Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells
2007; Springer Nature; Volume: 26; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601557
ISSN1460-2075
AutoresBjörn Stork, Konstantin Neumann, Ingo Goldbeck, Sebastian Alers, Thilo Kähne, Michael Naumann, Michael Engelke, Jürgen Wienands,
Tópico(s)Ion Channels and Receptors
ResumoArticle8 February 2007free access Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells Björn Stork Björn Stork Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Konstantin Neumann Konstantin Neumann Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Ingo Goldbeck Ingo Goldbeck Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Sebastian Alers Sebastian Alers Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Thilo Kähne Thilo Kähne Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany Search for more papers by this author Michael Naumann Michael Naumann Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany Search for more papers by this author Michael Engelke Michael Engelke Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Jürgen Wienands Corresponding Author Jürgen Wienands Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Björn Stork Björn Stork Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Konstantin Neumann Konstantin Neumann Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Ingo Goldbeck Ingo Goldbeck Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Sebastian Alers Sebastian Alers Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Thilo Kähne Thilo Kähne Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany Search for more papers by this author Michael Naumann Michael Naumann Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany Search for more papers by this author Michael Engelke Michael Engelke Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Jürgen Wienands Corresponding Author Jürgen Wienands Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany Search for more papers by this author Author Information Björn Stork1,‡, Konstantin Neumann1,‡, Ingo Goldbeck1,‡, Sebastian Alers1, Thilo Kähne2, Michael Naumann2, Michael Engelke1 and Jürgen Wienands 1 1Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany 2Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany ‡These authors contributed equally to this work *Corresponding author. Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Humboldtallee 34, 37073 Göttingen, Germany. Tel.: +49 (0)551 39 5812; Fax: +49 (0)551 39 5843, E-mail: [email protected] The EMBO Journal (2007)26:1140-1149https://doi.org/10.1038/sj.emboj.7601557 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok-3 (downstream of kinase-3) as a key component of negative feedback regulation in Ca2+ signaling from the B-cell antigen receptor. Dok-3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok-3 inhibits antigen receptor-induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Bruton's tyrosine kinase. This leads to diminished activation of phospholipase C-γ2 and reduced production of soluble inositol trisphosphate. Hence, the Dok-3/Grb2 module is a membrane-associated signaling organizer, which orchestrates the interaction efficiency of Ca2+-mobilizing enzymes. Introduction Development, survival and activation of B lymphocytes are tightly controlled by intracellular Ca2+ ions, which act as second messengers in a wide range of signaling pathways (Gallo et al, 2006). The regulation of Ca2+ concentrations is a key function of the B-cell antigen receptor (BCR). BCR ligation triggers elevation of intracellular Ca2+ concentrations through activation of spleen tyrosine kinase Syk and subsequent phosphorylation of the adaptor protein SLP-65 (Wienands et al, 1998) (alternatively called BLNK, (Fu et al, 1998) or BASH, (Goitsuka et al, 1998)). Phosphorylated SLP-65 recruits Bruton's tyrosine kinase (Btk) and phospholipase C-γ2 (PLC-γ2) into a trimolecular Ca2+ initiation complex (Hashimoto et al, 1999; Ishiai et al, 1999a, 1999b; Su et al, 1999; Chiu et al, 2002). This allows phosphorylation-mediated activation of PLC-γ2, which in turn hydrolyzes membrane phospholipids to yield soluble inositol trisphosphate (IP3) (Kurosaki and Tsukada, 2000). IP3 receptors are ligand-gated Ca2+ channels located in the membrane of the endoplasmic reticulum (ER), which stores intracellular Ca2+. Hence, IP3 production causes the release of Ca2+ from the ER into the cytosol. The IP3-driven intracellular Ca2+ flux is followed by entry of Ca2+ from the extracellular space through weakly characterized membrane channels (Parekh and Putney, 2005; Putney, 2005). This biphasic character of the Ca2+ response allows shaping of the Ca2+ signal in the dimensions space and time, which is thought to contribute to cell fate determination during B-cell differentiation (Dolmetsch et al, 1997, 1998). Indeed, Koncz et al (2002) and Hoek et al (2006) reported differential Ca2+ signaling in BCR-activated splenic B-cell populations, which represent distinct developmental stages and are known to respond to antigen stimulation with induction of either anergy, clonal deletion or proliferation (Niiro and Clark, 2002). Several negative regulators of the Ca2+ activation cascade have been described. Most prominently, the SH2 domain-containing 5′-inositol phosphatase (SHIP) interferes with membrane recruitment and concomitant activation of Btk or PLC-γ2 by disrupting the lipid binding motifs for the enzyme's pleckstrin homology (PH) domains at the inner leaflet of the plasma membrane (Ono et al, 1997; Bolland et al, 1998; Okada et al, 1998; Kim et al, 1999; Brauweiler et al, 2000). Also the protein tyrosine phosphatase SHP-1 and the inhibitory C-Src kinase (Csk) are implicated in the attenuation of BCR-regulated Ca2+ elevation and inhibition of cellular activation (Ono et al, 1997; Adachi et al, 2001). Our group has recently described the downmodulation of intra- and extracellular Ca2+ fluxes by the adaptor protein Grb2 (growth factor receptor-bound protein 2) (Stork et al, 2004). Grb2 is expressed in all cell types and throughout the B-cell lineage. It is composed of a central Src homology (SH) 2 domain flanked on either side by one SH3 domain (Lowenstein et al, 1992). DT40 B-cell mutants, which were rendered deficient for Grb2 expression by gene targeting (Hashimoto et al, 1998), but not their wild-type counterparts, showed a sustained biphasic Ca2+ response following BCR engagement (Stork et al, 2004). This raised the question how Grb2-positive B cells of the peripheral lymph organs mount a full Ca2+ response, which is mandatory for their antigen-mediated activation and differentiation. It turned out that stimulation-induced recruitment of cytosolic Grb2 into the lipid raft fraction of the plasma membrane prevents Ca2+ inhibition (Stork et al, 2004). Relocalization can be achieved by transmembrane adaptor proteins such as NTAL (non-T-cell activation linker), which upon tyrosine phosphorylation bind the SH2 domain of Grb2. NTAL expression is low in developing B cells showing a weak Ca2+ response, but high in mature B cells with robust Ca2+ elevation (Stork et al, 2004; Hoek et al, 2006). A number of additional transmembrane adaptor proteins with consensus binding sites for the Grb2 SH2 domain exist (Horejsi et al, 2004) and may subrogate NTAL function, for example, in NTAL-deficient mouse mutants, which possess immunocompetent B cells (Wang et al, 2005). The effector proteins that execute Grb2-mediated Ca2+ inhibition are unknown. Here we report the identification of the critical Grb2 partner for Ca2+ inhibition as the hematopoietic adaptor protein Dok-3 (Cong et al, 1999; Lemay et al, 2000). SH2-mediated recruitment of Grb2 to tyrosine-phosphorylated Dok-3 at the plasma membrane attenuates Btk-mediated PLC-γ2 phosphorylation independently of SHIP and Csk. Unlike positive Grb2 regulators with transmembraneous and palmitoylated polypeptide anchors, Dok-3 is tethered at the inner side of the plasma membrane through its PH domain. Hence, Dok-3 appears to direct Grb2 into a distinct membrane compartment. In this location Grb2 acts as a negative regulator of Btk, resulting in diminished PLC-γ2 activity. These findings exert a molecular basis for differential Ca2+ signals in B cells and moreover, directly enforce the concept that precise membrane compartmentalization of signaling elements determines positive versus negative cellular responses. Results Grb2 controls inducible phosphorylation of Dok-3, the main tyrosine kinase substrate protein in DT40 B cells To assess the signaling role of Grb2 in B cells, we analyzed BCR-induced tyrosine phosphorylation in wild-type and Grb2-deficient DT40 cells. Anti-phosphotyrosine (pTyr) immunoblotting of cleared cellular lysates revealed that the main tyrosine kinase substrate protein, migrating with an apparent molecular mass of approximately 50 kDa (p50), remains almost unphosphorylated in the absence of Grb2 (Figure 1A). The association of phosphorylated p50 with the Grb2 SH2 domain (data not shown) was employed to affinity-purify large amounts of p50 from stimulated DT40 cells in order to determine the peptide profile of tryptic digestion products by mass spectrometry (Figure 1B). The obtained peptide amino-acid sequences matched to a partial chicken EST (GenBank accession number XP_427516), which shows highest homology to the murine adaptor protein downstream of kinase-3 (Dok-3). Murine Dok-3 encompasses one PH and one PTB domain at its N-terminal end, followed by consensus tyrosine phosphorylation motifs in the C-terminal half (Lemay et al, 2000). Our cloning of the full-length avian dok-3 cDNA revealed that this overall structure is evolutionary conserved and that avian Dok-3 shares 68% and 62% amino-acid sequence homology to its murine and human orthologs, respectively (Supplementary Figure S1). The identity of p50 and Dok-3 was confirmed by anti-Dok-3 immunopurification (data not shown). Further reconstitution experiments with Grb2-deficient cells showed that efficient Dok-3 phosphorylation is independent of the N-terminal SH3 domain of Grb2, but requires the SH2 and C-terminal SH3 domains (Figure 1C, lanes 7–12). Similar to avian Dok-3, efficient tyrosine phosphorylation of murine Dok-3 is also dependent on Grb2 expression, as revealed by our analysis of Grb2-deficient mouse B-cell line Bal-17.TR and its Grb2-reconstituted transfectants (Figure 1D). As shown in Figure 1E, inducible tyrosine phosphorylation of Dok-3 is detectable in the absence of Syk (lanes 3 and 4) and Btk (lanes 5 and 6), but requires expression of the Src family kinase Lyn (lanes 7 and 8). Collectively, these data identify the intracellular adaptor protein Dok-3 as a major substrate of Src family kinases in activated B cells. The efficiency of Dok-3 tyrosine phosphorylation is, however, critically dependent on the additional presence of Grb2, which we have previously described as a negative regulator of BCR-induced Ca2+ mobilization. Figure 1.Grb2 controls Lyn-mediated phosphorylation of the adaptor protein Dok-3. (A) Wild-type (wt) and Grb2-deficient (grb2−/−) DT40 cells (lanes 1, 2 and 3, 4) were left untreated (−) or stimulated through their BCRs for 3 min (+). Equal amounts of proteins from cleared cellular lysates (CCL) were analyzed by anti-phosphotyrosine (α-pTyr) immunoblotting. (B) The major phosphotyrosine-containing protein, p50, was affinity-purified (AP) by GST-Grb2[SH2] from stimulated DT40 cells (lane 2), silver-stained, excised, digested by trypsine and peptide products were analyzed by ESI-Trap mass spectrometry. Purified proteins from unstimulated cells served as negative control (lane 1). The obtained amino-acid sequences are shown (single-letter code) with lysine (K) and arginine (R) being inferred from trypsine cleavage specificity (indicated by dots). These sequences matched a partial chicken EST (GenBank accession number XP_427516). Full-length chicken cDNA was isolated and submitted to GenBank with the accession number EF051736 (see also Supplementary Figure S1). (C) Wild-type (lanes 1 and 2) and grb2−/− DT40 cells (lanes 3 and 4) reconstituted with either wild-type Grb2 (lanes 5 and 6) or Grb2 variants, in which one of the three SH domains has been inactivated by single amino-acid substitution (N-terminal SH3 domain, P49L; SH2 domain, R86K; C-terminal SH3 domain, W193K; lanes 7–12), were left untreated (−) or stimulated through their BCRs (+). Equal amounts of proteins from CCL were subjected to anti-pTyr immunoblotting. To confirm equal loading, phospho-SLP-65 was detected separately by anti-SLP-65 immunoblotting (data not shown). (D) Murine Bal17.TR B cells, deficient for Grb2 expression, were transfected with an expression vector for Grb2 (lanes 1 and 2) or the empty vector as control (lanes 3 and 4) and left untreated (−) or stimulated through their BCRs (+). CCL were subjected to anti-Dok-3 immunoprecipitation and purified proteins were analyzed by immunoblotting with antibodies to pTyr and Dok-3 (upper and lower panels, respectively). (E) Resting (−) or BCR-activated (+) wild-type DT40 cells (lanes 1 and 2) or variants deficient for the protein tyrosine kinase Syk (lanes 3 and 4), Btk (lanes 5 and 6) or Lyn (lanes 7 and 8) were lysed and subjected to affinity purification with GST-Grb2[SH2]. Phosphorylated Dok-3 was detected by anti-pTyr immunoblotting. Relative molecular masses of marker proteins are indicated on the left in kDa. Download figure Download PowerPoint Dok-3 is a negative regulator of BCR-induced Ca2+ mobilization To functionally characterize Dok-3, we generated a Dok-3-deficient DT40 variant by gene targeting (see Materials and methods and Supplementary Figure S2A for details). Successful inactivation of dok-3 alleles and ablation of protein expression was confirmed by genomic PCR analysis (Supplementary Figure S2B) and anti-pTyr immunoblotting of cleared cellular lysates, Grb2[SH2]-purified proteins and anti-Dok-3-immunoprecipitates (Figure 2A and B; Supplementary Figure S2C). Note that Dok-3 tyrosine phosphorylation is considerably reduced in heterozygous dok-3+/− cells (Figure 2B, lanes 1–4). Figure 2.Gene targeting reveals a negative regulatory role of Dok-3. (A) Dok-3-deficient DT40 B cells were generated by targeted disruption of both dok-3 alleles (dok-3−/−, see Materials and methods for details), and absence of tyrosine-phosphorylated p50/Dok-3 in cleared cellular lysates (CCL) of resting (−) and BCR-activated (+) cells was tested by anti-pTyr immunoblotting (lanes 3 and 4). As control, wild-type DT40 and Dok-3-reconstituted dok-3−/− cells were analyzed in parallel (lanes 1 and 2 and 5 and 6, respectively). (B) Wild-type DT40 cells (lanes 1 and 2), heterozygous dok-3+/− (lanes 3 and 4) and homozygous dok-3−/− mutants (lanes 5 and 6) were left untreated (−) or stimulated through their BCRs (+). Cell lysates were subjected to affinity purification with the GST-Grb2[SH2] fusion protein and proteins so obtained were analyzed by anti-pTyr immunoblotting. Relative molecular mass of marker protein is indicated in (A) and (B) on the left in kDa. (C, D) BCR-induced intra- and extracellular Ca2+ mobilization of the indicated DT40 cells was recorded by flow cytometry as described in detail in Materials and methods. Briefly, cells were loaded with Indo-1 and release of intracellular Ca2+ was measured for 6 min in the presence of EGTA. Subsequently, extracellular Ca2+ was restored to 1mM in order to monitor Ca2+ entry across the plasma membrane. Lines represent wild-type DT40 (orange), dok-3−/− mutants (black), Dok-3-reconstituted dok-3−/− cells (gray), grb2−/− mutants (blue) and wild-type and dok-3−/− transfectants expressing the dominant-negative W193K version of Grb2 (brown and green, respectively). Data are representative of at least three independent measurements. Download figure Download PowerPoint Given the reported role of Grb2 for BCR-induced Ca2+ signaling (Stork et al, 2004), we next tested this response in various DT40 cell lines, which are positive or negative for Dok-3 or Grb2 (Figure 2C). In marked contrast to wild-type DT40 cells, Dok-3-deficient cells show a biphasic Ca2+ profile, which is almost identical to that of Grb2-deficient cells (Figure 2C, orange, black and blue lines). The monophasic Ca2+ response of wild-type DT40 cells, which is characteristic for B cells with an immature phenotype (Koncz et al, 2002; Stork et al, 2004; Hoek et al, 2006), was restored in the Dok-3 mutant cells upon reconstitution with wild-type Dok-3 (gray line). These results show that similar to Grb2, Dok-3 is a negative regulatory element of BCR-induced Ca2+ mobilization. Moreover, both adaptor proteins appear to function in a common signaling pathway. To further confirm the latter notion, we employed a dominant-negative Grb2 mutant protein, which harbors an inactivated C-terminal SH3 domain (W193K). Expression of Grb2 W193K in DT40 cells overwrote the inhibitory function of endogenous wild-type Grb2 and allowed extracellular Ca2+ influx (Figure 2D, brown and orange lines). In marked contrast, expression of the Grb2 W193K protein in Dok-3-deficient DT40 cells had no effect on the Ca2+ profile (black and green lines), which strongly suggests that Dok-3 and Grb2 build a functional unit to attenuate BCR-induced Ca2+ mobilization. Plasma membrane tethering and association to Grb2 are sufficient for Dok-3 to inhibit Ca2+ signaling To elucidate the structural requirements of Dok-3 for Ca2+ inhibition, we expressed a series of HA-tagged Dok-3 mutants (see Figure 3A) in dok-3−/− cells. Inactivation of the PTB domain (R197A) had no effect on the ability of Dok-3 to prevent extracellular Ca2+ entry (Figure 3B, left panel, green and orange lines). Deletion of the PH domain (ΔPH) abolished Dok-3-mediated Ca2+ inhibition, which resulted in a biphasic response that was indistinguishable from that observed in cells with no Dok-3 expression (left panel, red and black lines). Single and dual Y-to-F amino-acid substitutions revealed that among the consensus tyrosine phosphorylation motifs of Dok-3, only that at Y331 is essential and sufficient for Ca2+ inhibition, whereas those at Y140 and Y307 are dispensable (right panel). Immunoprecipitation with anti-HA antibodies and subsequent immunoblot analysis showed that wild-type and mutant Dok-3 proteins are expressed by the transfectants at similar levels (Figure 3C, upper panel). This setting was also used to investigate the tyrosine phosphorylation status of the various Dok-3 proteins by anti-pTyr immunoblotting (Figure 3C, second panel). Inducible phosphorylation was easily and at almost identical levels detectable for wild-type Dok-3 (lanes 3 and 4) and Dok-3 mutants R197A and Y140F and Y307F (lanes 7–12), which all promoted the same biphasic Ca2+ profile (see above). In marked contrast, the Dok-3ΔPH protein did not become phosphorylated (lanes 5 and 6), and that of the Y331F mutant was strongly diminished (lanes 13 and 14). Both of these Dok-3 mutants were unable to support Ca2+ inhibition (see above). A strongly reduced tyrosine phosphorylation was also observed for the Y331 only protein (lanes 15 and 16) that, however, was fully capable of attenuating BCR-induced Ca2+ flux (see above). Collectively, we conclude that PH domain-mediated plasma membrane localization of Dok-3 is a requisite for Ca2+ inhibition, which itself is tightly associated with Dok-3 tyrosine phosphorylation. The latter event per se appears to be necessary but not sufficient for Ca2+ regulation. Rather, specific phosphorylation at Y331 is the second key element of Dok-3-mediated Ca2+ regulation. Figure 3.Dok-3 and Grb2 build a functional unit, that inhibits Ca2+ flux independent of SHIP. (A) Schematic representation of expression constructs encoding HA-tagged versions of wild-type Dok-3, a PH domain deletion mutant (ΔPH) or mutants encompassing amino-acid exchanges depicted in single-letter code. (B) Expression vectors were introduced by retroviral transduction in dok-3−/− mutants and BCR-induced Ca2+ mobilization of the transfectants was measured by flow cytometry, as described in the legend to Figure 2. Wild-type DT40 cells and empty vector transfectants of dok-3−/− mutants served as control (see inlay for color code). (C) Wild-type and DT40 variants described in (B) were left untreated (−) or BCR-activated (+) and lysates were subjected to anti-HA immunoprecipitation. Expression and tyrosine phosphorylation of Dok-3 proteins, as well as their association to Grb2 and SHIP, were detected by sequential immunoblotting with antibodies to HA, pTyr, Grb2 and SHIP (upper to lower panels, respectively). (D) BCR-induced Ca2+ fluxes were analyzed as described in the legend to Figure 2 in SHIP-deficient DT40 cells (ship−/−, brown) and ship−/− transfectants expressing a Dok-3 Y331F variant that counteracts Ca2+ inhibition by endogenous wild-type Dok-3 (orange). As control, parental DT40 cells, which are positive for endogenous SHIP and Dok-3 (black), and the Dok-3 Y331F transfectants (gray) were analyzed in parallel, demonstrating the dominant-negative function of Dok-3 Y331F. Download figure Download PowerPoint Phosphorylation of Y331 creates a consensus binding site for the Grb2 SH2 domain. Indeed, the inducible association of Dok-3 with Grb2 was lost in cells expressing the Y331F mutant of Dok-3 (Figure 3C, third panel, lanes 13 and 14). Also the signaling-inactive ΔPH domain mutant did not co-immunoprecipitate with Grb2 (Figure 3C, third panel, lanes 5 and 6). For all other Dok-3 mutants, which retained their inhibitory capacity, BCR-induced complex formation with Grb2 was preserved (lanes 7–12). Hence, the biochemical property of inducible Grb2 association directly correlates with the functional ability of Dok-3 to downmodulate Ca2+ signals. This further demonstrates that Dok-3 and Grb2 together constitute a Ca2+-regulating signaling module. Dok-3 has been previously reported to associate with SHIP and Csk via the PTB domain and phospho-Y307, respectively (Lemay et al, 2000; Robson et al, 2004). Indeed, the R197A amino-acid exchange in the PTB domain of Dok-3 abolished SHIP binding, which moreover appeared to require specific phosphorylation at Y331 (Figure 3C, lower panel). SHIP, however, is a well-known inhibitor of BCR-induced Ca2+ elevation, and it was therefore unexpected that disruption of the Dok-3/SHIP complex had no effect on the Ca2+ response. Hence, we wanted to confirm the missing role of SHIP with a second experimental setting. For this purpose, we employed the Y331F mutant of Dok-3, which counteracted Ca2+ inhibition by wild-type Dok-3, and when expressed in DT40 cells allowed for entry of extracellular Ca2+ (Figure 3D, black and gray lines). We reasoned that if Dok-3 controls Ca2+ through SHIP, expression of the Y331F dominant-negative version in SHIP-deficient cells should have no effect on the extracellular Ca2+ influx observed in these cells (Figure 3D, brown line). However, and consistent with our mutational analysis described above, expression of the Y331F mutant in ship−/− cells strongly augmented intra- and extracellular Ca2+ mobilization (orange line). This result demonstrates that inhibition of Ca2+ signals by endogenous wild-type Dok-3 is independent of SHIP expression. Final proof that SHIP is not a major downstream effector of Dok-3 came from the biochemical analysis of SHIP itself and its downstream target, the kinase Akt (alternatively called PKB). Neither phosphorylation of SHIP nor of Akt/PKB was drastically altered in the absence of Dok-3 expression (Supplementary Figure S3A). Similar to SHIP, also the catalytic activity of the Dok-3 binding partner Csk appeared unaltered in dok-3−/− cells (Supplementary Figure S3B), which further supports our mutational analysis. In summary, SHIP and Csk are both dispensable for Dok-3-mediated regulation of Ca2+, demonstrating that these proteins do not function together in a common Ca2+ signaling pathway. PLC-γ2 is a target of Dok-3 In search for an enzymatic activity that is under the control of Dok-3, we focused on PLC-γ2. First, we tested the overall tyrosine phosphorylation status of PLC-γ2, which appeared to be very similar in the presence and absence of Dok-3 (Figure 4A). Using a site-specific antibody that detects phosphorylation of Y759 (in human PLC-γ2), we observed drastic differences between Dok-3-positive and Dok-3-negative cells (Figure 4B). The kinetic and extent of PLC-γ2 phosphorylation at this specific residue was substantially upregulated in dok-3−/− cells (lanes 5–8) compared with wild-type parental cells (lanes 1–4) or Dok-3-reconstituted transfectants (lanes 9–12). Phosphorylation of Y759 is known to be dependent on Btk and directly correlates with the enzymatic activity of PLC-γ2 (Humphries et al, 2004; Kim et al, 2004). Indeed, the hydrolysis of membrane phospholipids was more rapid and efficient in dok-3−/− cells than in reconstituted transfectants, as shown by monitoring the intracellular levels of the PLC-γ2 product IP3 (Figure 4C, left panel). The same was also true for grb2−/− cells (Figure 4C, right panel). These data identify PLC-γ2 as an effector protein of the Dok-3/Grb2 signaling module. Figure 4.The Dok-3/Grb2 module attenuates PLC-γ2 activity. (A) Dok-3-deficient DT40 mutants (lanes 4–6) and reconstituted cells expressing HA-tagged wild-type Dok-3 (lanes 1–3) were left untreated (0) or stimulated through their BCRs for the indicated times (min). Lysates were subjected to anti-PLC-γ2 immunopurification and proteins obtained were analyzed by anti-pTyr and anti-PLC-γ2 immunoblotting (upper and lower panels, respectively). (B) Parental DT40 cells (lanes 1–4), dok-3−/− mutants (lanes 5–8) and HA-Dok-3-reconstituted transfectants (lanes 9–12) were left untreated (0) or stimulated through their BCRs for the indicated times (min). Cleared cellular lysates (CCL) were subjected to immunoblot analysis with antibodies that specifically detect PLC-γ2 phosphorylation at the Btk-dependent phospho-acceptor site corresponding to Y759 in human PLC-γ2 (upper panel). Equal protein loading was confirmed by reprobing the membrane with anti-PLC-γ2 antibodies (lower panel). Relative molecular mass of marker protein is indicated in (A) and (B) on the left in kDa. (C) DT40 mutant cells deficient for either Dok-3 (left panel) or Grb2 (right panel) and the empty vector control transfectants (open and filled bars, respectively) were left untreated (0) or BCR-activated for 0.5 or 3 min. IP3 levels in these cells were measured using a competitive binding assay with radiolabelled IP3-binding proteins. Error bars represent s.e.m. of three independent experiments with double preparation. Download figure Download PowerPoint Membrane-bound Dok-3 controls BCR-induced relocalization of Grb2 Stimulation-dependent plasma membrane anchoring is a critical event for PLC-γ2 function (Nishida et al, 2003). This led us to investigate the in vivo subcellular localization of Dok-3 and Grb2 in resting and BCR-activated cells by confocal laser scanning microscopy (Figure 5). Dok-3-deficient DT40 cells were reconstituted with GFP-tagged versions of either wild-type or mutant Dok-3. Wild-type Dok-3 was constitutively and almost exclusively localized at the plasma membrane (Figure 5A, upper row). Nonetheless, BCR activation appeared to induce intra-membraneous relocalization of Dok-3, as indicated by the shift from uniform plasma membrane staining in resting cells to dotted fluorescence signals in stimulated cells. Membrane tethering was completely lost for the ΔPH mutant of Dok-3, which was homogeneously distributed in the cytoplasm of the cells (Figure 5A, middle row). The Y331F mutant of Dok-3 behaved like the wild-type protein (Figure 5A, lower row). Figure 5.Dok-3 is permanently localized at the plasma membrane and is essential for stimulation-dependent recruitment of Grb2. (A) Dok-3-deficient DT40 mutants were transfected with expression constructs encoding fusion proteins between the green fluorescence protein (GFP) at the C terminus and either wild-type Dok-3 (upper row), Dok-3ΔPH (middle row) or Dok-3 Y331F (lower row) at the N terminus. Subcellular localization of Dok-3/GFP fusion proteins in restin
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