Down-regulation of B Cell Receptor Signaling by Hematopoietic Progenitor Kinase 1 (HPK1)-mediated Phosphorylation and Ubiquitination of Activated B Cell Linker Protein (BLNK)*

Background: HPK1 is a hematopoiesis-specific Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. Results: HPK1 knock-out B cells show loss of Thr-152 phosphorylation, 14-3-3 binding, and Lys-37/38/42-ubiquitination of BLNK. Conclusion: HPK1 attenuates BCR signaling via inducing phosphorylation and ubiquitination of BLNK. Significance: This is the first report of BLNK ubiquitination that is initiated by HPK1-induced phosphorylation. Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. B cell receptor (BCR) signaling activates HPK1 by inducing BLNK/HPK1 interaction. Whether HPK1 can reciprocally regulate BLNK during BCR signaling is unknown. Here, we show that HPK1-deficient B cells display hyper-proliferation and hyper-activation of IκB kinase and MAPKs (ERK, p38, and JNK) upon the ligation of BCR. HPK1 attenuates BCR-induced cell activation via inducing BLNK threonine 152 phosphorylation, which mediates BLNK/14-3-3 binding. Furthermore, threonine 152-phosphorylated BLNK is ubiquitinated at lysine residues 37, 38, and 42, leading to attenuation of MAPK and IκB kinase activation in B cells during BCR signaling. These results reveal a novel negative feedback regulation of BCR signaling by HPK1-mediated phosphorylation, ubiquitination, and subsequent degradation of the activated BLNK.

B lymphocytes produce antibodies that play an important role in eliminating pathogenic challenge or, conversely, causing autoimmune diseases (1). B cells are activated by B cell receptor (BCR) 3 signaling via a highly ordered signaling cascade (2). The recognition of antigens by BCR rapidly induces the activation of cytoplasmic Src family protein-tyrosine kinases FYN and LYN. Followed by the activation of FYN and LYN, immunoreceptor tyrosine kinase-based activation motifs of Ig␣ and ␤ chains are phosphorylated, which in turn recruit the tyrosine kinase SYK. Activated SYK then induces tyrosine phosphorylation of B cell linker protein (BLNK, also named SLP-65), which acts as a central adaptor to assemble multiple signaling molecules, including phospholipase C␥2 (PLC␥2), Bruton tyrosine kinase, GRB2, and hematopoietic progenitor kinase 1 (HPK1) (3). Bruton tyrosine kinase activates PLC␥2, which in turn stimulates the PKC␤-IKK-NF-B cascade (4). GRB2 facilitates the activation of RAS-RAF-MEK-ERK signaling (5), but the role of HPK1 in BCR downstream signaling remains unclear.
HPK1 (also named MAP4K1) is a Ste20-like serine/threonine kinase and is mainly expressed in hematopoietic cell compartments (6,7). Because HPK1 specifically activates JNK through the signaling cascade MAP3K (e.g. MEKK1, TAK1, or MLK3) to MAP2K (e.g. MKK4 or MKK7) in overexpression systems (6 -11), HPK1 belongs to the MAP4K subfamily that also includes the other five members GCK/MAP4K2, GLK/ MAP4K3, HGK/MAP4K4, GCKR/MAP4K5, and MINK/ MAP4K6 (12)(13)(14). In addition to regulating the JNK pathway, HPK1 negatively regulates AP-1 and ERK activation in T cell receptor signaling (7,10). The role of HPK1 in NF-B activation is complex. HPK1 binds to and phosphorylates CARMA1, resulting in the activation of IKK␤/NF-B (15,16). In addition, HPK1 can be processed by caspases into the N-terminal kinase domain and C-terminal regulatory domain in apoptotic cells (17); the cleaved HPK1 C-terminal fragment has a suppressive activity on NF-B activation (18). The in vivo roles of HPK1 are demonstrated using HPK1-deficient mice that show enhanced immune responses upon antigen immunization and are more susceptible to experimentally induced autoimmune encephalo-myelitis (10). Furthermore, HPK1 attenuation may also promote human autoimmune diseases, as the HPK1 expression level is down-regulated in peripheral blood mononuclear cells of human patients with psoriatic arthritis or systemic lupus erythematosus (19 -21).
We investigated the role of HPK1 in the regulation of BCR signaling. Our results demonstrate that HPK1 attenuates BCRinduced B cell activation by inducing BLNK Thr-152 phosphorylation and subsequent BLNK/14-3-3 binding; more importantly, we show that Thr-152 phosphorylation induces ubiquitination and proteasomal degradation of the activated BLNK. Furthermore, Lys-37, Lys-38, and Lys-42 are identified as BLNK ubiquitination sites, which attenuate ERK, JNK, and IKK activation during BCR signaling.

EXPERIMENTAL PROCEDURES
Mice-C57BL/6 (B6) WT and HPK1-deficient mice were bred in a specific pathogen-free environment within the Transgenic Mouse Facility at Baylor College of Medicine. All animal experiments were done according to institutional guidelines and regulations.
B Cell Purification, Stimulation, and in Vitro Proliferation-B cells were purified from splenocytes using MACS columns (Miltenyi Biotec) according to the manufacturer's instructions. The purity of B cells (CD19 ϩ cells) was more than 95% as determined by flow cytometry. For in vitro B cell proliferation, purified B cells (1 ϫ 10 5 or 2 ϫ 10 5 cells/well) were stimulated with different doses of anti-IgM F(abЈ) 2 or LPS for 40 -64 h in 96-well plates. In the final 16 h of cell culture, the cells were pulsed with [ 3 H]thymidine (1 Ci/well). To study BCR signaling, purified B cells were either untreated or incubated with 10 g/ml biotin-labeled goat anti-mouse IgM F(abЈ) 2 for 15 min on ice and then stimulated by BCR cross-linking by using 20 g/ml streptavidin and incubating in a 37°C water bath. The stimulation was immediately stopped by cold spin, and cells were lysed using the lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyro-phosphate, 1 mM ␤-glycerophosphate, 0.5 mM PMSF, 5 g/ml leupeptin, 5 g/ml aprotinin, and 1 mM Na 3 VO 4 ) for 30 min on ice.
Cell Transfections-HEK293T cells were cultured in DMEM plus 10% FBS and transfected using calcium phosphate precipitation (Specialty Media Inc.) according to the manufacturer's instructions. HEK293T cells were harvested for experiments 40 h after transfection. BLNK-deficient chicken DT40 B cells (RIKEN BRC, Japan) were cultured in RPMI 1640 medium plus 10% FBS, 1% chicken serum, and 50 M 2-mercaptoethanol at 39.5°C. BLNK-deficient DT40 B cells were transfected using Neon TM transfection system (Invitrogen) with the setting of 1435 V, 30 ms, and 1 pulse. WEHI-231 B cells were cultured in RPMI 1640 medium plus 10% FBS and transfected using Neon TM (1375 V, 40 ms, 1 pulse).
GST Pulldown, Co-immunoprecipitations, and Western Blotting-GST-14-3-3 or GST proteins were incubated with 400 -800 g of cell lysates at 4°C for 2 h and immobilized on glutathione-Sepharose 4B beads for 45 min at 4°C. Beads were washed three times in cell lysis buffer and then boiled in 2ϫ SDS loading buffer for 5 min. For co-immunoprecipitation, 2-5 g of antibody and 400 -600 g cell lysate were mixed and rotated on a rocker at 4°C for 2 h, followed by adding 30 l of protein-A/G plus conjugated agarose beads (Sant Cruz Inc.) and incubating for 1 h. Beads were washed four times with cell lysis buffer and boiled in 20 l of 2ϫ SDS loading buffer. Samples were resolved by SDS-polyacrylamide gel, transferred to a PVDF filter, and subjected to Western blotting analyses for the detection of interested molecules.
Protein Denaturation and Re-immunoprecipitation-To determine BLNK ubiquitination, HEK293T cells overexpressing FLAG-BLNK and HA-ubiquitin were lysed and boiled in the presence of 1% SDS. Boiled cell lysates were diluted 10-fold to reduce SDS concentration, followed by immunoprecipitation with anti-FLAG antibody. The ubiquitination of BLNK was determined by Western blotting using anti-HA antibody.
Liquid Chromatography-Mass Spectrometry (LC-MS/MS)-Specific protein bands from silver-stained SDS-polyacrylamide gels were excised, destained, and digested with trypsin. The resulting peptide mixtures were loaded onto a 75-m ϫ 250-mm nanoACQUITY UPLC BEH130 column packed with C18 resin (Waters, Milford, MA) and separated using a segmented gradient in 110 min from 5 to 85% solvent B (95% acetonitrile with 0.1% formic acid) at a flow rate of 300 nl/min. Solvent A was 0.1% formic acid in water. The samples were maintained at 4°C in the autosampler. The LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) was operated in the positive ion mode with the following acquisition cycle: a full scan (m/z 350 -1600) recorded in the orbitrap analyzer at resolution R 60,000 was followed by MS/MS of the 20 most intense peptide ions in the LTQ analyzer. All MS and MS/MS raw data were processed by Raw2MSM and searched against a target protein sequence data base using the Mascot Daemon 2.2 server. Search criteria used were as follows: trypsin digestion; variable modifications set as carbamidomethyl (Cys), oxidation (Met), phosphorylation (Ser, Thr, Tyr), and ubiquitination (Lys); up to two missed cleavages allowed; and mass accuracy of 15 ppm for the parent ion and 0.60 Da for the fragment ions.
Statistical Analysis-Statistical analysis was performed using the unpaired Student's t test.

HPK1 Attenuates BCR-induced B Cell
Activation-To study whether HPK1 regulates B cell development, different B cell populations in the spleen were analyzed. There was no significant difference between WT mice and HPK1-deficient mice in transitional 1 (T1), T2, marginal zone, and follicular B cells (Fig.  1A), suggesting that HPK1 is not essential for B cell development. To study the role of HPK1 in peripheral B cell activation, we isolated splenic B cells and studied in vitro B cell proliferation following anti-IgM and LPS treatment, which activates BCR and Toll-like receptor 4 signaling, respectively. B cell pro- liferation was significantly enhanced in HPK1-deficient B cells upon anti-IgM but not LPS stimulation (Fig. 1B). Similarly, B cell activation markers, including CD69 and CD25, were significantly increased in HPK1-deficient B cells by anti-IgM but not LPS stimulation (Fig. 1C). The enhanced B cell activation in HPK1-deficient B cells was not due to the increased expression of BCR in the cell surface (supplemental Fig. 1A). These data suggest that HPK1 regulates BCR intracellular signaling. Thus, we further determined the activation of intracellular BCR sig-naling in wild-type (WT) and HPK1-deficient splenic B cells. The phosphorylation of MAPKs (ERK, p38, and JNK) was all significantly enhanced in HPK1-deficient B cells upon anti-IgM stimulation; IKK phosphorylation and IB degradation, which lead to NF-B activation, were also significantly enhanced in HPK1-deficient B cells (Fig. 2, A and B, and supplemental Fig. 1, B and C). In addition, we determined the phosphorylation of BCR proximal signaling molecules SYK, BLNK, and PLC␥2. SYK phosphorylation was not affected in HPK1-deficient B  Fig. 1, D and E); however, BLNK, which is downstream of SYK, displayed significantly enhanced tyrosine phosphorylation in HPK1-deficient B cells (Fig. 2, C and D). PLC␥2, a downstream molecule of BLNK, also showed some enhancement of tyrosine phosphorylation in HPK1-deficient B cells (Fig. 2, C and D). An important outcome of PLC␥2 activation is calcium flux, which leads to activation of MAPKs, NF-AT, and NF-B. Consistently, HPK1-deficient B cells showed increased calcium flux in response to anti-IgM stimulation (Fig. 2E). Taken together, these data suggest that HPK1 may target BLNK to attenuate BCR signaling and peripheral B cell activation.
We further studied the functional significance of BLNK Thr-152 phosphorylation using DT40 B cells, which is a transformed chicken B cell line with a high frequency of homologous gene targeting (25). Anti-chicken IgM stimulation of DT40 B cells induces signaling cascades that are analogous to BCR signaling cascades in murine and human B cells (26). BLNK-deficient DT40 mutant B cells were further generated by gene targeting (27,28); these BLNK-deficient DT40 mutant B cells display a defective activation of MAPKs (ERK, p38, and JNK) upon antichicken IgM stimulation (27,28). We transfected murine FLAG-tagged BLNK WT or T152A mutant, both of which were subcloned into pIRES2-EGFP vector simultaneously expressing GFP, into BLNK-deficient DT40 mutant B cells. Transient expression of murine BLNK WT partially restored BCR-induced activation of MAPKs and IKK in BLNK-deficient DT40 mutant B cells (Fig. 5, A and B). As expected, BLNK-deficient DT40 mutant B cells that were transfected with the BLNK T152A mutant showed significantly enhanced phosphorylation of all three MAPKs and IKK (Fig. 5, A and B). BLNK T152A mutant also displayed significantly enhanced tyrosine phosphorylation in the transfected BLNK-deficient DT40 mutant B cells (Fig. 5, C and D). Consistent with the role of BLNK Thr-152 phosphorylation in mediating 14-3-3 interaction in HEK293T cells, the BLNK T152A mutant showed drastically reduced binding with 14-3-3 in BLNK-deficient DT40 mutant B cells upon anti-IgM stimulation (Fig. 5, C and D).
Thr-152 Phosphorylation Induces BLNK Ubiquitination in B Cells-Next, we aimed to delineate the underlying mechanism of BCR signaling attenuation by HPK1-induced Thr-152 phosphorylation and subsequent 14-3-3 binding of BLNK. 14-3-3 proteins may regulate the functions of its binding partners through different mechanisms as follows: (i) change the conformation of its binding proteins, leading to the change of enzymatic properties or protein interactions of the binding part-

. Thr-152 is phosphorylated by HPK1 and mediates BLNK/14-3-3 interaction.
A, schematic diagram of identification of BLNK/14-3-3 binding region. B, interaction of BLNK fragments and 14-3-3 in HEK293T cells was determined by GST-pulldown assays. C, T152A mutation abolishes HPK1-dependent BLNK/14-3-3 interaction in HEK293T cells. Full-length FLAG-BLNK mutants containing S141A, S151A, or T152A mutation were transfected into HEK293T cells with HPK1. The interaction of FLAG-BLNK mutants with GST-14-3-3 was detected by GST-pulldown assays. D, HPK1 induces BLNK Thr-152 phosphorylation in HEK293T cells. FLAG-tagged BLNK WT or T152A mutants were transfected into HEK293T cells with HPK1. FLAG-BLNK was immunoprecipitated with anti-FLAG antibody and Western-blotted with anti-p-BLNK(Thr-152) antibody. E, BCR signaling induces BLNK Thr-152 phosphorylation in WT but not HPK1-deficient B cells. Purified splenic B cells were stimulated with anti-IgM. The phosphorylation of BLNK Thr-152 was detected by Western blotting (WB) with anti-p-BLNK(Thr-152) antibody. Data are representative of three independent experiments. ners; (ii) physically block the interaction of other proteins with 14-3-3 binding partners; and (iii) function as an obligatory dimer and may bridge an interaction between two proteins, each associated with a 14-3-3 monomer (29,30). As BLNK interactions with PLC␥2 and other signaling molecules are important for the activation of BCR downstream signaling (3), we first studied whether HPK1 regulates BLNK and PLC␥2 signaling complexes. The anti-IgM-induced BLNK-and PLC␥2interacting complexes were not changed in HPK1-deficient B cells upon anti-IgM stimulation (Fig. 6A). We also studied whether the recruitment of 14-3-3 to the activated BLNK led to BLNK degradation. The proteasomal inhibitor MG132 Statistical analysis was conducted on the data derived from three independent experiments. *, p Ͻ 0.05, Student's t test. C, BLNK T152A mutant shows reduced 14-3-3 binding and increased tyrosine phosphorylation in BLNK-deficient DT40 B cells. BLNK-deficient DT40 B cells were transfected as described above. BLNK was immunoprecipitated by anti-FLAG antibody followed by Western blotting (WB) with anti-14-3-3 or anti-p-BLNK(Y84) antibody. D, quantification of BLNK/14-3-3 interaction and BLNK tyrosine phosphorylation for C.
increased the protein levels of the phosphorylated BLNK but not total BLNK in splenic B cells upon anti-IgM stimulation (Fig. 6, B and C). This suggests that the phosphorylated BLNK is targeted for ubiquitination and degradation during BCR signaling.
As BLNK ubiquitination has not been previously reported, we studied whether FLAG-BLNK can be ubiquitinated in HEK293T cells by co-transfection with HA-tagged ubiquitin. Anti-FLAG-BLNK immunoprecipitation from the transfected HEK293T cells resulted in the co-immunoprecipitation of ubiquitinated proteins above 78 kDa that likely represented multiubiquitinated or polyubiquitinated BLNK species (supplemental Fig. 2). Similar results were shown when utilizing GST-tagged BLNK, which is 26 kDa higher than FLAG-tagged BLNK (supplemental Fig. 2). To confirm that these ubiquiti-nated species were BLNK, we denatured anti-FLAG-BLNK immunoprecipitates to dissociate BLNK-interacting proteins and then re-immunoprecipitated FLAG-BLNK after renaturation. Ubiquitinated bands were detected both in the first and second anti-FLAG-BLNK immunoprecipitates (Fig. 6D), indicating that BLNK itself is ubiquitinated. To study whether BLNK was ubiquitinated in primary B cells during BCR signaling, we immunoprecipitated endogenous BLNK from WT and HPK1-deficient splenic B cells upon anti-IgM stimulation. BLNK ubiquitination was detected in WT splenic B cells but not HPK1-deficient B cells after anti-IgM stimulation for 3 min (Fig. 6E).
Because HPK1 induces BLNK Thr-152 phosphorylation and subsequent BLNK/14-3-3 binding, we next studied whether BLNK Thr-152 phosphorylation regulated BLNK ubiquitina-FIGURE 6. Characterization of BLNK ubiquitination. A, HPK1 deficiency has no discernible effect on the assembly of BLNK-and PLC␥2-interacting complexes. Splenic B cells were isolated from WT and HPK1-deficient mice, stimulated with anti-IgM, and subjected to immunoprecipitation (IP) using anti-BLNK or anti-PLC␥2 antibody. The activation of BLNK and PLC␥2 complexes was determined by Western blotting (WB) with anti-phosphotyrosine antibody. ERK phosphorylation in the whole cell lysate was detected as the control for induction of BCR signaling. B, MG132 stabilizes the Thr-152-phosphorylated BLNK proteins in B cells upon anti-IgM stimulation. WT splenic B cells were cultured in the presence of DMSO or MG132 (20 M) for 2 h. The cells were then stimulated with anti-IgM. Tyr-84 and Thr-152 phosphorylation of BLNK was determined by Western blot. C, quantification of relative BLNK phosphorylation levels for B. Statistical analysis was conducted on the data derived from three independent experiments. *, p Ͻ 0.05, Student's t test. D, detection of BLNK ubiquitination using sequential immunoprecipitation. FLAG-tagged BLNK was transfected into HEK293T cells with HA-ubiquitin. FLAG-BLNK was immunoprecipitated with anti-FLAG antibody (1st IP); half of the 1st anti-FLAG immunoprecipitates was denatured and followed by second round of immunoprecipitation (2nd IP) with anti-FLAG antibody. The ubiquitination of 1st and 2nd anti-FLAG immunoprecipitation was detected by Western blotting analyses with anti-HA antibody. E, BCR stimulation induces endogenous BLNK ubiquitination in WT B cells but not HPK1-deficient B cells. WT and HPK1-deficient B cells were stimulated with anti-IgM stimulation. BLNK was immunoprecipitated by anti-BLNK antibody followed by Western blotting with anti-ubiquitin antibody. Data are representative of three independent experiments. tion. We co-transfected FLAG-tagged BLNK WT or T152A mutant with HPK1 and HA-ubiquitin into HEK293T cells. HPK1 co-expression significantly enhanced ubiquitination of BLNK WT, whereas T152A mutation abrogated the effect of HPK1 on BLNK ubiquitination (Fig. 7A). To study whether Thr-152 phosphorylation induced BLNK ubiquitination in B cells, we co-transfected FLAG-BLNK WT or T152A mutant with HA-ubiquitin into BLNK-deficient DT40 mutant B cells. FLAG-BLNK WT but not T152A mutant was polyubiquitinated through Lys-48 linkage in the transfected BLNK-deficient DT40 mutant B cells treated with anti-IgM and MG132 (Fig. 7B). Consistently, immunoprecipitates of anti-phospho-BLNK(Thr-152) from anti-IgM-stimulated WT splenic B cells displayed significant ubiquitination, which was detected by both anti-ubiquitin antibody and anti-pBLNK(Thr-152) antibody (Fig. 7C). These data indicate that Thr-152 phosphorylation targeted BLNK for ubiquitination during BCR signaling.
Lys-37, Lys-38, and Lys-42 Are BLNK Ubiquitination Sites That Attenuate BCR Signaling in B Cells-To identify BLNK ubiquitination site(s), we isolated transfected BLNK from WEHI-231 mouse B cell line treated with anti-IgM and MG132, followed by mass spectrometry analyses. Three lysine residues Lys-37, Lys-38, and Lys-42 were identified as BLNK ubiquitination sites in anti-IgM-stimulated B cells (Fig. 8A). Mass spectrometry data also confirmed that BLNK was phosphorylated at Thr-152 site in B cells (Fig. 8A). To characterize BLNK ubiquitination sites, we generated a FLAG-tagged BLNK K37R/ K38R/K40R/K42R mutant containing triple Lys-to-Arg muta-tions at Lys-37, Lys-38, and Lys-42. Compared with BLNK WT, which displayed a significant Lys-48-linked ubiquitination, BLNK K37R/K38R/K40R/K42R mutant did not show any detectable ubiquitination in BLNK-deficient DT40 mutant B cells treated with anti-IgM stimulation and MG132. These data indicate that Lys-37, Lys-38, and Lys-42 are indeed BLNK ubiq- Following BCR stimulation, BLNK is tyrosine-phosphorylated and activated. Activation of BLNK initiates the formation of the BLNK-interacting protein complex, leading to activation of downstream BCR signaling. HPK1 is also activated by interacting with BLNK. The activation of HPK1 in turn induces phosphorylation of BLNK Thr-152, resulting in the binding of 14-3-3. 14-3-3 dimers recruit a putative E3 ubiquitin ligase to BLNK. The putative E3 ligase subsequently induces BLNK ubiquitination at Lys-37, Lys-38, and Lys-42 residues, leading to degradation of the activated BLNK and subsequent attenuation of BCR signaling. uitination sites (Fig. 8B). Furthermore, anti-IgM-induced phosphorylation of ERK, JNK, and IKK was significantly enhanced in BLNK-deficient DT40 mutant B cells transfected with BLNK K37R/K38R/K40R/K42R mutant (Fig. 8C). These data indicate that loss of BLNK ubiquitination enhances activation of BCR downstream signaling.

DISCUSSION
BCR signaling is delicately controlled by positive and negative regulatory mechanisms. Imbalance of BCR signaling may result in autoimmune diseases or immune deficiency disorders. In this study, we have found that HPK1 plays a negative role in BCR signaling. HPK1-deficient B cells are hyperactive in response to BCR ligation, resulting in enhanced B cell proliferation and activation (Figs. 1, B and C, and 2). The enhanced BCR signaling in HPK1-deficient B cells may contribute to increased antibody productions and experimental autoimmune encephalomyelitis induction in HPK1-deficient mice as shown previously (10).
Previous studies show that HPK1 activates IKK (15,16,19) and JNK (6 -9). In contrast, our data show an inhibitory role of HPK1 for IKK and JNK activation in B cells (Fig. 2, A and B). The difference may be due to using different experimental systems. The positive role of HPK1 in JNK and IKK activation was mainly demonstrated using overexpression systems. One caveat of these overexpression systems is that these cells have limited or no expression of the target proteins (such as BLNK) required for the HPK1-mediated feedback inhibition. Therefore, HPK1 may be skewed to activate JNK and IKK in these cells. HPK1-deficient primary B cells show enhanced JNK and IKK phosphorylation during BCR signaling, indicating that the inhibitory roles of HPK1 in JNK and IKK activation overcome the positive roles HPK1 in JNK and IKK activation in primary B cells.
Our study shows a negative feedback regulation of BLNK by HPK1-induced Thr-152 phosphorylation, which recruits 14-3-3 to BLNK and subsequently attenuates BCR downstream signaling. Previous studies show that HPK1 attenuates T cell receptor signaling via inducing SLP-76 Ser-376 phosphorylation and SLP-76/14-3-3 interaction in T cells (10,31). Thus, HPK1 negatively regulates BCR and T cell receptor signaling via a similar mechanism. However, in contrast to partial loss of SLP-76/14-3-3 binding in HPK1-deficient T cells (10), BLNK/ 14-3-3 interaction was undetectable in HPK1-deficient B cells (Fig. 3C). This suggests that HPK1 plays an exclusive role in B cells. It is notable that the transfected BLNK WT is weakly tyrosine-phosphorylated in BLNK-deficient DT40 mutant B cells (Fig. 5C). Consistently, transient expression of BLNK WT in BLNK-deficient DT40 mutant B cells does not strongly induce downstream MAPK and IKK activation (Figs. 5A and 8C). One potential explanation is that murine BLNK has only 56% identity as chicken BLNK. The transfected murine BLNK may not fully reconstitute BCR signaling of BLNK-deficient DT40 chicken B cells. However, after transfection with murine BLNK WT or T152A, chicken DT40 cells displayed a remarkable difference in BLNK tyrosine phosphorylation and downstream MAPK and IKK phosphorylation (Fig. 5). Future generation and characterization of knock-in or transgenic mice carrying BLNK(T152A) would provide more insights on in vivo functions of BLNK(Thr-152) phosphorylation in immune responses and autoimmune diseases.
A novel finding in this report is that the activated BLNK is ubiquitinated at Lys-37, Lys-38, and Lys-42 residues in B cells during BCR signaling and that the loss of BLNK ubiquitination results in enhanced BCR signaling. Ubiquitination has emerged as a key mechanism regulating the immune function (32). We showed that BLNK was regulated by polyubiquitination via Lys-48 linkage in BCR signaling (Figs. 6, D and E, and 7). The proteasomal inhibitor MG132 stabilized the phosphorylated BLNK proteins but not the total BLNK proteins (Fig. 6B), suggesting that only the phosphorylated BLNK is targeted for ubiquitination and degradation. As phosphorylation and ubiquitination events of a protein only account for a small portion of the total protein, this could explain why the stabilization of total BLNK proteins by MG132 was not discernible. More interestingly, BLNK ubiquitination is initiated by HPK1-induced Thr-152 phosphorylation as HPK1 deficiency or loss of BLNK Thr-152 phosphorylation abolished BLNK ubiquitination (Figs. 6E and 7, B and C). We further identify that Lys-37, Lys-38, and Lys-42 residues are BLNK ubiquitination sites in mouse B cells. These three lysine residues and Thr-152 are also conserved in human BLNK protein sequence (supplemental Fig. 3), suggesting that mutations or post-translational modification of these residues could regulate B cell activation in humans as well. As HPK1 phosphorylates SLP-76 in T cells, our findings in B cells suggest that HPK1-induced SLP-76 phosphorylation may also regulate SLP-76 ubiquitination. Our study has implied that an E3 ligase is recruited to BLNK via 14-3-3-mediated binding and induces BLNK ubiquitination. Although c-Cbl is shown to interact with 14-3-3 (33, 34), BLNK ubiquitination was not reduced in c-Cbl-deficient splenic B cells (data not shown), suggesting that an E3 ligase other than c-Cbl is responsible for BLNK ubiquitination. The identification of the elusive E3 ubiquitin ligases for BLNK is not the focus of this report and will be studied in the future.
In summary, our studies have uncovered a novel negative feedback regulation of the activated BLNK mediated by HPK1induced Thr-152 phosphorylation, 14-3-3 binding, and subsequent ubiquitination of BLNK at Lys-37, Lys-38, and Lys-42 (Fig. 8D). The activated BLNK is tagged with Thr-152 phosphorylation and subsequently targeted for ubiquitination and degradation. BLNK ubiquitination results in the subsequent attenuation of ERK, JNK, and IKK activation, which in turn prevents B cell overactivation and hyperproliferation. This report in combination with our previous studies of HPK1-deficient mice (10) suggest that HPK1 down-regulation, BLNK mutations (at Thr-152, Lys-37, Lys-38, and Lys-42), and dysregulation of BLNK phosphorylation and ubiquitination in B cells may contribute to the pathogenesis of autoimmune diseases.