Tyrosine phosphorylation of MB-1, B29, and HS1 proteins in human B cells following receptor crosslinking

Immunology Letters, 40 (1993) 65-71 Elsevier Science B.V. IMLET 02108

Tyrosine phosphorylation of MB-1, B29, and HS1 proteins in human B cells following receptor crosslinking Daisuke Hata a, Tetsuya N a k a m u r a b, Toshiaki KawakamiC, Yuko Kawakami c, Bettie Herren a and Mitsufumi Mayumi a' aDepartment of Pediatrics, Faculty of Medicine, Kyoto University, Kyoto 606, Japan; bFirst Department of lnternal Medicine, Faculty of Medicine, Tokyo University, Tokyo 113, Japan; CLa Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA; and dDivision of Developmental and Clinical Immunology, Department of Medicine, Pathology, Pediatrics and Microbiology, University of Alabama, Birmingham, AL 35294, USA (Received 5 November 1993; accepted 3 February 1994)

I. Summary

Recent studies of murine and human B lymphocytes have shown that crosslinking of surface IgM (slgM) and slgD stimulates tyrosine phosphorylation of a set of proteins involved in signal transduction. We investigated tyrosine phosphorylation of the slg-associated proteins MB-1 and B29, and p75 Iasl (HS1), and the association of HS1 with MB1/B29 heterodimers in normal human B cells and a human B lymphoma cell line, B104. Using immunoprecipitation with anti-phosphotyrosine antibodies (Abs) followed by immunoblotting with anti-MB-1 Abs, anti-B29 Abs or anti-HS1 Abs, we demonstrated that MB-1, B29 and HS1 were tyrosine-phosphorylated after slgM or slgD crosslinking. Immunoprecipitation with anti-B29 Abs followed by antiHSI Abs immunoblotting revealed that HS1 was associated with MB-1/B29 heterodimers after slgM or slgD crosslinking. The results showed that HS 1 may play an important role in signal transduction through slgM and slgD on human B cells.

2. Introduction

It has been shown in murine and human B cells that Ig-~ (MB-1) and Ig-fl (B29), the products of MB-1 and B29 genes, respectively, are non-covalently associated with sIg [1-4], forming the B-cell antigen-receptor Key words: MB-I; B29; HS1; IgM; IgD; Tyrosine phosphorylation *Corresponding author: Mitsufumi Mayumi, M.D., Department of Pediatrics, Faculty of Medicine, Kyoto University, Shogoin, Kawahara-cho 54, Sakyo-ku, Kyoto 606, Japan. SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) 0 0 0 2 4 - L

complex. This multi-molecular complex is associated with protein tyrosine kinases including p53/p56 tyn, p55 btk, p59fyn and a 72 kDa tyrosine kinase (PTK72 or Syk). These tyrosine kinases have been shown to be activated by slg crosslinking [5-10] and phosphorylate the tyrosine residues of a set of proteins including phospholipase C~a [11,12], Cv2[12-14], phosphatidylinositol 3-kinase [15,16], p21 ras GTPase-activating protein (GAP) [17], MB-1 and B29 [18] and p75 us1 (HS1), a 75 kDa protein encoded by the HS1 gene expressed only in hematopoietic cells [19-21]. These proteins have thus been considered to play important roles in signal transduction through sIg. We previously reported tyrosine phosphorylation of sIgM- and sIgD-associated molecules in a human mature B cell line, B104 [22], which express both sIgM and sIgD. However, only sIgM crosslinking by anti-IgM Abs induces B104 cell death, although both sIgM and sIgD appear to have signal transducing abilities [23,24]. We also reported that sIgM and sIgD transduce different signals in the cell division of normal mature B cells [25]. In the present study, we examined tyrosine phosphorylation of MB-1, B29 and HS1 following crosslinking of sIgM or sIgD in B104 cells and normal human B cells to determine the participation of MB-1, B29 and HS1 in the signal transduction through sIgs.

3. Materials and Methods

3.1. Reagents and cells The following mouse hybridomas were purchased from American Type Culture Collection (Rockville, MD): HB57 (anti-human IgM), HB138 (anti-human

IgM), HB70 (anti-human IgD) and HB104 (anti-human Ia). The mAbs were purified from ascitic fluids with ammonium sulfate and DEAE column chromatography. The murine anti-human B29 mAb (CB3-1) has been described previously [26]. The rabbit antiserum against human MB-1 was produced by immunizing rabbits with the :extracellular domain of recombinant human MB-1. The recombinant human MB-1 protein was made by amplifying the extracellular portion of the cDNA using PCR. N-terminal and C-terminal primer sequences were 5'-CTGTGGATCCACAAGGTCCCAG and 5'-CCATAAGCTTATCGGTTCTTGGTGCCC, respectively. The PCR product was cloned into an expression vector, pQE-11 (Qiagen, Chatsworth, CA), and recombinant proteins were expressed and purified according to manufacturer's instructions. A murine monoclonal phosphotyrosine-specific antibody, PY20 [27,28], was obtained from Seikagaku Kogyo (Tokyo, Japan). Non-reactive mouse myeloma IgG1 protein was purchased from Cappel (West Chester, PA). Anti-HS1 antibody was described previously [21]. The human B lymphoma cell line, B104 (slgM-, slgD ÷) has been described previously [23,29]. The B104 cells were maintained at 2 × 105-2 × 10 6 cells/ml in RPMI-1640 medium (Nissui, Tokyo, Japan) supplemented with 10% FCS (Hazleton Biologics, Lenexa, KS), 5 × 10 -5 M 2-ME, and antibiotics (100 U/ml of penicillin and 100 #g/ml of streptomycin). Tonsillar B cells were isolated and purified according to the purification method of peripheral blood B lymphocytes as previously described [25]. 3.2. Stimulation of cells and preparation of cell lysates B104 or tonsillar B cells (2 x 107/10 ml of growth medium) were stimulated with various monoclonal antibodies (mAbs) (5 #g/ml) at 37°C, pelleted immediately by centrifugation and lysed in 1% NP-40 lysis buffer [22]. Insoluble materials were removed by centrifugation. 3.3. Immunoprecipitation with anti-phosphotyrosine mAbs followed by immunoblotting with anti-MB-1, anti-B29 or anti-HS1 Abs This procedure was carried out a s described previously [22]. Briefly, NP-40 lysaJ~es of B cells were incubated with PY20 and Pansorbin (Calbiochem, La Jolla, CA), and bound phosphotyrosine-containing proteins were eluted with elution buffer containing p-nitrophenyl phosphate. The eluted proteins were resolved b y SDS-PAGE and transferred to Im66

mobilon-P (Millipore) membranes. After blocking, the membranes were incubated with rabbit anti-MB1 antiserum (1:200 diluted in blocking solution) or biotinylated anti-hUman B29 mAb (CB3-1) (5 #g/ ml), and then with 125I-labeled protein A or 125I-labeled streptavidin (Amersham, Arlington Heights, IL), respectively. Alternatively, the membranes were incubated sequentially with affinity-purified rabbit anti-HS1 Abs (1 #g/ml) and 125I-labeled protein A. 3.4. Immunoprecipitation with anti-B29 mAbs followed by immunoblotting with anti-phosphotyrosine or anti-HS1 Abs NP-40 lysates of unstimulated or anti-Ig Abs-stimulated B104 cells were incubated with excess protein G-Sepharose (Pharmacia, Piscataway, N J). The cleared lysates were incubated with CB3-1 and protein G-Sepharose. Bound molecules were dissociated with Laemmli's SDS sample buffer, resolved by SDSPAGE and transferred to Immobilon-P membranes. The membranes were incubated with PY20 or anti~ HS1 Abs, and then 125I-labeled protein A. 3.5. Immunoprecipitation of B cell antigen receptor complexes followed by immunoblotting with anti-MB-1 or anti-B29 Abs One percent digitonin lysates [22] of unstimulated B cells were incubated with the anti-IgM or anti-IgD mAbs and protein: G-Sepharose. Bound molecules were eluted with solubilizing buffer containing 1% NP-40, 0.1% sodium deoxycholate and 0.1% SDS. Samples were analyzed by immunoblotting with the use of anti-MB-1 or anti-B29 Abs as described above. 3.6. N-glycosidase treatment Samples eluted with the above-mentioned solubilizing buffer from immunoprecipitates with anti-IgM or anti-IgD Abs were boiled for 5 min in the presence of 0.5% SDS and 100 mM 2-ME. Solutions were adjusted to a final concentration of 20 mM phosphate, pH 7.5, 1.25% NP-40, and 10 U/ml N-gl3/canase T M (Genzyme, Boston, MA) and the mixtures were incubated overnight at 37°C.

4. Results

4.1. Immunoreactivity of anti-B29 and anti-MB-1 Abs The reactivity of anti-B29 and anti-MB-1 Abs with

sIgM- and sIgD-associated proteins was assessed by Western blotting. Anti-IgM or anti-IgD immunoprecipitates from 1% digitonin lysates of unstimulated B cells were resuspended in 1% NP-40 solubilizing buffer to dissociate the Ig-associated proteins from Ig and resolved by SDS-PAGE. The Ig-associated proteins were transferred to Immobilon-P membranes and probed with anti-B29 and anti-MB-1 Abs. AntiB29 Abs reacted with sIgM-associated proteins of 50-37 kDa and sIgD-associated proteins of 46-39 kDa in BI04 cells (lanes 5 and 6, Fig. 1A), and with sIgM-associated proteins of 43-35 kDa and sIgD-associated proteins of 41-35 kDa in tonsillar B cells (lanes 5 and 6, Fig. 2A). Anti-MB-1 Abs reacted with sIgM-associated proteins of 60-45 kDa and sIgD-associated proteins of 58-47 kDa in B104 cells (lanes 5 and 6, Fig. 1B), and with sIgM-associated proteins of 55-42 kDa and sIgD-associated proteins of 57-45 kDa in tonsillar B cells (lanes 5 and 6, Fig. 2B). No immunoreactivity was observed when anti-Ia immunoprecipitates were probed with anti-B29 and anti-MB-1 Abs (lane 7, Figs. 1A,B and 2A,B). After deglycosylation by N-glycosidase, all the above-mentioned anti-B29 Abs-reactive and anti-MB-1 Abs-reactive proteins were detected as a single band at 27 and 26 kDa, respectively (data not shown), indicating that heterogeneity of MB-1 and B29 in B cell types

A

and Ig isotypes is caused by differences in N-glycosylation. These results are in good agreement with the predicted molecular weights of human MB-1 and B29 [30,31]. With N-glycosidase treatment under suboptimal conditions, anti-MB-1 Abs-reactive proteins gave rise to two bands of 29 and 26 kDa (data not shown), which may account for our previous observation by in vitro kinase assay of three deglycosylated slgM- or slgD-associated phosphoproteins with molecular weights of 29, 27, and 26 kDa [22]. The specificity of anti-B29 Abs to B29 in the whole cell lysates of human B cells in immunoprecipitation experiment has previously been shown [26]. Anti-MB-1 Abs did not work well in the immunoprecipitation experiment (data not shown).

4.2. Tyrosine phosphorylation of MB-1, B29 and HS1 after slg crosslinking NP-40 lysates of unstimulated and anti-Ig Abs-stimulated B104 and tonsillar B cells were immunoprecipitated with anti-phosphotyrosine Abs and analyzed by immunoblotting with anti-B29 and antiMB-1 Abs. In B104 cells, stimulation with anti-IgM Abs caused tyrosine phosphorylation of proteins of 5239 kDa which reacted with anti-B29 Abs (lane 2,

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Anti-B29 blot

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Fig. 1. Tyrosine phosphorylation of MB-I and B29 after slg crosslinking in BI04 cells. Immunoprecipitates with anti-phosphotyrosine Abs from 1% NP-40 lysates of unstimulated (lane 1), anti-Ig Abs-stimulated (lanes 2 and 3), or anti-Ia Abs-stimulated (lane 4) BI04 cells were analyzed by 10% SDS-PAGE under reducing conditions, and transferred to polyvinylidene difluoride sheets followed by immunoblotting with biotinylated anti-B29 Abs (A: lanes 1-4) or rabbit an ti-MB-1 antiserum (B: lanes 1-4) and detection with 1251-labeled streptavidin or IzSI-labeled protein A, respectively. Immunoprecipitates with anti-lgM Abs (lane 5), anti-lgD Abs (lane 6) or anti-la Abs (lane 7) from 1% digitonin lysates of unstimulated B104 cells were resuspended in 1% NP-40 solubilizing buffer to dissociate the Ig-associated proteins from Ig. The Ig-associated proteins were analyzed by immunoblotting with anti-B29 Abs (lanes 5-7, Fig. IA) or anti-MB-1 Abs (B: lanes 5-7) as described above. Anti-P-T ippt: anti-phosphotyrosine immunoprecipitates. Positions of molecular weight standards (kDa) are indicated on the left side of the figure.

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Fig. 2. Tyrosine phosphorylation of MB-1 and B29 after slg crosslinking in tonsillar B cells. NP-40 lysates of unstimulated (lane 1), anti-Ig Abs-stimulated (lanes 2 and 3), or anti-Ia Abs-stimulated (lane 4) tonsillar B cells were immunoprecipitated with anti-phosphotyrosine mAbs (anti-P-T ippt) and analyzed by immunoblotting with anti-B29 Abs (A: lanes 1-4) or anti-MB-1 Abs (B: lanes 1-4). IgM- and IgD-associated proteins of tonsillar B cells were also analyzed by immunoblotting with anti-B29 Abs (A: lanes 5-7) or anti-MB-1 Abs (B: lanes 5-7) as described in Fig. 1. Positions of molecular weight standards (kDa) are indicated on the left side of the figure.

Fig. 1A) and of 60-45 kDa which reacted with antiMB-1 Abs (lane 2, Fig. 1B). Stimulation with antiIgD Abs also caused tyrosine phosphorylation of the anti-B29 Abs-reactive and the anti-MB-1 Abs-reactive proteins (lane 3, Fig. 1A and lane 3, Fig. 1B) with the similar molecular weights as those detected in the anti-IgM Abs treatment. In tonsillar B cells, treatment with anti-IgM Abs induced tyrosine phosphorylation of proteins of 43-35 kDa and 57-45 kDa identified by anti-B29 Abs (lane 2, Fig. 2a) and by anti-MB-1 Abs (lane 2, Fig. 2B), respectively. Treatment with anti-IgD Abs induced similar patterns of tyrosine phosphorylation to those detected with antiIgM Abs (lane 3, Fig. 2A and lane 3, Fig. 2B). Tyrosine phosphorylation of MB-1 and B29 was induced by another anti-IgM Ab (HB138) (data not shown) but not by HB104 Ab against human Ia (lane 4 in Figs. 1A,B and 2A,B), which was expressed on B104 cells [23] and tonsillar B cells (data not shown). To examine the kinetics of tyrosine phosphorylation of MB-1, B29, and HS1, NP-40 lysates of unstimulated and anti-IgM Abs-stimulated B104 cells for planned intervals were immunoprecipitated with PY20 and analyzed by immunoblotting with antiB29, anti-MB-1 and anti-HS1 Abs (Fig. 3). Treatment with anti-IgM Abs induced tyrosine phosphorylation of MB-1, B29 and HS1 rapidly and with the same kinetics. Increased tyrosine phosphorylation was visible immediately after addition of anti-IgM Abs, was maximal at 5 rain, and then decreased to 68

almost basal levels after 60 min (Fig. 3). Our observations did not exclude the possibility that MB-1, B29 and HS1 were not tyrosine-phosphorylated but were associated with some unknown proteins that were tyrosine-phosphorylated by slg crosslinking. Therefore, NP-40 lysates from unstimulated or anti-Ig Abs-stimulated B104 cells were immunoprecipitated with anti-B29 Abs and analyzed by immunoblotting with anti-phosphotyrosine Abs (Fig. 4). Crosslinking of slgM and slgD induced rapid tyrosine phosphorylation of a 75 kDa protein and 60-39 kDa proteins that were immunoprecipitated with anti-B29 Abs (lanes 2 and 3, Fig. 4). From the molecular weights of MB-1 (60-45 kDa) and B29 (52-39 kDa) observed in B104 cells (Fig. 1), proteins of 60-39 kDa were most likely tyrosine-phosphorylated MB-1 and B29. However, proteins of 60-39 kDa may include s r c family tyrosine kinases associated with MB-1/B29 heterodimers. Addition of 0.5% SDS to the NP-40 lysates from anti-Ig Absstimulated B104 cells resulted in disappearance of the 75 kDa protein but not of the 60-39 kDa proteins (data not shown). The results showed that the 75 kDa protein did not react with the anti-B29 Abs but Was physically associated and co-immunoprecipitated with B29 or MB-1/B29 heterodimers. The level of tyrosine phosphorylation of B29 and its associated proteins after slgD stimulation was lower than that after slgM stimulation. This is presumably due to the higher expression of slgM than slgD in B104 cells

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Fig. 3. Time course of tyrosine phosphorylation of MB-I, B29 and HS1 after slgM crosslinking in B104 cells. NP-40 lysates from B104 cells that had been incubated with 5 #g/ml anti-IgM Abs for the indicated times were immunoprecipitated with anti-phosphotyrosine Abs and analyzed by immunoblotting with anti-B29 Abs (A), anti-MB-1 Abs (B) or anti-HS1 Abs (C) as described in Fig. 1. Positions of molecular weight standards (kDa) are indicated on the left side of the figure.

Anti-B29 ippt Anti-P-T blot

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[23]. To examine whetherthe 75 kD a protein in antiB29 immunoprecipitates was HS1, anti-B29 immunoprecipitates from NP-40 lysates of B104 cells were analyzed by anti-HS 1 immunoblotting (Fig. 5). AntiHS1 Abs-reactive protein with 75 kDa molecular weight was detected in anti-B29 immunoprecipitates only after slgM or slgD crosslinking (lanes 3 and 4, Fig. 5). The addition of 0.5% SDS to NP-40 lysates of antiLIgM Abs-stimulated B104 cells resulted in dissociation of p75"from B29 (lane 6, Fig. 5).

97 5. Discussion

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Fig. 4. slg-stimulated tyrosine phosphorylation of MB-1, B29 and a 75 kDa protein associated with MB-1/B29 heterodimers in B104 ceils. NP-40 lysates from unstimulated (lane 1), anti-Ig Abs-stimulated (lanes 2 and 3) or anti-Ia Abs-stimulated (lane 4) BI04 cells were immunoprecipitated with anti-B29 Abs (anti-B29 ippt) and analyzed by immunoblotting with anti-phosphotyrosine Abs (antiP-T blot). Positions of molecular weight standards (kDa) are indicated on the left.

Surface Ig crosslinking induces tyrosine phosphorylation of a set of proteins. It has been shown that two components of the B cell antigen receptor complex, Ig-ct (MB-1) and Ig-fl (B29), are phosphorylated on tyrosine residues after slg crosslinking in murine B cells [18]. In human B cells, however, ther~ have only been reports which show that MB-1 and B29 are associated with slgM and slgD, and their tyrosine phosphorylation has only been demonstrated by in vitro kinase assay [4]. In the present study, we pre 7 sent evidence that crosslinking of slgM or slgD stimulates tyrosine phosphorylation of MB-1 and B29 in human B cells. We also demonstrated that both MB-1 and B29 have broad molecular weight spectra due to differences in the degree of N-glycosylation, in agreement with previous reports [26,32]. The cytoplasmic tails of MB-1 and B29 both contain a conserved sequence motif which is considered 69

Anti-HS 1 blot

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Fig. 5. Association of HS1 with MB-1/B29 heterodimers after slg crosslinking. NP-40 lysates from unstimulated (lane 1), anti-Ig Abs-stimulated (lanes 2, 3, 4, and 6) or anti-Ia Abs-stimulated (lane 5) B104 cells were immunoprecipitated with anti-B29 Abs (anti-B29 ippt) and analyzed by immunoblotting with anti-HS1 Abs (anti-HSl blot). In lane 6, 0.5% SDS was added to NP-40 lysates from anti-lgM Abs-stimulated B104 cells. Positions of molecular weight standards (kDa) are indicated on the left. as a potential target for tyrosine phosphorylation. The same motif has also been found in a number of other signal transducer chains, including components of CD3, FceRIfl, ~ chains and others [33], suggesting the possible signal transducing ability o f MB-1 and B29. Several proteins have now been identified which possess SH2 and/or SH3 domains that enable them to bind to other proteins containing tyrosine phosphoproteins (SH2) and proline-rich motifs (SH3) [34,35]. Recent studies have shown that a 75 k D a protein, designated as HS1, which is expressed in human hematopoietic cells and has an SH3 domain and repetitive helix-turn-helix sequences [20], is tyrosine phosphorylated after crosslinking of slgM as well as crosslinking of FceRI and T C R / C D 3 [19,21]. In the present study, we present evidence that HS1 is tyrosine-phosphorylated after slgM or slgD crosslinking, and is associated with B29 or MB-1/B29 heterodimers only after slg-crosslinking. However, 70

neither HS1 nor MB-1/B29 heterodimer has an SH2 domain, suggesting that HS1 is not directly associated with MB-1/B29 heterodimers. It has been shown that MB-1/B29 heterodimer is associated with src family tyrosine kinases (blk, lyn or fyn) in B cell lysates containing 1% NP-40 [7]. Thus, tyrosinephosphorylated HS1 m a y be associated with MB-1/ B29 heterodimers via the SH2 domain of blk, lyn or fyn in B104 cells. This is consistent with the findings demonstrating that HS1 is associated with the SH2 domain of lyn in slgM-mediated signaling [19]. It would be interesting to know whether HS1 binds to downstream signal-transducing molecules through its SH3 domain or other portions, and regulates p21 ras activity like an epidermal growth factor regulates p21 ras through the formation of a complex of the receptor with an SH2 and SH3 domain-containing adaptor protein (Grb2), and proline-rich domaincontaining protein, SOS (guanidine nucleotide exchange factor) [36,37]. The slg-induced tyrosine phosphorylation of HS1 and its association with MB-1/B29 heterodimers may be an important signal-transducing event in antigenstimulated B cells, but our data of HS1 do not explain the differences between slgM- and slgDmediated signals. Further studies are required to clarify the differences in signals through slgM and slgD.

Acknowledgements

We appreciate the help of Dr. Max D. Cooper, University of Alabama at Birmingham, in this study. This work was supported by a grant from the Ministry of Health and Welfare of Japan.

References

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