(Received for publication, September 1, 1995; and in revised form, November 20, 1995)
From the
Evidence from a large body of studies indicates that CD44 is
involved in a number of important biological processes, including
lymphocyte activation and homing, hematopoiesis, and tumor progression
and metastasis. A proper understanding of the role of CD44 in these
processes has been severely hampered by a lack of insight into the mode
in which CD44 communicates with intracellular signal transduction
pathways. In this report, we have addressed this aspect of CD44
functioning by studying CD44 signaling in T lymphocytes. We show that
ligation of CD44 by monoclonal antibodies (mAbs) transduces signals to
T cells which lead to tyrosine phosphorylation of ZAP-70 and other
intracellular proteins. In vitro kinase assays demonstrate
that cross-linking of CD44 induces an increase in the intrinsic
activity of p56. Furthermore,
immunoprecipitations show that CD44 is physically associated with
p56
. Our findings suggest that tyrosine kinases,
particularly p56
, play a central role in CD44
mediated signaling.
CD44 is a broadly distributed family of cell surface glycoproteins involved in cell-cell and cell matrix adhesion(1, 2, 3, 4, 5, 6, 7) . Although the exact spectrum of functions of CD44 is presently unknown, members of the CD44 family have been implicated in a number of important biological processes, including lymphocyte functioning, hematopoiesis, and tumor progression and metastasis(1, 6, 8, 9, 10, 11, 12, 13, 14, 15) . The CD44 gene consists of 20 exons(16) . Due to alternative RNA splicing which involves at least 10 exons encoding domains of the extracellular portion of the CD44 molecule, a large number of CD44 isoforms are generated. In addition to variable exon usage, variations in glycosylation contribute to the structural and functional diversity of CD44(1) .
A widely expressed CD44 isoform is the
``standard'' or ``hematopoietic'' CD44 (CD44s)
molecule(1, 4, 10) . On hematopoietic cells
and lymphocytes this 85-95-kDa molecule is the principle CD44
isoform(4, 10, 17, 18, 19) .
Larger CD44 variants that contain different combinations of
alternatively spliced exons are preferentially expressed on epithelial
cells(17, 18, 19) , but they can also be
found on activated lymphocytes (10, 20) and high grade
malignant lymphomas(10) . During lymphocyte ontogeny and
activation the expression of CD44 is strictly regulated, suggesting an
important functional role for
CD44(1, 8, 21) . Indeed, CD44 has been
reported to be involved in a variety of lymphocyte functions including
lymphopoiesis(11) , lymphocyte homing(6) , and
lymphocyte activation (22, 23, 24, 25, 26) . In
TCR()
CD3- and CD2-mediated T cell activation, CD44 can
function as an important costimulatory molecule leading to enhanced
proliferation and cytokine release (22, 23, 24, 25) . Furthermore,
engagement of CD44 can lead to activation of the integrin LFA-1
(CD11a/18) on the cell surface of T lymphocytes resulting in enhanced
adhesiveness(26) . Taken together, these data suggest that CD44
functions as an important signaling molecule in immune interactions;
however, the signal transduction pathways involved in this
CD44-mediated signaling are unknown.
Phosphorylation of proteins on
tyrosine residues through protein tyrosine kinases is a key event in
the regulation of cell growth and differentiation(27) . In T
lymphocytes, they play a pivotal role in antigen-specific activation
and proliferation(28) . In the present study, we have therefore
explored the possible role of protein tyrosine kinases in CD44-mediated
signaling. The results show that triggering of CD44 transduces signals
across the plasma membrane that lead to tyrosine phosphorylation of
ZAP-70 and other intracellular proteins. Furthermore, we demonstrate a
physical association between the protein tyrosine kinase
p56 and the CD44 molecule, suggesting its
importance in the CD44 signaling pathway.
For triggering or precipitation of
CD44, the mAbs Hermes-3(29) , J173 (Immunotech S.A., Marseille,
France), and NKI-P1 (5) directed against epitopes on the
standard part of CD44 were used. Biotinylated Leu-4 (Becton Dickinson,
Mountain View, CA) was used for triggering of CD3. Polyclonal
antibodies against p56(30) alone or together
with swine anti-rabbit Ig (S
R) were used for immunoprecipitation
of the p56
. Polyclonal antibodies against ZAP-70 (kindly
provided by Dr. Arthur Weiss, University of California, San Francisco,
CA) were used for immunoprecipitation and immunoblotting of ZAP-70.
G
M Ig (Southern Biotechnology Inc. Birmingham, AL) and Steptavidin
(Sigma) were used for cross-linking of CD44 or CD3 receptors.
Horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine (PY)
(PY20-HRP) (Affiniti, Nottingham, UK) was used for the detection of
tyrosine phosphorylated proteins. mAbs against p56
(Santa
Cruz Biotechnology Inc.) or Hermes-3, together with HRP-conjugated
rabbit anti-mouse Ig were used for detecting the p56
or
CD44 proteins on blots. Polyclonal antibody against p56
together with HRP-conjugated goat anti-rabbit Ig-HRP were also
used to detect p56
. mAb against CD27, 2E4 (kindly
provided by Dr. Rene van Lier, CLB, Amsterdam, The Netherlands) were
used for immunoprecipitation of CD27.
Triggering of CD44 on resting peripheral blood T cells with either of two anti-CD44 mAbs that recognize epitopes on CD44s (Hermes-3 and J173) induced a rapid and important increase of tyrosine phosphorylation of several intracellular proteins, including substrates with a molecular mass of approximately 140, 125, 100, 70, 55, and 45 kDa (Fig. 1). This indicated that CD44 might be physically associated with intracellular kinase(s). To assess this possibility, the kinase activity of CD44 immunoprecipitates of Nonidet P-40-treated T cell lysates was studied by means of the in vitro immune complex kinase assay. As is shown in Fig. 2, the anti-CD44 precipitates yielded a major phosphorylated protein, migrating at approximately 55-60 kDa.
Figure 1:
Induction of
tyrosine phosphorylation by CD44 triggering in resting peripheral blood
T lymphocytes (T-PBL). Anti-PY stained Western blot of purified T cells
that were triggered with anti-CD44 mAbs (Hermes-3 and J173) or anti-CD3
mAb (Leu-4) and cross-linking. At two (lanes 2, 3, 5, and 6) or 5 min (lane 4) after the
addition of the cross-linker, the cells were lysed in Nonidet P-40
sample buffer. 2 10
cells/lane were separated on 8%
SDS-PAGE, transferred to nitrocellulose, and then analyzed by Western
blot using mAb PY20-HRP followed by ECL chemiluminescence detection. T
cells were incubated with lane 1, PBS alone; lane 2,
cross-linker (G
M) alone; lane 3, Hermes-3 plus G
M; lane 4, Hermes-3 plus G
M; lane 5, J173 plus
G
M; lane 6, biotinylated Leu-4 plus
streptavidin.
Figure 2:
CD44 is associated with kinase activity.
Kinase activity of CD44 immunoprecipitates from T-PBL (with mAb
Hermes-3) was assessed by means of the immune complex kinase assay as
detailed in materials and methods. lane 1, Control precipitate
(beads coupled to normal mouse serum (NMS)); lane 2,
precipitate of CD27; lane 3, precipitate of CD44 from
unstimulated T-PBL; lane 4, control precipitate (uncoated
beads); lane 5, precipitate of CD44 from CD44 (with mAb
J173)-triggered T-PBL. The arrow points at a 55-60-kDa P-labeled protein detected only in the immunoprecipitates
of CD44.
To identify the kinase(s) associated
with CD44, a series of immunoprecipitations using antibodies against
CD44 and Src-family tyrosine kinases were performed.
Immunoprecipitation with anti-CD44 co-precipitated a 55-60-kDa
protein reacting with mAb PY20 against PY (not shown). Further studies
showed that this protein reacted also with monoclonal and polyclonal
antibodies against p56 (Fig. 3). Hence,
precipitation of CD44 leads to co-precipitation of p56
,
strongly suggesting a physical association between these two molecules.
To ascertain this finding, reverse precipitations were performed using
antibodies against p56
. These studies demonstrated that
precipitation of p56
leads to co-precipitation of CD44 (Fig. 4). Furthermore, experiments using COS cells that had been
co-transfected with CD44 and p56
cDNAs also confirmed the
association between CD44 and p56
; immunoprecipitation of
CD44 from these cells co-precipitated p56
(Fig. 5). Hence, CD44 is physically associated with
p56
a finding which suggests that p56
might
be functionally involved in the signal transduction via CD44.
Figure 3:
Co-precipitation of p56 with
CD44. Western blot analysis of CD44 immunoprecipitates from T-PBL
stained either with mAb (lanes 1, 2, and 3)
or polyclonal antibody (lanes 4, 5, 6, and 7) against p56
. Lanes 1 and 4, antibody controls; lanes 2 and 5, control
precipitates (unlabeled beads); lanes 3 and 6,
immunoprecipitates of CD44; lane 7, control precipitate (beads
coupled to normal mouse serum (NMS)). The arrow points at the p56
band in the
immunoprecipitates of CD44. The strong band of approximately 60 kDa in lanes 1 and 3 represents the Ig heavy chain of
Hermes-3.
Figure 4:
Co-precipitation of CD44 with
p56. Western blot analysis, of lane 1, the total
cell lysate of T-PBL; lane 2, p56
; and lane 3, control (normal rabbit serum (NRS))
immunoprecipitates from T-PBL, using anti-CD44 mAb (Hermes-3). The arrow points at the CD44 molecule (about 90 kDa). The bands at
55 kDa and 28-35 kDa represent the Ig chains of the Swine
antibodies.
Figure 5:
Co-precipitations of P56 with CD44 from COS cells co-transfected with CD44 and p56
cDNAs. A, B, and C, COS7 cells were
transfected with plasmid DNA containing CD44 and p56
inserts (lane 1), p56
inserts alone (lane 2), and CD44 inserts alone (lane 3). A, anti-CD44 (Hermes-3) stained Western
blot of the lysates of COS-7; CD44 is detected in lanes 1 and 3. The arrows point at different forms of CD44. B, anti-p56
-stained Western blot of the
lysates of COS7; p56
is detected in lanes 1 and 2. C,
anti-p56
-stained Western blot of the CD44
immunoprecipitate from COS7. The arrow points at the
p56
molecule in the
immunoprecipitate.
To
substantiate the notion of a functional association between CD44 and
p56, we next measured the effect of CD44 triggering on
the intrinsic kinase activity of p56
and on the
phosphorylation state of ZAP-70, a substrate of
p56
(31) . As is shown in Fig. 6,
cross-linking of CD44 induced a time-dependent increase in the tyrosine
kinase activity of p56
, as measured by the in vitro kinase assay, with an optimum at 2-5 min after
cross-linking. Only in the precipitates of p56
, obtained
after cross-linking of CD44, prolonged exposure of the gel revealed the
presence of additional phosphorylated proteins (data not shown). This
finding is in agreement with the fact that several intracellular
proteins complex with p56
upon its activation. ZAP-70, a
tyrosine kinase that becomes tyrosine-phosphorylated in lck-dependent manner, was found to become
tyrosine-phosphorylated after cross-linking of CD44 (Fig. 7).
Together, these findings establish that CD44 is functionally linked to
p56
.
Figure 6:
Cross-linking of CD44 induces an increase
in the intrinsic activity of p56. The immunoprecipitates
of p56
were collected by using protein
A-Sepharose beads coupled to polyclonal antibodies against
p56
and subjected to in vitro kinase
assay. A, lane 1, immunoprecipitate of
p56
from unstimulated T-PBL; lanes
2-5, the immunoprecipitates of p56
from CD44-stimulated T-PBL for 0.5 (lane 2), 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. B, a diagrammatic representation of the intensity of the
p56
bands before and after cross-linking of
CD44. The black column represents the upper band of
p56
, whereas the striped column represents the lower band. The optimum kinase activity
(approximately 2.5-fold increase in the tyrosine kinase activity of
p56
) was detected after CD44-triggering for
2-5 min.
Figure 7:
Cross-linking of CD44 induces tyrosine
phosphorylation of ZAP-70. A, immunoprecipitates of ZAP-70
were collected using protein A-Sepharose beads coupled with
anti-ZAP-70. Lane 1, ZAP-70 immunoprecipitate from
unstimulated T-PBL; lane 2, ZAP-70 immunoprecipitate from
CD44-triggered cells for 3 min; lane 3, ZAP-70
immunoprecipitate from CD3-triggered cells; lane 4, molecular
mass marker (from top to bottom 200, 116, 97 and 66 kDa); lane
5, total cell lysate of unstimulated cells; lane 6, total
cell lysate of CD44-triggered cells; lane 7, total cell lysate
of CD3-triggered cells. The filter was subjected to immunoblotting
using PY antibodies and rabbit anti-mouse-HRP. The arrows point to the tyrosine-phosphorylated ZAP-70 detected after
cross-linking of CD44 and was highly phosphorylated after cross-linking
of CD3. B, the same filter was stripped and then incubated
with antibodies against
ZAP-70 antibodies and goat
anti-rabbit-HRP. The arrowheads point to ZAP-70 where the
amounts of ZAP-70 precipitated are equal in lanes 1, 2, and 3, and the presence of ZAP-70 is detected in
the cell lysates.
p56 plays a key role in thymocyte
development and TCR
CD3-mediated
signaling(32, 33) . In a mutant clone of the Jurkat T
leukemia line deficient in lck, signaling through the
TCR
CD3 complex was severely defective; it was restored upon
reconstitution with wild-type lck(33) . Furthermore,
mice lacking functional p56
show a block in early
thymocyte development with a dramatic reduction of the double positive
(CD4
CD8
) thymocyte population and
absence of mature single (CD4
or CD8
)
positive thymocytes(34) . Of the molecules involved in
antigen-specific recognition via the TCR
CD3 complex, CD4 and CD8
are associated with p56
(35) . In addition,
several other receptors on the T cell membrane including CD2, IL-2R,
CD5, and CD50 have more recently also been shown to be associated with
p56
(36, 37, 38, 39) .
Although lck plays a key role in TCR
CD3-mediated T cell
activation and lck can be recruited into the TCR complex via
CD4 and CD8, recent studies have indicated that binding of lck to CD4 is not required for the strongly potentiating effect of CD4
engagement on TCR
CD3-mediated T cell activation(40) .
Presumably, lck, being essential for TCR-mediated signaling,
may also be recruited into the TCR
CD3 complex from other sources
than CD4 (or CD8). lck bound to CD44 might be one of these
sources and CD44 might directly or indirectly interact with components
of the TCR
CD3 complex. Since CD44 is also abundantly expressed on
hematopoietic stem cells, prothymocytes and early
(CD4
, CD8
, and
CD25
) thymocytes it will be of interest to determine
whether CD44 on these cells is associated with p56
. If
so, CD44-lck-mediated signaling, triggered by hitherto
undefined CD44 ligands in the bone marrow and/or thymus might play an
important role in early lymphocyte development.
Our observation that
CD44 in T lymphocytes is associated with p56 raises the
important question how CD44 interacts with lck. In principle,
it is possible that the intracytoplasmic domain of CD44 is directly
involved in lck binding. Since CD44 contains serine
phosphorylation sites, the interaction of CD44 with lck could
be regulated through CD44 serine phosphorylation, in a way similar to
that described for the CD4 molecule. In the CD4 molecule, the serines
are, however, placed next to the p56
recognition site
which contains a the characteristic cysteine motive CXCP (41) that is not present in CD44. Hence, CD44 either uses
another unknown lck-binding motif or is indirectly associated
with lck via a multimolecular complex.
Whether various CD44 splice variants show differential association with lck and with other components of the cell's signal transduction system will be another intriguing question to be answered. On resting T lymphocytes, as used in our present study, CD44 isoforms other than the standard ``hematopoietic'' form of CD44 are virtually absent. However, activation of T lymphocytes leads to a transient expression of several CD44 splice variants(10, 20) . Although the cytoplasmic tail of these variants is identical to that of CD44s, the variations in the extracellular domain might alter CD44 association with lck either by affecting the conformation of the putative cytoplasmic lck binding domain or by modulating its interactions with molecular partners on the T cell surface. In addition to binding to specific (hitherto undefined) ligands, the CD44 splice variants might thus act by modulating signal transduction. In this way they might regulate lymphocyte activation. In other cell types including tumor cells, they might also interact with tyrosine kinases and act as regulators of cell growth, differentiation, and tumor progression. Moreover, it will be important to determine the cascade of the signaling through CD44 and also the role of ZAP-70 in this signaling pathway.