Tyrosine phosphorylation of the c-cbl proto-oncogene product mediated by cell surface antigen CD38 in HL-60 cells.

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120. These results indicated that tyrosine phosphorylation of cellular proteins, including p120, is possibly involved in transmembrane signaling mediated by CD38.

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD ؉ glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosinephosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl protooncogene product, p120 c-cbl . These results indicated that tyrosine phosphorylation of cellular proteins, including p120 c-cbl , is possibly involved in transmembrane signaling mediated by CD38.
The human cell surface antigen CD38, originally termed T10 (1), is a 46-kDa type II glycoprotein with a single transmembrane domain. However, CD38 exhibits no significant homology with other known cell surface molecules (2,3). Cytochemical studies revealed that CD38 is predominantly produced on the cell surface in both the early and late stages of T and B lymphocyte maturation but not in intermediate ones (1,4,5). We recently demonstrated that the extracellular domain of CD38 exhibits NAD ϩ glycohydrolase (NADase) 1 activity and that the ecto-form NADase activity induced by RA in HL-60 cells is indeed due to CD38 (6). Moreover, it has been reported that CD38 catalyzes not only the hydrolysis of NAD, but also the formation and hydrolysis of cyclic ADP-ribose (7-9), which is a novel candidate that mediates Ca 2ϩ release from intracellular Ca 2ϩ stores (see Refs. 10 and 11 for reviews). Besides these enzyme activities, CD38 has the ability to bind hyaluronate, which is a large glycosaminoglycan existing in the extracellular matrix and on the cell surface (12). Recent studies revealed that stimulation of CD38 with anti-CD38 mAbs induces various cell responses including DNA synthesis by human thymocytes in the presence of accessory cells (13), the proliferation of mouse B cells in the presence of IL-4 (14), and the rescue of germinal center cells from apoptosis (15). Moreover, anti-CD38 mAb inhibited lymphocyte adhesion to endothelial cells (16) and suppressed the growth of immature B lymphoid cells in the bone marrow microenvironment (17). Regardless of these observations, little is known concerning the intracellular signaling mediated by CD38.
In hematopoietic cells, the stimulation of T and B cell antigen receptors activates multiple protein kinases, resulting in the phosphorylation of numerous intracellular substrate proteins. Extensive research on these protein kinases and their substrates has revealed that protein-tyrosine phosphorylation plays a crucial role in transmembrane signaling via hematopoietic receptors (18 -20). In the present study, we examined the possibility that the tyrosine phosphorylation of cellular proteins might be involved in the CD38-mediated signaling pathway. We found that stimulation of RA-differentiated HL-60 cells with anti-CD38 mAb induces rapid tyrosine phosphorylation of cellular proteins. One of the prominent phosphorylated proteins was identified as the c-cbl proto-oncogene product, p120 c-cbl .

EXPERIMENTAL PROCEDURES
Cell Culture-HL-60 cells were cultured and caused to differentiate by various inducers as described previously (21). Mouse hybridoma HB136 cells, which produce an anti-CD38 mAb (HB-7, subclass IgG1), were obtained from the American Type Culture Collection and cultured in serum-free Cosmedium-001 (Cosmo Bio Co., Ltd., Tokyo, Japan) containing insulin and transferrin.
Materials-The anti-CD38 mAb, HB-7, was purified from the culture medium of HB136 cells by means of column chromatography on protein A-Cellulofine (Seikagaku-Kogyo, Tokyo, Japan). Another anti-CD38 mAb, T16, and the subclass-matched control IgG1 were purchased from Cosmo Bio (IOB6) and ICN Biochemicals, Inc. (64-335), respectively. The anti-PY mAb PY-20 and rabbit anti-PY pAb were obtained from Leinco Technologies Inc. (Ballwin, MO) and Chemicon International Inc. (Temecula, CA), respectively. An anti-p120 c-cbl pAb was purchased from Santa Cruz Biotechnology (SC-170). Sepharose 4B and protein G-Sepharose 4-FF were from Pharmacia Biotech. (Uppsala, Sweden). The tyrosine kinase substrate, Raytide, and P-81 ion exchange chromatography paper were purchased from Oncogene Science Inc. (Uniondale, NY) and Whatman (Maidstone, United Kingdom), respectively. [␥-32 P]ATP and 125 I-protein A were from DuPont NEN. All other reagents were of analytical grade and from commercial sources.
Stimulation of HL-60 Cells with Anti-CD38 mAbs and Analysis of Tyrosine-phosphorylated Proteins-HL-60 cells which had been cultured with or without various inducers were washed three times with ice-cold phosphate-buffered saline and then resuspended in serum-free RPMI 1640 containing 10 mM Na-Hepes (pH 7.4) at a cell density of 3 ϫ 10 7 cells/ml. After preincubation at 37°C for 5 min, the cells (1.2 ϫ 10 7 * This work was supported by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed. Tel.: 81-3-3812-2111 (ext. 4750); Fax: 81-3-3815-9604; E-mail: katada@mol.f.u-tokyo.ac.jp. 1 The abbreviations used are: NADase, NAD ϩ glycohydrolase; mAb, monoclonal antibody; pAb, polyclonal antibody; PAGE, polyacrylamide electrophoresis; TBS, Tris-buffered saline; PY, phosphotyrosine; RA, retinoic acid. cells/400 l) were stimulated with various concentrations of antibodies (or the control IgG1) at 37°C for the indicated times. The reaction was terminated by adding the same volume (400 l) of a lysis buffer consisting of 40 mM Na-Hepes (pH 7.4), 150 mM NaCl, 30 mM NaF, 2 mM Na 3 VO 4 , 20 mM Na 4 P 2 O 7 , 4 mM EDTA, 2% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, and 4 g/ml of aprotinin. The cell lysate was kept at 4°C for 30 min with occasional stirring, before being centrifuged at 14,000 ϫ g for 20 min. The supernatant was precleared by incubation with 20 l of Sepharose 4B resin (50% suspension) at 4°C for 30 min. After removal of the Sepharose resin by brief centrifugation, the supernatant was transferred to a new tube and incubated with 1 g (10 l) of PY20 at 4°C for 2 h. Twenty l of protein G-Sepharose resin (50% suspension) was added to the reaction mixture, followed by further incubation at 4°C for 2 h. The immune complex precipitated with the resin was washed three times with 800 l of a washing buffer consisting of 20 mM Na-Hepes (pH 7.4), 75 mM NaCl, 15 mM NaF, 1 mM Na 3 VO 4 , 10 mM Na 4 P 2 O 7 , 2 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 2 g/ml aprotinin and then resuspended in 30 l of the same buffer. The samples were subjected to SDS-PAGE (10% of acrylamide), and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane, after being shaken at room temperature for 30 min in TBS buffer (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 5% bovine serum albumin, was incubated with rabbit anti-PY pAb at room temperature for 2 h and then washed with TBS buffer containing 0.2% (w/v) Tween 20. The membrane was further incubated with 125 I-protein A at room temperature for 2 h in TBS containing 5% (w/v) bovine serum albumin and then washed with TBS buffer containing 0.2% (w/v) Tween 20. The radioactivity retained on the membrane was visualized with a Fuji BAS2000 bioimaging analyzer.
In Vitro Tyrosine Kinase Assay-Tyrosine kinase activity in the cell lysate was determined by measuring the phosphorylation of a broad specificity peptide substrate, Raytide (Oncogene Science Inc., Manhasset, NY), as described previously (22). A lysate was prepared from HL-60 cells which had been stimulated with HB-7 or the control IgG1 and then subjected to immunoprecipitation with PY20 as described above. The resultant immune complex precipitated with protein G-Sepharose was further washed twice with 800 l of a kinase buffer consisting of 50 mM Na-Hepes (pH 7.4), 75 mM NaCl, 0.1 mM EDTA, and 0.075% (w/v) Brij 35 and then resuspended in 30 l of the same buffer. The kinase reaction was initiated by adding 10 l of the kinase buffer containing 60 M Raytide, 44 mM MgCl 2 , 12 mM MnCl 2 , 100 M ATP, and 10 Ci of [␥-32 P]ATP, the reaction mixture being incubated at 30°C for 30 min. After brief centrifugation, the supernatant (20 l) was transferred to a new tube containing 40 l of 15% (v/v) phosphoric acid to stop the reaction and then the mixture was applied onto P-81 ion exchange chromatography paper (2 ϫ 2 cm). The paper was washed seven times with 0.5% (v/v) phosphoric acid and once with acetone, before being dried and subjected to the associated radioactivity counting with a scintillation counter.

Stimulation of Protein Tyrosine Phosphorylation by Anti-
CD38 mAbs in RA-differentiated HL-60 Cells-We first investigated the possibility that the tyrosine phosphorylation of cellular proteins might be induced on stimulation with anti-CD38 Abs. HL-60 cells that had been cultured with RA to produce CD38 were stimulated with an anti-CD38 mAb, T16, and then a lysate was prepared from the cells. Tyrosine-phosphorylated proteins in the cell lysate, after being immunoprecipitated with PY20, were separated by SDS-PAGE and visualized by means of immunoblotting with an anti-PY pAb. As shown in Fig. 1A, the addition of T16 to RA-differentiated cells stimulated tyrosine phosphorylation of cellular proteins with M r of 120,000, 87,000, and 77,000 (lane 3). For simplicity, the major tyrosine-phosphorylated proteins are henceforth referred to as p120, p87, and p77, respectively. In addition, several other proteins with M r of 65,000 to 45,000 appeared to be tyrosine-phosphorylated by T16 to a lesser extent. Another anti-CD38 mAb, HB-7, also stimulated protein tyrosine phosphorylation, the pattern being the same as that observed for T16 (lane 4). The stimulatory effect of HB-7 on the tyrosine phosphorylation was observed at a concentration as low as 0.3 g/ml and was saturated at about 3 g/ml (data not shown).
However, the subclass-matched control mAb (IgG1) had no effect on the tyrosine phosphorylation (lane 2). The specificity of the action of HB-7 was confirmed by its inability to stimulate the tyrosine phosphorylation in undifferentiated or dibutyryl cAMP-differentiated HL-60 cells (Fig. 1B), in which CD38 is not produced (6). These results clearly indicated that the anti-CD38 mAb stimulates the tyrosine phosphorylation of cellular proteins through CD38 produced on the surface of HL-60 cells. NAD ϩ and hyaluronate, which are an enzyme substrate and a potential ligand for CD38 (12), respectively, did not stimulate the tyrosine phosphorylation of cellular proteins (data not shown). Fig. 2A shows the time course of protein tyrosine phosphorylation after the addition of HB-7 to RA-differentiated HL-60 cells. All the tyrosine phosphorylation of p120, p87, and p77 occurred within 1 min, the maximal levels being reached at 1-2 min, followed by gradual decreases in the phosphorylation levels (Fig. 2B). The overall pattern of tyrosine phosphorylation induced by HB-7 was not altered by further cross-linking of the membrane-bound anti-CD38 mAb with an anti-mouse IgG antibody (data not shown). This may imply that the aggregation of CD38 molecules is not essentially required for the CD38induced tyrosine phosphorylation in cells.
Stimulation by Anti-CD38 mAb of Tyrosine Kinase Activity in a Cell Lysate Immunoprecipitated with Anti-PY mAb-It has been generally observed in tyrosine kinase-induced signaling pathways that the activation of tyrosine kinases induces the autophosphorylation of tyrosine residues and/or association with tyrosine-phosphorylated proteins. Thus, we next measured the tyrosine kinase activity in the lysate of HL-60 cells that had been stimulated with HB-7. Tyrosine-phosphorylated proteins and/or proteins associated with them in the cell lysate were immunoprecipitated with PY20, and the in vitro tyrosine kinase assay was carried out with Raytide as the substrate (22) . Fig. 3 shows the time course of the tyrosine kinase activity in the PY20-immunoprecipitated fraction after the addition of HB-7 to RA-differentiated HL-60 cells. HB-7 rapidly stimu- lated the tyrosine kinase activity in the fraction; the maximum stimulation was observed at 2.5 min. The tyrosine kinase activity in the fraction prepared from undifferentiated HL-60 cells was, however, not affected by the addition of HB-7. An effect of the control IgG1 on the tyrosine kinase activity was not observed in undifferentiated or RA-differentiated HL-60 cells. These results suggested that stimulation of CD38 activates a cellular tyrosine kinase(s), which may be responsible for the tyrosine phosphorylation of p120, p87, and p77.
Identification of p120 as the c-cbl Proto-oncogene Product-Recent reports revealed that tyrosine phosphorylation of the c-cbl proto-oncogene product, p120 c-cbl , may play a critical role in signal transduction mediated by the T cell receptor (23), Fc receptor for IgG (24), and epidermal growth factor receptor (25,26). These observations led us to examine whether or not p120 ccbl is involved in transmembrane signaling mediated by CD38. To investigate this possibility, a lysate prepared from RAdifferentiated HL-60 cells that had been treated with HB-7 or the control IgG1 was subjected to immunoprecipitation with anti-p120 c-cbl pAb, followed by immunoblotting with anti-PY pAb. The addition of HB-7 to the cells stimulated the tyrosine phosphorylation of p120 c-cbl (Fig. 4A, lane 2), although the immunoprecipitated amounts of p120 c-cbl were the same for the control IgG1-and HB-7-treated HL-60 cells (lanes 3 and 4). This clearly indicated that p120 c-cbl is tyrosine-phosphorylated on stimulation of CD38 in RA-differentiated HL-60 cells.
The relationship between this p120 c-cbl and p120 previously identified as the tyrosine-phosphorylated protein in the same RA-differentiated HL-60 cells was further investigated as follows. A cell lysate obtained from HB-7-stimulated HL-60 cells was first treated with the anti-p120 c-cbl antibody to deplete p120 c-cbl from the lysate. The treated lysate, after being immunoprecipitated with PY20, was separated by SDS-PAGE and then subjected to immunoblotting with the anti-p120 c-cbl pAb or anti-PY pAb (Fig. 4B). As expected, the depletion of p120 c-cbl from the cell lysate was almost completely achieved (lanes 1 and 2). When the cell lysate was analyzed by immunoblotting with the anti-PY pAb, a marked decrease in the immunoreactivity of p120 to the pAb was observed without significant changes in any other tyrosine-phosphorylated proteins (lanes 3  and 4). These results indicated that the major fraction of the tyrosine-phosphorylated p120 observed on the stimulation of CD38 represents the phosphorylated p120 c-cbl in the HL-60 cells. DISCUSSION We demonstrated that stimulation with anti-CD38 mAbs induces tyrosine phosphorylation of cellular proteins in HL-60 cells caused to differentiate into granulocytes by RA. The action of the anti-CD38 mAb, HB-7, was specifically observed in CD38-producing HL-60 cells; there was no stimulatory effect of the mAb on undifferentiated or dibutyryl cAMP-differentiated HL-60 cells, in which CD38 is not produced. Protein tyrosine phosphorylation induced by anti-CD38 mAb has been also reported in mouse B cells (27). However, none of the phosphorylated proteins was analyzed in the B cells, although some of the anti-PY-immunoreacted proteins appeared to be similar to those observed in the RA-differentiated HL-60 cells in terms of molecular weight.
In the present study, we found that one of the major tyrosinephosphorylated proteins stimulated by anti-CD38 mAb is the c-cbl proto-oncogene product with M r of 120,000, p120 c-cbl . The cbl gene was initially identified as a transforming component of Cas NS-1 retrovirus that induces early B-lineage lymphoma (28). The c-cbl proto-oncogene is mainly expressed in hematopoietic cells (29). Recent studies revealed that p120 c-cbl is tyrosine-phosphorylated in response to T cell receptor, Fc receptor, and epidermal growth factor receptor activation (23)(24)(25)(26). It was also shown that tyrosine-phosphorylated p120 c-cbl has an ability to bind the SH2 domains of Fyn, Lck, and Blk proteintyrosine kinases, GTPase-activating protein and phospholipase C␥, and that p120 c-cbl also binds to the SH3 domain of Nck, Lyn, and the N-terminal SH3 domain of Grb-2 (23, 30). Moreover, the conversion of c-cbl to a transforming gene involves tyrosine phosphorylation of its protein products (31). Thus, FIG. 2. Time course of the anti-CD38 mAb-stimulated tyrosine phosphorylation of cellular proteins in RAdifferentiated HL-60 cells. A, RA-differentiated HL-60 cells were incubated with 7.5 g/ml of HB-7 at 37°C for the indicated times and then tyrosine-phosphorylated proteins in the cell lysate were visualized as described in Fig. 1. B, the intensities of tyrosine phosphorylation of p120, p87, and p77 were measured with a Fuji BAS2000 bioimaging analyzer and expressed as the -fold increases in the control levels at zero time.
FIG. 3. Tyrosine kinase activity in the PY20-immunoprecipitated fraction, as stimulated by the addition of HB-7 to differentiated HL-60 cells. RA-differentiated (E, q) or undifferentiated (Ç, å) HL-60 cells were incubated with 7.5 g/ml of HB-7 (q, å) or the control IgG1 (E, Ç) at 37°C for the indicated times. The cell lysate was immunoprecipitated with PY20 and then subjected to the in vitro kinase assay as described under "Experimental Procedures." The kinase activities are expressed as picomoles of phosphorylated Raytide/assay tube. p120 c-cbl appears to play an important role in the signal transduction in hematopoietic cells.
In the case of T cell receptor or Fc receptor stimulation (23,24), p120 c-cbl might be tyrosine-phosphorylated via intracellular protein-tyrosine kinases which bind to a common cytoplasmic motif, termed ITAM (immune tyrosine-based activation motif, which has been referred to as ARAM or TAM), in those receptors (19,32). Such a sequence motif is not found in the cytoplasmic domain of CD38 (2); however, we can't totally rule out the possibility that the short cytoplasmic domain of CD38 contains an unidentified motif that is capable of interacting with a cellular tyrosine kinase(s). We have not determined what kind of protein-tyrosine kinase(s) is activated upon stimulation of CD38 in RA-differentiated HL-60 cells. In this regard, an interesting finding has been reported that B cells from X-linked immunodeficient mice, which have a defect in Btk protein-tyrosine kinase (33), could not proliferate in response to anti-CD38 mAb (34), suggesting that Btk might be involved in the signal transduction via CD38 in B cells. However, considering that CD38 is also present in several types of hematopoietic cells (1,(3)(4)(5), including thymocytes in which Btk is absent (35), there might be some distinct pathways for CD38mediated intracellular signaling. Although the mechanism whereby p120 c-cbl , together with p87 and p77, is tyrosine-phosphorylated after the stimulation of CD38 remains unknown, identification of such tyrosine kinase(s) leading to the phosphorylation of p120 c-cbl in RA-treated HL-60 cells would provide useful information for understanding the transmembrane signaling mediated by CD38.