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Laboratory of Molecular Oncology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7,1 Departments of Biochemistry,2 Microbiology and Immunology,3 Medicine, McGill University, Montreal, Quebec, Canada H3G 1Y6,4 Department of Medicine, University of Montreal, Montreal, Quebec, Canada H3C 3J75
Received 22 October 2003/ Returned for modification 6 December 2003/ Accepted 11 December 2003
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INTRODUCTION |
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and Igß, two subunits bearing immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs function by recruiting several classes of cytoplasmic protein tyrosine kinases (PTKs), which phosphorylate intracellular enzymes and adaptor molecules. Such phosphorylation events cause increased levels of intracellular calcium, activation of phosphatidylinositol (PI) 3-kinase, cytoskeletal reorganization, transcriptional activation, and, finally, B-cell maturation, proliferation, and antibody secretion.
Given the high sensitivity of B cells to BCR triggering, several mechanisms exist to prevent inappropriate B-cell activation and avoid autoreactive antibodies and autoimmune diseases (7, 34, 45). These regulatory mechanisms include a large group of receptors carrying intracytoplasmic tyrosine-based inhibitory motifs termed ITIMs (immunoreceptor tyrosine-based inhibitory motifs). Such inhibitory receptors make up PD-1, which recruits Src homology 2 (SH2) domain-containing protein tyrosine phosphatases (PTPs), as well as Fc
RIIB, which binds the SH2 domain-bearing 5' inositol phosphatase SHIP-1. These two classes of phosphatases prevent B-cell activation by inhibiting critical steps in the BCR signaling cascade.
SHIP-1 is expressed mostly in hemopoietic cells, including cells of lymphoid and myeloid lineages (6, 24, 37). It acts by hydrolyzing inositol metabolites phosphorylated at the 5' position of the inositol ring, namely, PI(3,4,5)P3 and I(1,3,4,5)P4. The membrane-bound PI(3,4,5)P3 is critical for binding and membrane recruitment of pleckstrin homology (PH) domain-containing molecules like the PTK Btk, a pivotal effector of B-cell activation, and the serine-threonine-specific protein kinase Akt/PKB, a prosurvival factor. By converting PI(3,4,5)P3 to PI(3,4)P2, SHIP-1 precludes activation of these PH domain-bearing effectors and can prevent B-cell activation. In support of this idea, it has been reported that B cells freshly isolated from SHIP-1-deficient mice exhibited augmented BCR-induced proliferation (5, 12, 27). Moreover, in vivo B-cell maturation is accelerated in SHIP-1-/- animals.
The primary mode of recruitment of SHIP-1 in activated B cells is believed to involve FcRIIB (31, 32). Engagement of FcRIIB by the Fc portion of immunoglobulin G (IgG) present in immune complexes (which are generated as a consequence of productive B-cell activation) results in tyrosine phosphorylation of the ITIM of FcRIIB, thus triggering binding of the SHIP-1 SH2 domain and membrane translocation of SHIP-1. Analyses of ex vivo B cells or B-cell lines lacking SHIP-1 have provided evidence that FcRIIB-associated SHIP-1 inhibits B-cell activation by preventing BCR-induced PI(3,4,5)P3 accumulation, activation of Btk and Akt/PKB, calcium fluxes, and Erk activation (2, 4, 20, 27, 32, 39). There are also FcRIIB-independent mechanisms for recruiting SHIP-1 in B cells. In agreement with this, it has been reported that SHIP-1-deficient B cells display enhanced BCR-elicited PI(3,4,5)P3 generation and Akt activation even in the absence of FcRIIB coligation (5, 20, 27). While the exact mechanism of recruitment of SHIP-1 in this setting is not known, it likely involves interactions with other molecules. This view is also consistent with the finding that SHIP-1 can associate with intracellular adaptor molecules like Shc and Dok-related polypeptides (13, 26).
Cong et al. (10) and Lemay et al. (26) previously reported the identification of Dok-3, a member of the Dok family of adaptors expressed in B cells and macrophages. Like its relatives Dok-1 and Dok-2, Dok-3 possesses an amino-terminal PH domain, a phosphotyrosine-binding (PTB) region, and a long carboxyl-terminal segment with potential sites of tyrosine phosphorylation. Dok-3 becomes rapidly tyrosine phosphorylated in response to B-cell activation and associates by way of tyrosines in its carboxyl-terminal segment with the SH2 domains of SHIP-1 and the PTK Csk, an inhibitor of Src-related PTKs (26). Our studies demonstrated that overexpression of Dok-3 in the A20 B-cell line caused an inhibition of BCR-induced release of interleukin (IL)-2. An opposite effect was seen with expression of a mutant of Dok-3 (Dok-3 4F), in which the four carboxyl-terminal tyrosines were replaced by phenylalanines. Since this mutant was also incapable of binding SHIP-1 and Csk, it was estimated that Dok-3 4F is a dominant-interfering form of Dok-3 that blocks the action of endogenous wild-type Dok-3. Coupled with the findings that wild-type Dok-3 and Dok-3 4F were also able to regulate BCR-induced proliferation of normal B cells (J. D. Robson and A. Veillette, unpublished results), these findings led to the idea that Dok-3 is an inhibitor of B-cell activation on the basis of its capacity to recruit SHIP-1, Csk, or both.
In this study, we examined the mechanism of Dok-3-mediated inhibition in B cells. The results of our investigation showed that the inhibitory effect of Dok-3 is mediated through SHIP-1 rather than Csk. We found that the Dok-3-SHIP-1 complex functions by selectively suppressing the Jun N-terminal protein kinase (JNK) signaling cascade without affecting the activation of known targets of SHIP-1 like Akt/PKB. Finally, we demonstrated that BCR-triggered activation of JNK is enhanced in B cells lacking SHIP-1, implying that Dok-3-mediated recruitment of SHIP-1 or an analogous mechanism is a physiologically relevant mode of JNK inhibition.
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MATERIALS AND METHOD |
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For transient transfections, cDNAs were cloned into the vector pXM139, which contains the simian virus 40 origin of replication and the adenovirus major late promoter. For stable transfection in A20 cells, cDNAs were inserted into the mammalian expression vector pNT-Neo, which bears an SR-based promoter and the neo gene.
Cells and transfections Cos-1 cells were grown in alpha minimal essential medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. They were transfected by the DEAE-dextran method as described elsewhere (16). A20 B cells were propagated in RPMI medium supplemented with 10% FBS and antibiotics. Stable and transient transfections of A20 were achieved as outlined previously (26). Splenic B cells were purified by negative selection (Stem Cell Technologies, Vancouver, Canada) from 4- to 8-week-old C57BL/6 mice. The purity of the preparations was confirmed by flow cytometry, using surface IgM and IgD as markers. In all experiments, greater than 90% of cells were positive for surface immunoglobulins (data not shown). Purified B cells were then propagated in RPMI medium supplemented with 10% FBS, antibiotics, and lipopolysaccharide (LPS; 20 µg/ml) prior to experimentation (see below).
Antibodies Rabbit antisera reacting against Dok-3, SHIP-1, Csk, and FynT have been reported elsewhere (9, 14, 26). A mouse monoclonal antibody (MAb) directed against the FLAG epitope tag (M2) was purchased from Sigma-Aldrich (Oakville, Canada). Polyclonal rabbit antibodies directed against Erk-1 and Erk-2, JNK, p38, phospho-Erk (pT180/pY182), phospho-JNK (pT183/pY185), phospho-p38 (pT180/pY182), ATF-2, phospho-ATF-2 (pT69/pT71), Rac-1, and phospho-Btk (pY223) were purchased from Cell Signaling Technologies (Beverly, Mass.). Anti-phosphotyrosine MAb 4G10 was obtained from Upstate Biotechnologies (Lake Placid, N.Y). Anti-Btk MAb G149-11 was purchased from Pharmingen.
Cell stimulation A20 B cells (2 x 107 cells/ml) were stimulated with the indicated concentrations of F(ab')2 fragments of sheep anti-mouse (SAM) IgG (Jackson Immunoresearch Laboratories, West Grove, Pa.). After stimulation, cells were lysed in TNE buffer (1x TNE is 50 mM Tris [pH 8.0], 1% Nonidet P-40, and 2 mM EDTA) supplemented with protease and phosphatase inhibitors, as described in an earlier report (26). Lysates were processed for immunoprecipitation, in vitro binding assays, or immunoblotting. Mouse splenic B cells (2 x 107 cells/ml) were stimulated for the indicated periods of time with F(ab')2 fragments of goat anti-mouse (GAM) IgM (5 or 10 µg/ml) (Jackson Immunoresearch Laboratories) and lysed by addition of boiling 2x sodium dodecyl sulfate sample buffer. In some cases, A20 B cells or ex vivo B cells were stimulated with phorbol myristate acetate (PMA; 50 ng/ml).
Immunoprecipitations and immunoblots Immunoprecipitations and immunoblots were performed as described in earlier reports (14, 44). Immunoreactive products were detected using 125I-protein A (Amersham Pharmacia Biotech), horseradish peroxidase-coupled protein A (Amersham Pharmacia Biotech), 125I-rabbit anti-mouse IgG (ICN Biochemicals), or horseradish peroxidase-SAM IgG (Amersham Pharmacia Biotech). Radioactive signals were quantitated using a phosphorimager (BAS2000; Fuji).
In vitro binding assays Glutathione S-transferase (GST) fusion proteins encompassing the SH2 domain of SHIP-1 or Csk have been described elsewhere (26). A construct coding for GST fused to the Rac-binding domain of Pak was obtained from Sylvain Meloche, IRCM, Montreal, Canada. Production and purification of fusion proteins, as well as in vitro binding assays, were performed according to previously described protocols (33).
BCR-triggered IL-2 production Pools of three independent A20 B-cell clones (105 cells in 200 µl) were stimulated for 24 h at 37°C in 96-well plates in the presence of various concentrations of F(ab')2 fragments of SAM IgG. As a control, cells were stimulated with a combination of PMA (100 ng/ml) and ionomycin (1 µM). The resulting production of IL-2 was measured using a bioassay, in agreement with an established protocol (1). All cytokine secretion assays were performed in triplicate and were repeated at least three times. Representative results are shown.
BCR-induced proliferation Ex vivo splenic B cells were stimulated for 48 h in the presence of either F(ab')2 fragments of GAM IgM or LPS (20 µg/ml). They were then labeled for 6 h with tritiated thymidine to measure proliferation according to standard procedures (1).
BCR-induced activation of IL-2 promoter A20 cells (10 x 106) were transfected by electroporation with 20 µg of pIL-2 promoter-luciferase in combination with 5 µg of the indicated plasmids and 1 µg of pXM139-ß-galactosidase (to standardize for transfection efficiency). After 40 h, 2 x 106 cells were stimulated for 6 h with 10 µg of F(ab')2 fragments of SAM IgG/ml or with a combination of 100 ng of PMA/ml and 1 µM ionomycin. Cells were then lysed and assayed for luciferase activity using the luciferase reporter assay system (Promega) and a luminometer (EG&G Berthold). Luciferase activity was calculated either as the percentage of PMA plus ionomycin-induced activity or as the increase (n-fold) over what was seen with unstimulated cells. ß-Galactosidase activity was determined using a ß-gal assay system (Tropix, Bedford, Mass.). To verify expression of the Dok-3 polypeptides, equal numbers of viable cells were lysed in boiling sample buffer and probed by immunoblotting with anti-Dok-3 or anti-FLAG antibodies.
BRC-induced calcium fluxes A20 cells were loaded with the calcium indicator dye fura-2/AM. They were subsequently resuspended in calcium assay buffer (140 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl2, 1 mM MgCl2, 2 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.4]), and intracellular calcium levels were measured using a Perkin-Elmer LS50 luminescence spectrometer. After 30 s of incubation at 37°C to establish baseline values, cells were activated by addition of F(ab')2 fragments of SAM IgG (10 µg/ml). The response of cells to the calcium ionophore ionomycin (1 µM) was later tested as a control.
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RESULT |
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RIIB, CD45, and CD40 (data not shown).
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To ascertain whether Y325, Y343, or both were responsible for the inhibitory influence of Dok-3, single point mutants were analyzed (Fig. 1D). We found that mutation of Y325 alone did not prevent the inhibitory effect of Dok-3. Whereas alteration of Y343 alone alleviated to some extent the inhibitory influence of Dok-3, the impact of this mutation was not as marked as that of mutation of both Y325 and Y343. Therefore, these findings indicated that both Y325 and Y343 were involved in the inhibitory function of Dok-3 in A20 cells. It is noteworthy, though, that mutation of Y325 alone seemed to augment the capacity of Dok-3 to inhibit IL-2 production (Fig. 1D) (data not shown). While the basis for this effect is not known, this observation raised the possibility that Y325 may also carry out a stimulatory function under certain conditions.
The inhibitory effect of Dok-3 in B cells correlates with binding to SHIP-1 but not Csk To clarify the biochemical mechanism implicated in the inhibitory function of Dok-3, the tyrosine residues of Dok-3 responsible for mediating the interaction with the SH2 domain of SHIP-1 or Csk were identified. The various Dok-3 mutants detailed above were transiently expressed in Cos-1 cells in the presence or absence of an activated version of the Src-related PTK FynT to allow Dok-3 tyrosine phosphorylation. After cell lysis, the ability of the Dok-3 variants to interact with the SHIP-1 (Fig. 2A) or Csk (Fig. 2B) SH2 domain was revealed by in vitro binding assays. As reported elsewhere (26), wild-type Dok-3 interacted with the SH2 domain of SHIP-1 (Fig. 2A) or Csk (Fig. 2B) when expressed in the presence (lanes 2), but not in the absence (lanes 1), of FynT. Mutation of all four carboxyl-terminal tyrosines (lanes 3) abolished these associations. In addition, these experiments revealed that replacement of Y325 (Fig. 2A, lane 6) or Y343 (lane 7) alone was insufficient to abolish binding of Dok-3 to the SHIP-1 SH2 domain. This interaction was eliminated only when both residues were replaced (lane 4). Mutation of the two other tyrosines (Y378 and Y399, lane 5) had no effect on binding. In the case of the Csk SH2 domain, substitution of Y325 alone (Fig. 2B, lane 6), but not Y343 alone (lane 7), was found to eliminate the association with Dok-3. Therefore, these results showed that while the SHIP-1 SH2 domain bound to Y325 and Y343, the Csk SH2 region interacted only with Y325. Since mutation of Y325 alone was insufficient to abolish the inhibitory effect of Dok-3 (Fig. 1D), it is unlikely that the inhibitory function of Dok-3 in B cells was mediated by Csk. Rather, it correlated with the capacity of Dok-3 to bind SHIP-1 through Y325 and Y343.
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A) or to the kinase domain of Csk (Dok-3-Csk), were produced. All fusions carried a FLAG epitope tag at the carboxyl terminus in order to allow comparison of levels of expression.
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To expand on these findings, the active Dok-3-SHIP-1 chimera and N-Dok-3 were stably expressed in A20 cells (Fig. 5). An immunoblot analysis of representative clones by using anti-FLAG antibodies showed that the two polypeptides were expressed in equal amounts in these cells (Fig. 5A). Upon stimulation with F(ab')2 fragments of anti-IgG (Fig. 5B), cells expressing Dok-3-SHIP-1 exhibited a reduced BCR-triggered cytokine secretion in comparison to Neo cells in a manner analogous to that of clones overexpressing wild-type Dok-3. In contrast, reminiscent of cells bearing Dok-3 4F, those containing N-Dok-3 had a moderately augmented response. Nevertheless, all cells responded equally to PMA and ionomycin (Fig. 5C). Thus, combined with the results shown in Fig. 4, these findings indicated that the inhibitory impact of Dok-3 in A20 cells was mimicked by replacing the carboxyl-terminal region of Dok-3 with the active catalytic domain of SHIP-1 but not with that of Csk.
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) tyrosine phosphorylation, calcium fluxes, and cell survival. To ascertain whether Dok-3 is acting on these targets, we examined the impact of wild-type Dok-3, Dok-3 4F, and Dok-3-SHIP-1 on these parameters (Fig. 6 and data not shown). In repeated experiments, we were unable to demonstrate that Dok-3 or Dok-3-SHIP-1 had an effect on BCR-induced tyrosine phosphorylation of Btk, PLC-2, or Vav-1, induction of calcium fluxes, or activation of Akt/PKB. Likewise, no impact was noted on BCR-triggered clustering and internalization of the antigen receptor complex (data not shown). Thus, these findings indicate that the Dok-3-SHIP-1 complex may inhibit BCR signaling by acting on targets distinct from those previously shown to be regulated by SHIP-1.
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Enhanced JNK activation in B cells derived from SHIP-1-deficient mice To examine the physiological relevance of these findings, we assessed whether lack of SHIP-1 in B cells had an impact on the extent of JNK activation induced by BCR stimulation by using B cells derived from SHIP-1-deficient mice (Fig. 8). It should be pointed out first that a relative increase in the abundance of mature (IgMlo/IgDhi) over immature (IgMhi/IgDlo) splenic B cells is found in SHIP-1-/- animals (5, 20). One potential problem stemming from this difference is that the B-cell populations isolated from SHIP-1-deficient mice are not exactly comparable to those from wild-type animals. To circumvent this problem, splenic B cells from SHIP-1+/+ and SHIP-1-/- mice were subjected to an initial in vitro stimulation with LPS. Our preliminary studies had indicated that this treatment corrected the difference in relative abundance of immature and mature B cells between the two types of animals (Fig. 8A) (Robson and Veillette, unpublished). Whereas the basis for this effect remains to be clarified, it allows a more adequate comparison of B cells from wild-type and SHIP-1-deficient mice.
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RIIB-independent function of SHIP-1, B cells were stimulated with F(ab')2 fragments of anti-IgM, which trigger the antigen receptor without engaging FcRIIB. After 48 h, proliferation was determined by measuring tritiated thymidine incorporation. This analysis showed that lack of SHIP-1 caused an enhancement of B-cell proliferation in response to BCR engagement. The magnitude of the increase seen in these LPS-propagated B cells was similar to that previously described for freshly isolated SHIP-1-deficient B cells (5, 20, 27). By contrast, SHIP-1 deficiency had no impact on the proliferative response to LPS (Fig. 8C). Therefore, these findings confirmed that SHIP-1 had an inhibitory role in B cells in the absence of ligation of FcRIIB and established that LPS pretreatment did not alleviate this effect.
Next, LPS-induced B cells were stimulated with F(ab')2 fragments of anti-IgM and the activation of JNK, Erk, and p38 was monitored as outlined above (Fig. 8D). This study revealed that BCR-evoked activation of p54JNK and, to a lesser extent, p46JNK (top block) was noticeably augmented in B cells from SHIP-1-/- mice (lanes 5 to 7) in comparison to what was seen with cells obtained from SHIP-1+/+ animals (lanes 1 to 3). However, both cell types responded in an equivalent manner to PMA (lanes 4 and 8). Contrary to the impact of SHIP-1 deficiency on JNK activation (Fig. 8D, top block), there was no appreciable effect on Erk (middle block) or p38 (bottom block) activation. The lack of impact of SHIP-1 deficiency on Erk activation was in keeping with the finding of Liu et al. (27). These various effects were not caused by differences in the levels of expression of the kinases, as demonstrated by immunoblotting of parallel cell lysates with the relevant antibodies (Fig. 8E). Thus, in conclusion, these results supported the idea that recruitment of SHIP-1 by one or more mechanisms independent of FcRIIB is implicated in selective inhibition of JNK in normal activated B cells.
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DISCUSSION |
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These data indicated that the inhibitory role of Dok-3 correlated with the capacity of Dok-3 to bind SHIP-1. To provide more direct evidence supporting this model, various Dok-3-based chimeric proteins were created and studied. We found that fusion of the PH and PTB regions of Dok-3 (the presumed intracellular targeting domains of Dok-3) to the active catalytic domain of SHIP-1, but not to the catalytic region of Csk, was able to reestablish efficiently the inhibitory effect of Dok-3 in A20 cells. Because the Dok-3-SHIP-1 chimera did not encompass regions outside the catalytic domain of SHIP-1, such as its SH2 domain or its sites of tyrosine phosphorylation, it is likely that the impact of Dok-3-SHIP-1 was due to Dok-3-mediated recruitment of the catalytic activity of SHIP-1. In agreement with this, we found that the effect of Dok-3-SHIP-1 was abolished by mutation of a conserved residue required for SHIP-1 catalytic activity (D672A mutation) (22). Furthermore, the biochemical consequences of expression of wild-type Dok-3 and Dok-3-SHIP-1 in A20 cells were identical.
Indeed, our biochemical studies demonstrated that Dok-3 and the Dok-3-SHIP-1 chimera inhibited very specific targets in BCR signaling. Both polypeptides had no effect on BCR-induced overall protein tyrosine phosphorylation or tyrosine phosphorylation of PLC-2 and Vav-1 (Fig. 6 and data not shown). Likewise, they failed to have an impact on the BCR-triggered activation of Btk and Akt/PKB or the induction of calcium fluxes, which are known to be regulated by FcRIIB-associated SHIP-1 (2, 4, 31, 32, 39). Surprisingly, however, Dok-3 and Dok-3-SHIP-1 caused an inhibition of BCR-induced phosphorylation and activation of JNK, while they had no effect on other MAPKs like Erk-1, Erk-2, and p38. Conversely, the dominant-negative Dok-3 4F induced an increase in JNK activity in activated B cells. Similar effects were seen on ATF-2, a known downstream target of JNK. Therefore, these results implied that the Dok-3-SHIP-1 complex was acting by selectively inhibiting the JNK pathway. This notion is also supported by the finding that B cells derived from SHIP-1-deficient mice exhibited an augmentation of BCR-elicited activation of JNK but not Erk-1, Erk-2, and p38. While this last observation did not establish that Dok-3 per se was responsible for recruitment of SHIP-1 leading to JNK regulation in normal B cells, this was nevertheless consistent with the notion that non-FcRIIB-interacting SHIP-1 was implicated in selective down-regulation of JNK activity.
Like other MAPKs, JNKs are activated through phosphorylation by upstream kinases consisting of MAPK kinases MKK-4 and MKK-7 as well as several MAPK kinase kinases like MEKKs and MLKs (15, 23). However, we were unable to detect an impact of Dok-3 on the phosphorylation state of MKK-4 (data not shown). It has also been reported that PLC-2 and Rac-1 can be involved in the activation of JNK in BCR-stimulated B cells (11, 18, 21). Once again, though, we observed no effect of Dok-3 on PLC-2 tyrosine phosphorylation or Rac-1 activity. Whereas it is possible that the Dok-3-SHIP-1 complex acts on a restricted pool of these JNK regulators that cannot be identified with the currently available assays, we favor the idea that Dok-3-SHIP-1 affects less conventional or as-yet-unknown activators of the JNK pathway. In particular, it may prevent the PI(3,4,5)P3-dependent membrane recruitment of a PH domain-containing enzyme or scaffold molecule involved in JNK activation. This possibility will deserve future consideration.
The ability of SHIP-1 to bind various types of docking proteins suggests that it carries out diverse functions in immune cells. As reported elsewhere, by interacting with FcRIIB, SHIP-1 can inhibit the BCR-induced activation of Btk and Akt/PKB, thus causing diminished PLC-2 tyrosine phosphorylation and calcium fluxes, as well as reduced cell proliferation and survival (2, 4, 20, 27, 32, 39). Once soluble antibody secretion is initiated during the course of an immune response, this signal is presumed to prevent sustained B-cell activation in vivo. In striking contrast, by way of its association with Dok-3, SHIP-1 selectively inhibits the JNK cascade in activated B cells without measurably affecting the Btk and Akt/PKB pathways (this report). This effect is accompanied by an inhibition of BCR-induced cytokine production and proliferation (26; Robson and Veillette, unpublished). Thus, the Dok-3-SHIP-1 complex constitutes an alternative negative feedback mechanism that may be important for terminating B-cell activation in the absence of FcRIIB coligation. It is also possible that Dok-3 qualitatively modifies the outcome of B-cell activation. Along these lines, Healy et al. reported that JNK activation is selectively deficient in tolerant, but not naïve, B cells (19). Hence, Dok-3-associated SHIP-1 may promote B-cell tolerance.
One of the most surprising results observed in the present study is that activation of Btk and Akt was not detectably inhibited by Dok-3-associated SHIP-1. Given that binding of the PH domain of Btk and Akt to PIP3 appears to be critical for their activation in response to BCR stimulation, this finding suggests that Btk and Akt may be physically protected from the action of Dok-3-SHIP-1. In all likelihood, Dok-3 and its associated SHIP-1 are targeted to an intracellular compartment distinct from those occupied by Btk and Akt. Although less likely, it is also conceivable that the substrate specificity of Dok-3-associated SHIP-1 is distinct from that of FcRIIB-interacting SHIP-1. For example, while the SHIP-1 molecules recruited by FcRIIB seem to target PI(3,4,5)P3, Dok-3-associated SHIP-1 may hydrolyze the 5' phosphate of an alternative inositol phospholipid implicated in regulation of the JNK pathway. Obviously, future studies are needed to address these issues.
In addition to identifying SHIP-1 as a negative regulator of JNK signaling in B cells, our results provide evidence of the functional importance of JNK in B-cell activation. While little is known about the role of JNK in B cells, others have reported that B-cell differentiation and functions seemed to be unaffected in mice lacking expression of single jnk genes (38, 46). While one interpretation of these findings is that JNK proteins have no significant role in B cells, it is likely that there is functional redundancy between the various JNKs as well as JNK-related molecules such as p38. JNKs may also have a more specialized function in B cells. For instance, as suggested above, they may be involved in protecting B cells against tolerance induction. Clearly, based on these and our findings, more should be done to address the role of JNK in B cells.
SHIP-1 interacts not only with Dok-3 but also with other adaptors such as Dok-1, Dok-2, and Shc (13, 26). Interestingly, it has been reported that, like Dok-3, Dok-1 and Dok-2 can inhibit receptor-mediated signaling events in immune cells, including T cells and B cells (17, 30, 40, 41). In light of this, it is appealing to propose that the inhibitory effect of Dok-1 and Dok-2 in these cells is caused at least in part by SHIP-1 and inhibition of JNK signaling. Although additional evaluations are required to test this possibility, the strong sequence similarities between the Dok-related molecules suggest that this may well be the case.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the National Cancer Institute of Canada. J.D.R. held a Studentship from the CIHR, while A.V. is a Senior Scientist of the CIHR and holds a Canada Research Chair in Immune Cell Signaling.
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FOOTNOTES |
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