affected the activity of other members of the family as well, leading to a more dramatic phenotype. The report from Thien et al. (56) may also seem to contradict our data as they reported constitutive activation of Rac in thymocytes isolated from Cbl–/– mice. However, this apparent discrepancy could be due to a difference in Cbl-dependent signaling between B and T cells. Alternatively, Rac activation could be regulated differently depending on the stage of maturation of the cells. Considering our data that show (i) maximal Rac activation early after BCR ligation, (ii) no Rac activation in Cbl–/– cells and (iii) impaired BCR capping in Cbl–/– cells, we conclude that Rac is involved in the process of BCR capping, which is consistent with a previous report of defective TCR capping in Rac-deficient T cells (48). Also, despite a report suggesting an effect of Cbl on the cytoskeleton in B cells (57), this paper focused on integrin-mediated signaling instead of BCR-induced signaling. Therefore, our data provide the first mechanistic evidence we are aware of linking Cbl to the regulation of CrkII by tyrosine phosphorylation, activation of Rac and cytoskeletal remodeling in the context of BCR-mediated signaling. Our data also indicate that Cbl plays a dual role in BCR signaling leading to endocytosis. In one case, the ring finger is important for Cbl to regulate signaling events occurring at the plasma membrane (ubiquitylation of BCR or BCR-associated molecules) whereas its role in modulating the cytoskeleton involves an association with CrkII and is independent of the ring finger domain. In fact, the Cbl–CrkII association depends on the same phosphorylated residues that were shown to be involved in the formation of Cbl–CrkL complexes (Y700 and Y774) in fibroblasts (50). These data imply that the SH2 domain of CrkII mediates the interaction with Cbl.

Although there may be trivial reasons as to why human Cbl was not as efficient as avian Cbl in reconstituting BCR endocytosis at early time points (e.g. different levels of over-expression), sequence divergence between species may also explain this difference. The N-terminal region of Cbl contains the ring finger domain (responsible for Cbl's ubiquitin ligase activity) and is extremely well conserved among species. By comparison, the C-terminal region of Cbl is less well conserved. In particular, the distribution of the Pro-rich motifs appears to be quite variable between species [reviewed in (58)]. Sequence alignment analysis revealed that, besides the ring finger, SH2 domain and Leu zipper that are highly conserved, all but one of the Tyr residues located in the C-terminal region of Cbl are conserved between both species (Fig. 7). These include Y700 and Y774, which we showed to be important for the association of Cbl with CrkII (Fig. 6). Similarly, the Ser residues that have been identified as phosphorylation sites in human Cbl (619/623 and 639/642) are conserved in avian Cbl. However, we found that a significant number of Pro residues are not conserved between human and avian Cbl, some of which are part of canonical PXXP motifs. Indeed, there is a PXXP motif at position 575 of avian Cbl that is not conserved in human Cbl whereas a similar motif located at position 632 of human is not found in avian Cbl. Also potentially interesting is the fact that the sequence between the conserved CrkII-binding sites in human Cbl (Y700–Y774) contains one PXXP motif whereas the corresponding region in avian Cbl (Y684–Y773) contains two overlapping PXXP motifs. Finally, avian Cbl contains 88 amino acids between the aforementioned tyrosine residues whereas human Cbl contains only 73. It is thus possible that differences in the primary structure of human Cbl reduced its interaction with the endogenous proteins.


Figure 7
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  		Fig. 7. Comparison of the amino acid sequence of human and avian Cbl. Divergent SH2 domain (blue); ring finger (red); Leu zipper (gray); Y (underlined, bold): Tyr residues involved in Cbl–CrkII association; S (brown): Ser residues known to be phosphorylated; Y (yellow): Tyr residue that is not conserved; P (green): conserved Pro residues; P (purple): Pro residues that are not conserved and PXXP (blocked sequence): Pro-rich motif that are not conserved.

 
Based on the data presented, we propose a model in which Cbl provides a critical link between receptor engagement and the re-organization of F-actin, a link that is required for prompt and efficient receptor endocytosis. According to our model (Fig. 8), BCR ligation activates the tyrosine kinases Syk and Lyn that coordinately phosphorylate Cbl (Jacob et al., unpublished observations). Tyrosine phosphorylated Cbl then binds to CrkII and—presumably—recruits it to the plasma membrane. Cbl–CrkII complexes in turn induce Rac in the proximity of BCR, which promotes re-organization of F-actin to facilitate BCR endocytosis by promoting BCR capping. Although we have not yet investigated this facet, the effect of Rac on the actin cytoskeleton could be mediated by PAK and LIMK, as was reported in other systems (58–61). It is also likely that activation of Rac in DT40 cells requires the GTP exchange factor Vav, as was described in human B cells. Vav has indeed been reported to physically associate with both Cbl and CrkII (8, 62), which further supports our proposed model.


Figure 8
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  		Fig. 8. Cbl promotes BCR endocytosis by coordinating receptor engagement to F-actin re-organization. CrkII is basally tyrosine phosphorylated in a Cbl-dependent manner. Basal levels of phospho-CrkII correlates with basal levels of Rac activity, which may poise the cytoskeleton to quickly respond to BCR ligation. Following BCR ligation, Cbl is tyrosine phosphorylated by Syk and Lyn. Phosphorylated Cbl ubiquitylates BCR (or BCR-associated signaling molecules) on one hand and associates with CrkII on the other. The association links BCR engagement to actin remodeling. ADFs, actin-depolymerizing factors; GEF, guanine nucleotide exchange factor and PTP, protein tyrosine phosphatase.

 
Our model implies that the N-terminal half of Cbl (which contains the ring finger) is required to ubiquitylate the BCR and/or proximal BCR-associated signaling molecules. On the other hand, the C-terminal half of Cbl (more specifically Y700/Y774) act as a binding site for members of the Crk family (51), thereby linking engaged BCR to the cytoskeleton. Intriguingly, the requirement for Cbl to maintain the basal phosphorylation of CrkII may be critical to keep the actin cytoskeleton poised to respond to BCR engagement once Cbl is tyrosine phosphorylated and associates with CrkII. Considering that Cbl, CrkII and Rac are ubiquitously expressed and that cytoskeletal-dependent receptor endocytosis is a hallmark of multiple cell types, this model is likely to prove relevant to many other cell types.

In summary, we showed that BCR endocytosis is delayed in Cbl-deficient B cells but not ablated as was previously reported (39). We also presented the first evidence that Cbl is essential for the regulation of the cytoskeletal adaptor CrkII by tyrosine phosphorylation, which has repercussions on the activation of Rac downstream of CrkII. Furthermore, we demonstrated that BCR ligation induces a physical association between Cbl and CrkII, as was reported in other cell systems, and more importantly, we showed that the Crk-binding sites in Cbl are as important to BCR endocytosis as Cbl's ring finger itself. This constitutes the first evidence of a direct link between receptor ubiquitylation, endocytosis and cytoskeletal re-organization. Our data thus identify Cbl as a linchpin at the junction of two pathways required for optimal receptor endocytosis and down-regulation of receptor-mediated signaling: receptor ubiquitylation and cytoskeletal rearrangement. Hopefully, these results will also help refine our understanding of the mechanisms by which de-regulation of Cbl and CrkII can lead to cell transformation.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Cancer Research Institute and National Science and Engineering Research Council of Canada to M.J.


   Acknowledgements
 
The authors are indebted to the "Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health" and the Wistar Institute Flow Cytometry Facility for their support.


   Abbreviations
 
BCR, B cell receptor
HA, hemagglutinin
MFI, mean fluorescence intensity
PTK, protein tyrosine kinase
RT, room temperature
wt, wild type

   Notes
 
Transmitting editor: D. T. Fearon

Received 8 January 2008, accepted 11 January 2008.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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