(Received for publication, May 2, 1995)
From the
CD20, a non-glycosylated cell-surface protein expressed
exclusively on B lymphocytes, is one of a family of 4-pass
transmembrane molecules that also includes the chain of the high
affinity receptor for IgE. The precise function of CD20 is unknown,
although in vitro effects of CD20-specific antibodies on
resting B cells indicate that it is able to transduce an extracellular
signal affecting the G
/G
cell cycle transition.
Previous studies have demonstrated that CD20-initiated intracellular
signals involve tyrosine kinase activation and that CD20 is tightly
associated with both serine and tyrosine kinases. Here, analysis of
CD20-associated molecules has revealed that CD20 is associated with the
Src family tyrosine kinases p56/53
,
p56
, and p59
and with
75/80-kDa proteins phosphorylated in vivo on tyrosine
residues. Mutagenesis of CD20 was performed to define regions of CD20
involved in intermolecular interactions. Mutants were analyzed in the
human T lymphoblastoid cell line Molt-4, in which ectopically expressed
wild-type CD20 associated with p59
,
p56
, and 75/80-kDa phosphoproteins. Deletion of
major portions of the cytoplasmic regions of CD20 did not abolish its
association with either p75/80 or tyrosine kinases. The interaction
between CD20 and the Src-related kinases is therefore likely to be
independent of CD20 cytoplasmic domains and may occur indirectly. The
interaction may be mediated by the p75/80 phosphoproteins, which were
found to be tightly associated with the Src family kinases isolated
from the CD20 complex.
Growth and differentiation of B lymphocytes occurs primarily in
the follicles and germinal centers of the spleen and lymph nodes and
depends not only on contact with specific antigen but also upon
co-stimulatory signals derived from accessory cell surface molecules.
Accessory signals may be mediated by soluble factors such as IL-4 or by
intercellular receptor-ligand interactions, such as the interaction
between CD40 on B cells and its ligand on the surface of activated T
cells(1) . Like CD40, CD20 is a B cell surface protein with the
capacity to initiate intracellular signals and modify cell growth and
differentiation(2, 3, 4, 5, 6, 7, 8) .
CD20 is a non-glycosylated 33-kDa protein expressed on all mature B
cells(9, 10, 11, 12, 13) .
The predicted amino acid sequence of CD20 suggests a structure that has
four transmembrane-spanning regions with both amino and carboxyl
termini located on the cytoplasmic side of the plasma
membrane(14, 15, 16) . CD20 is unrelated to a
large family of proteins with similar overall structure (CD9, CD37,
CD69, TAPA-1, and others) but does possess limited sequence homology,
particularly within the transmembrane regions, to a recently identified
molecule expressed in hemopoietic cells, HTm4, and to the chain
of the high affinity receptor for IgE
(Fc
RI)(17, 18) . The cytoplasmic region of
Fc
RI
contains an immunoreceptor tyrosine-based activation
motif (ITAM) (
)and is thought to be involved in relaying
signals for the IgE binding
chain(19) . HTm4 and CD20 do
not contain ITAMs and are not presently known to be part of receptor
complexes.
Recent studies have shown that treatment of resting B
cells with anti-CD20 mAb induces the accumulation of c-Myc mRNA via a
protein-tyrosine kinase (PTK)-dependent signaling pathway (20) . Furthermore, PTK activity was found to be associated
with CD20, and antibodies against CD20 induced tyrosine phosphorylation
of phospholipase C and other unidentified
substrates(20) . Tyrosine kinase activation is a critical early
component of signal transduction pathways initiated by the engagement
of several cell surface receptors, including the B cell antigen
receptor (BCR), CD19, and CD40(1) . These receptors lack
intrinsic kinase activity but are functionally, and in some cases
physically, associated with non-receptor tyrosine kinases. Several
tyrosine kinases expressed in B cells have been implicated in signal
transduction events. Specifically, p72
and the
Src family members p53/56
,
p59
, p55
and
p56
have been found in association with the BCR
complex, where they are thought to be involved in antigen-stimulated
signal transduction(21, 22, 23) . Lyn and Lck
have also been reported to associate with the CD19
complex(24) , and Lyn is activated by anti-CD40 treatment of a
B cell line(25) . An unrelated tyrosine kinase,
p77
, is required for normal B cell
development(26) , but its precise function is not yet known.
Tyrosine kinases can associate with the cytoplasmic regions of
cell-surface molecules by several mechanisms. In this study, we sought
to identify the tyrosine kinases associated with CD20 and to localize
the site of interaction. We found specific association of Lyn, Fyn, and
Lck tyrosine kinases with CD20, whereas no association with Blk, Btk,
or Syk was observed. CD20 also coprecipitated a
75/80-kDa doublet
that was phosphorylated in vivo on tyrosine residues. When
transfected into the human T lymphoblastoid line Molt-4, CD20
associated with Lck and Fyn, the only Src family kinases detected in
these cells, and also with
75-80-kDa proteins, suggesting
that p75/80 or a closely related molecule is also expressed in T cells.
Deletion of 49 of the 51 amino acids, which constitute the
amino-terminal cytoplasmic tail, and 45 of the
85 amino acids of
the carboxyl tail did not affect the ability of CD20 to associate with
either p75/80 or PTK in Molt-4 cells. Deletion of an additional 37
residues at the carboxyl terminus or deletion of 8 of the 13 amino
acids in the internal loop compromised the ability to detect CD20 at
the cell surface. Despite the low level of surface CD20 expression
caused by these deletions, CD20-associated kinase activity was
detected. These results demonstrate that the majority of the
cytoplasmic residues in CD20 are not required for its association with
Src family kinases and suggest that the association is mediated by an
additional molecule. p75/80 was coprecipitated by antibodies against
Fyn, Lyn and, to a lesser extent, Lck, from CD20 complexes following
their elution in 1% SDS, suggesting that p75/80 is tightly associated
with these tyrosine kinases and may mediate their interaction with
CD20.
Figure 1: Constitutive association of kinase activity with CD20. In vitro kinase assays were performed on CD20 complexes immunoprecipitated from Raji cells in the presence of 5 µg of acid-denatured enolase (A) (arrow). Cells were incubated at 37 °C for 10 min with medium alone (lane1) or with CD20 mAb 2H7 (lane2) prior to lysis. B, cells were incubated either at 37 °C for 10 min (lanes1-3) or at 0 °C for 30 min (lanes4-6), with medium alone (lanes1 and 4), with CD20 mAb 2H7 (lanes3 and 6), or with isotype matched control mAb LB2 (CT, lanes2 and 5). C, 2H7 or B1 mAb was added to whole cells at 0 °C for 30 min prior to lysis (lanes1 and 2), or B1 was added to cell lysates (lane3) for 2 h.
Figure 2:
CD20
association with 75/80- and 50-60-kDa proteins tyrosine
phosphorylated in vivo. Anti-phosphotyrosine immunoblots are
shown. A, 3 10
Raji cells were mixed with
medium alone (lane1), 20 µg of control mAb G28.1 (CT, lane2), or 20 µg of CD20 mAb 2H7 (lane3) and then washed, lysed, and subjected to
immunoprecipitation with protein A-Sepharose. Lane4 contains 1 µg of 2H7 mAb only. B, CD20 was
precipitated from 10
cells with 2H7 mAb (lane4); the p85 subunit of PI-3 kinase was precipitated from
lysates of 10
cells (lane2). NRS, nonimmune rabbit serum.
Figure 3:
p72 and
p77
are not detected in CD20 immune
precipitates. A, anti-Syk immunoblot. CD20 was precipitated
from whole cells with 2H7 mAb (lane2); Syk was
precipitated from lysates (lane3); no antibody was
added to the sample in lane1. The filter was probed
with anti-Syk antiserum and then stripped and reprobed with 4G10
anti-phosphotyrosine (B). C, in vitro kinase
assay performed on immunoprecipitated CD20 (lane2)
and Btk (lane4); no antibody was added to the sample
in lane1, normal rabbit serum was added in lane3. Exposure, 30 min. D, anti-Btk immunoblot;
samples are as in C. E, CD20 (lanes1-4) and Btk (lanes5 and 6) were immunoprecipitated and then released from the
Sepharose beads in 1% SDS. Samples were cleared with protein
A-Sepharose, and antibodies were added as follows: lane1, control mAb (CT); lane2,
4G10 anti-phosphotyrosine; lanes3 and 5,
nonimmune rabbit serum; lanes4 and 6,
anti-Btk antiserum. Complexes were recovered with protein A-Sepharose
and separated by SDS-PAGE. Exposure was 1.5
h.
Figure 4:
Association of p59,
p56
, and p56/53
with
CD20. A, Raji cell lysates were mixed with antisera against
Src family kinases as indicated, and in vitro kinase assays
were performed on recovered immune complexes. Exposure, 1 h at room
temperature. B, CD20 was precipitated from Raji cells with mAb
2H7 and released from the Sepharose beads in 1% SDS. Samples were
cleared with protein A-Sepharose, and antibodies were added as
indicated. Exposure, 24 h. Lane7 shows a 1-h
exposure of lane5. NRS, nonimmune rabbit
serum.
Figure 5:
Association of CD20 with p75/80,
p56, and p59
in
CD20-transfected Molt-4 T cells. A, the expression vector
BCMGSneo was transfected into Molt-4 cells either with (M20) or without
(MV) inserted CD20 cDNA, and 2 clones of each were analyzed. M0, untransfected Molt-4 cells. 2H7 was added to all samples,
and in vitro kinase assays were performed on the precipitated
material. Exposure, 30 min. B, CD20 complexes were
precipitated from Molt-4 transfectants, released from the beads, and
reprecipitated with 4G10 anti-phosphotyrosine (lane2), nonimmune rabbit serum (NRS, lane3), anti-Fyn (lane4), or anti-Lck (lane5). Exposure, 24 h.
Figure 6: Expression of CD20 deletion mutants. Indirect immunofluorescence staining with 2H7 anti-CD20 mAb on one representative clone for each deletion construct is shown. The toprow shows staining of Raji cells for comparison. All other rows show Molt-4 transfectants. Results of isotype-matched controls (not shown for simplicity) were the same as the peak shown for MV (Molt-4 cells transfected with the vector alone) stained with 2H7 (secondrow from top). M20, wild-type CD20.
In vitro kinase assays were performed on CD20 immune
complexes precipitated from Molt-4 transfectants expressing cytoplasmic
domain deletion mutants of CD20. The results, shown in Fig. 7,
demonstrate that tyrosine kinase activity was still associated with
CD20 in all cases. Both Lck and Fyn were detected by reprecipitation
from the in vitro phosphorylated CD20 complexes isolated from
transfectants expressing the N50 and C
252 deletions (data not
shown). The internal loop and C
222 deletions were expressed at too
low a level to allow reprecipitation experiments to be performed. Thus,
it is possible that one or both of these deletions could have affected
the association of one, but not both, of the PTKs. p75/80 was clearly
detected in association with each of the CD20 cytoplasmic domain
deletion mutants.
Figure 7:
Deletions involving the cytoplasmic
regions of CD20 do not eliminate associations with p75/80 or PTK. In vitro kinase reactions were performed on CD20 immune
complexes isolated from Molt-4 transfectants expressing N50 (amino
tail deleted) (A), C
252 (carboxyl tail acidic region
deleted) (B), C
222 (carboxyl tail deleted) (C),
or IL
106-115 (internal loop deleted) (D). M0, untransfected Molt-4. MV, Molt-4 transfected with
the vector alone. M20, wild-type CD20 transfectant. Two
independently derived clones of each transfectant are shown. 2H7 mAb
was added to all samples. Exposures were 30-60
min.
CD20-associated tyrosine kinases are shown in this report to
include p53/56, p59
, and p56
,
all members of the Src family. p56
and the non-Src family
kinases p77
and p72
were not detected. The
BCR, like CD20, is associated with multiple tyrosine kinases, including
Lyn, Fyn, Blk, Lck, and Syk(21, 22, 23) . It
is not clear whether each associated Src family kinase has distinct or
overlapping functions, but association of PTK with the BCR is required
for signal transduction upon antigen recognition. The high affinity
receptor for IgE, expressed on mast cells and basophils, is associated
with p56
in the rat basophilic leukemia cell line
RBL-2H3(37) . This association was specific since RBL-2H3 cells
also express p60
, but p60
did not associate
with Fc
RI. In the murine mast cell line, PT-18, Fc
RI is
associated with another Src family kinase,
p62
(37) . Thus, Fc
RI, which transmits
intracellular signals upon IgE binding, associates with some but not
all of the Src family tyrosine kinases, as does CD20 and the BCR.
In
some B cell lines, i.e. SKW6, T51, and a hairy cell leukemia
line, HCLL 7876, CD20 fails to coprecipitate kinase activity (20) , despite the fact that they all express Lyn. SKW6 and T51
also express Fyn, but none of these cell lines express Lck (data not
shown). Interestingly, Lck was expressed in all five human B cell lines
in which CD20 was observed to associate with PTK activity. This
correlation raises the possibility that the expression of Lck is
required for the association of Lyn and Fyn to CD20. Alternatively, the
PTK may associate with CD20 through an intermediate protein, which is
absent in these three cell lines. The addition of recombinant Lck to
CD20 immunoprecipitates isolated from T51, SKW6, and HCLL did not
reveal the presence of p75/80, although an exogenous substrate enolase
was phosphorylated (data not shown). This suggests that CD20
precipitates isolated from these lines may lack p75/80 as well as
p56, consistent with a model in which PTKs associate with
CD20 indirectly by binding to a co-associated molecule, p75/80.
Src
family tyrosine kinases can bind to other proteins via Src-homology 2
(SH2) domains, which recognize phosphorylated tyrosine residues within
specific amino acid sequences. Src-related PTKs also associate with
other proteins by mechanisms that are independent of tyrosine
phosphorylation, such as via SH3 domains or their unique amino-terminal
sequences(33) . Since the cytoplasmic domains of CD20 contain
no tyrosine residues, the PTK must either bind to CD20 indirectly or
via tyrosine-independent means. The association between FcRI and
Lyn has been shown to occur through the carboxyl tail of the Fc
RI
chain, a CD20-related molecule(38) . The carboxyl tail of
Fc
RI
includes an ITAM motif
((YXXL)X
(YXXL)),
although it is unclear whether this motif is involved in the binding of
Lyn. The cytoplasmic regions of CD20 do not include this motif; however
the carboxyl tail is highly charged with 16 acidic residues among the
40 COOH-terminal amino acids and also includes a potential SH3-binding
site (PEPP). Deletion of these acidic and proline-rich regions,
however, did not affect the ability of CD20 to bind to either PTK or
p75/80 in CD20-transfected Molt-4 T cells. Further deletions of the
carboxyl tail resulted in low or no surface expression of CD20.
Nevertheless, associated kinase activity was detected, albeit weakly,
after deletion of 75 COOH-terminal residues. This deletion left
potentially 10-18 amino acids protruding into the cytoplasm,
depending on the location of the fourth TM domain. Thus, although CD20
associations with PTK and p75/80 were independent of major portions of
all three cytoplasmic regions of CD20, it is possible that the region
of the carboxyl tail immediately proximal to the plasma membrane may be
involved in intermolecular associations. Since deletion of this region
results in the loss of CD20 expression, it is not possible to determine
if it is also involved in association to PTKs.
The identity of
p75/80 is not known at the present time. Neither is it known whether
the proteins represented by the two bands migrating at 75/80 kDa
are different proteins or variants of the same protein. Recently, it
was reported that an 85-kDa protein was phosphorylated in in vitro kinase assays performed on immunoprecipitated glycosyl
phosphatidylinositol-linked proteins, Thy-1 and CD48, molecules that
have been shown to associate with Src family kinases Lck and Fyn in T
cells(39) . Interestingly, this 85-kDa protein was also
reprecipitated by anti-Fyn and anti-Lck antibodies, as was the case for
CD20-associated p75/80. The tyrosine kinase Lyn has been found to
associate with the p85 subunit of PI-3 kinase and with HS1, a
75-kDa protein of unknown function(40, 41) .
Antisera against p85 and HS1, however, did not react with
CD20-associated p75/80 in immunoblots or in reprecipitation experiments
(data not shown).
CD20-associated kinase activity was detectable
using the B1 antibody to precipitate CD20 from cell lysates. This
indicates that at least some tyrosine kinase activity is associated
with CD20 in intact cells prior to the addition of antibody. Regulation
of the tyrosine kinase activity may occur by extracellular
interactions, by relocalization of the kinases, or by modifying access
to substrates. A possible alternative means of regulating CD20 function
may be related to the fact that CD20 is also associated in B cells with
serine/threonine kinase (PSK) activity, as well as with tyrosine
kinases(20) . In Molt-4 cells, transfected CD20 did not
coprecipitate detectable PSK activity for reasons that are not clear at
present. The CD20-associated PSK has not been identified, but there is
evidence that CD20 may be a substrate for
PKC(42, 43) . Interestingly, FcRI
has been
recently shown to be associated with the
isoenzyme of protein
kinase C(44) . CD20 is phosphorylated in vivo on
serine/threonine residues, and this phosphorylation is increased by
activation signals initiated by phorbol 12-myristate 13-acetate,
anti-CD40, or
anti-BCR(12, 13, 30, 42, 43, 45) .
Phosphorylation of CD20 may affect the activity or localization of the
associated tyrosine kinases. Alternatively, CD20-associated PSK may act
on the associated tyrosine kinases directly; serine phosphorylation of
Lck in an activated T cell line has been reported to down-regulate its
activity(46) .
In conclusion, CD20 is shown here to be a
component of a multimolecular complex that includes the tyrosine
kinases Lyn, Fyn, and Lck, and a 75/80-kDa doublet of proteins
phosphorylated on tyrosine residues in vivo. Mutational
analysis of the cytoplasmic domains of CD20 suggested that the PTKs do
not directly associate with the cytoplasmic regions of CD20 and
therefore more likely associate indirectly via an additional protein,
perhaps p75/80. As p75/80, or a closely related molecule, appears to be
expressed in both B and T cells, it may function as an adaptor protein
linking Src-like kinases to members of this 4-pass TM family in
lymphocytes. In this regard, as HTm4 mRNA is expressed in Molt-4 T
cells(18) , it will be important to determine if HTm4 protein
is also associated with p75/80 and Src family kinases. Both CD20 and
HTm4, like FcRI
, may be signal-transducing components of
complexes that include receptor subunits. Whether this receptor subunit
is p75/80 or whether p75/80s are adaptor molecules functioning to link
CD20 to downstream activation signals remains to be determined.
Characterization of the p75/80 components of the complex is in progress
to further elucidate the role of CD20 in B cell growth and
differentiation.