(Received for publication, September 1, 1995; and in revised form, November 9, 1995)
From the
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, 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 glycohydrolase (NADase) (
)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, 8, 9) , which is a
novel candidate that mediates Ca
release from
intracellular Ca
stores (see (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, 19, 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.
Figure 1:
Tyrosine phosphorylation of cellular
proteins after stimulation of RA-differentiated HL-60 cells with
anti-CD38 mAbs. A, HL-60 cells which had been cultured with 1
µM RA for 48 h were incubated at 37 °C for 2 min
without (lane 1) or with 7.5 µg/ml of the control IgG1 (lane 2) or the anti-CD38 mAb, T16 (lane 3) or HB-7 (lane 4). Tyrosine-phosphorylated proteins in the cell lysate,
after being immunoprecipitated with PY20, were separated by SDS-PAGE
and then visualized as described under ``Experimental
Procedures.'' The molecular weight markers used were obtained from
Bio-Rad and are indicated in kilodaltons. B, HL-60 cells which
had been cultured for 48 h without (lanes 1 and 2; Un) or with 0.5 mg/ml of dibutyryl cAMP (lanes 3 and 4; Bt-cAMP) or 1 µM RA (lanes 5 and 6; RA) were stimulated
at 37 °C for 2 min with the control IgG1 (C), or the
anti-CD38 mAb (H), and then tyrosine-phosphorylated proteins
were visualized as described above.
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 CD38-induced tyrosine phosphorylation in cells.
Figure 2: Time course of the anti-CD38 mAb-stimulated tyrosine phosphorylation of cellular proteins in RA-differentiated HL-60 cells. A, RA-differentiated HL-60 cells were incubated with 7.5 µg/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.
Figure 3:
Tyrosine kinase activity in the
PY20-immunoprecipitated fraction, as stimulated by the addition of HB-7
to differentiated HL-60 cells. RA-differentiated (,
) or
undifferentiated (
,
) HL-60 cells were incubated with 7.5
µg/ml of HB-7 (
,
) or the control IgG1 (
,
)
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.
Figure 4:
Tyrosine phosphorylation of the c-cbl proto-oncogene product upon stimulation of CD38. A, a
cell lysate was prepared from RA-differentiated HL-60 cells which had
been incubated with the control IgG1 (C) or anti-CD38 mAb HB-7 (H) at 37 °C for 2 min and then
p120 in the cell lysate was
immunoprecipitated with its pAb. The precipitated proteins, after being
separated by SDS-PAGE, were subjected to immunoblot analysis with the
anti-PY pAb (lanes 1 and 2) or
anti-p120
pAb (lanes 3 and 4), as described under ``Experimental Procedures.''
The position of p120
is indicated
by the arrow. B, a cell lysate prepared from HB-7-stimulated
HL-60 cells was first subjected to three rounds of immunoprecipitation
with the control IgG1 (lanes 1 and 3) or
anti-p120
pAb (lanes 2 and 4). The lysates, after being immunoprecipitated with PY20,
were separated by SDS-PAGE and then analyzed by immunoblotting with the
anti-p120
pAb (lanes 1 and 2) or anti-PY pAb (lanes 3 and 4).
The
relationship between this p120 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
antibody to
deplete p120
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
pAb or anti-PY pAb (Fig. 4B). As expected, the depletion of
p120
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
in the HL-60 cells.
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
tyrosine-phosphorylated proteins stimulated by anti-CD38 mAb is the
c-cbl proto-oncogene product with M of
120,000, p120
. 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
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
has an ability to bind the SH2 domains of Fyn, Lck, and Blk
protein-tyrosine kinases, GTPase-activating protein and phospholipase
C
, and that p120
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, p120
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 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 CD38-mediated intracellular signaling.
Although the mechanism whereby p120
,
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
in RA-treated HL-60 cells would provide useful information for
understanding the transmembrane signaling mediated by CD38.