(Received for publication, July 8, 1994; and in revised form, October 24, 1994)
From
the
CD45 is a protein-tyrosine
phosphatase expressed on all cells of hematopoietic origin. In an attempt
to further characterize CD45 function, we set out to identify molecule(s)
that specifically associate with CD45. A 116-kDa protein was detected in
immunoprecipitates from CD45 cells but not CD45
cells. The
association between CD45 and this 116-kDa protein can be reconstituted by
mixing lysates from CD45
cell lines with purified
CD45. p116 appears to associate with CD45 through the external,
transmembrane, or membrane-proximal region of CD45 since p116 is associated
with a mutant form of CD45 possessing a truncated cytoplasmic domain. The
association of p116 with CD45 is not isoform-specific as p116 associates
equally well with various CD45 isoforms. We have determined that p116 is a
tyrosine-phosphorylated glycoprotein and that it is associated with CD45 in
all hematopoietic cells examined. Because of its broad distribution, it is
possible that identification of p116 will provide additional insight into
the function of CD45 in lymphoid as well as non-lymphoid hematopoietic
cells.
CD45 is a high molecular weight glycoprotein abundantly expressed on nucleated cells of hematopoietic origin(1) . Isoforms of CD45 arise from differential splicing of 4 exons (exons 4-7) encoding sequences found near the amino terminus of the molecule, giving rise to structurally related molecules differing in molecular weight. Different cell lineages express different isoforms, and the isoform usage often changes as cell differentiate. All isoforms of CD45 share a large, highly conserved cytoplasmic domain containing two subdomains. These subdomains have significant homology with human placental protein-tyrosine phosphatase 1B(2) , and it is well established that CD45 has intrinsic protein-tyrosine phosphatase activity(3, 4) .
CD45 has
been shown to be important for both T- and B-lymphocyte
activation. CD45-deficient T-cell lines are unable to be activated to
proliferate, secrete interleukin-2, or mediate cytolytic events after
stimulation through the T-cell antigen receptor(5, 6, 7) . CD45 appears to exert its function early in the
activation cascade since tyrosine phosphorylation, generation of inositol
phosphates, and an increase in intracellular Ca after stimulation through the
T-cell or B-cell receptor for antigen are ablated in most CD45-deficient
cells (8, 9, 10, 11)
. Transfection of chimeric molecules containing the cytoplasmic domain of
CD45 into CD45
cells restores the signaling
capacity, suggesting that the external domain is not required for these
early activation events(12, 13, 14) . CD45 appears to
regulate the phosphorylation state and the enzymatic activity of
src-related tyrosine kinases including p56
and p59
(4, 15, 16, 17, 18, 19) , consistent with
it being required early in the activation cascade. It is not clear if CD45
has additional substrates or if the regulation of src-related
kinases is its only function during lymphocyte activation.
In an attempt to further define the role of CD45 in lymphocyte activation, a number of investigations have been undertaken to identify molecules that are specifically associated with CD45. Such associated molecules might regulate the enzymatic activity or modulate the physical proximity of CD45 with its specific substrate(s). The most commonly employed approaches include chemical cross-linking and cocapping. Using the same chemical cross-linking reagent, one group has demonstrated that CD45 immunoprecipitates contain CD2 (20) and another Thy-1 and the T-cell antigen receptor(21) . Cocapping studies suggest that the association of CD45 with CD4, CD8, LFA-1, and CD2 is isoform-specific(22, 23) . It has also been reported that CD45 is coimmunoprecipitated with CD26 (dipeptidyl peptidase IV) from digitonin lysates of human T-cells(24) . Since the expression of these various molecules is restricted to certain cell lineages, we speculate that there may be alternative molecules associated with CD45 in other cell types or that there may be more ubiquitously expressed molecules associated with CD45 in all hematopoietic cells.
Studies have also been performed to determine if there are protein interactions with the cytoplasmic domain of CD45 that may regulate its function. CD45 is associated with a 30-kDa protein (25) that has no homology with known sequences and appears to be expressed in all leukocytes(26) . This molecule has been postulated to be an adapter molecule for CD45-mediated signaling events. The cytoskeletal protein fodrin has also been shown to associate with CD45 and may stimulate its enzymatic activity(27) . The interaction of CD45 with the cytoskeleton, through fodrin, may also regulate its association with other cell-surface molecules(27) .
Our goal was to identify molecules
that are stably associated with CD45 in the detergent Nonidet P-40. Herein
we establish that a 116-kDa protein is associated with CD45 in
immunoprecipitates of CD45 but not CD45
T-lymphomas. We
also demonstrate that a 116-kDa protein appears to be specifically
associated with CD45 in all CD45-expressing cells examined. Initial
characterization of this molecule suggests that it is a
tyrosine-phosphorylated glycoprotein. Identification of this 116-kDa
protein might provide additional insight into the function of CD45 in
non-lymphoid cells as well as lymphoid cells.
The
monoclonal antibodies used in this study were I3/2 (29) and M1/9.34 HL2, which are both specific for a
portion of the ectodomain common to all isoforms of CD45, and M1/42, which
is specific for mouse major histocompatibility complex class I
molecules. Both M1/9.34 HL2 and M1/42 were obtained from ATCC. Cells
producing monoclonal antibodies were grown in serum-free media
(protein-free hybridoma medium II, Life Technologies, Inc.) and
concentrated by ammonium sulfate precipitation followed by dialysis. The
purity of the antibodies was verified by SDS-PAGE. ()The antibodies were then directly coupled to
cyanogen-activated Sepharose 4B. The polyclonal antiserum to recombinantly
expressed cytoplasmic domain of CD45 has been described(4) .
For the reconstitution
studies, membranes from CD45 SAKRTLS 12.1 cells were
isolated as described previously(31)
. Briefly, the cells were disrupted by nitrogen cavitation in PBS followed
by high speed centrifugation to separate cytosolic proteins from the
membrane fraction. Cell membranes were reconstituted at 1.6 mg/ml in lysis
buffer, and an additional ultracentrifugation step was performed to
eliminate the detergent-insoluble material. Samples were saved at each step
for I3/2 immunoprecipitation in the presence or absence of approximately 10
µg of purified CD45. Beads mixed with detergent-solubilized fractions
were prepared for SDS-PAGE by washing 5 times in lysis buffer containing
0.5 M NaCl and 3 times in regular lysis buffer. Beads treated with
cytosolic proteins in the absence of detergent were washed 8 times in
PBS.
Figure
1:SDS-PAGE of CD45 immunoprecipitated from
CD45
and CD45
cell lines. A,
silver-stained gel of I3/2 immunoprecipitates prepared from 4
10
CD45
(+) and CD45
(-) SAKRTLS
12.1 cells. B, as in A except isotype-matched control
antibody M1/42 was used in parallel and the proteins were detected by India
ink staining of Immobilon-P after transfer. C, as in A except cells
were metabolically labeled with [
S]methionine. D, as in B
except anti-CD45 antibody M1/9.34 HL2 was employed. Arrows indicate
the position of p116.
To ascertain if the
association of p116 with anti-CD45 immunoprecipitates is unique to the
anti-CD45 monoclonal antibody that we employed, we have also used a second
antibody to CD45, M1/9.34 HL2, and found that a 116-kDa protein is also
present in these immunoprecipitates (Fig. 1D). Taken together, these results
suggest that the 116-kDa protein specifically associates with CD45 and does
not nonspecifically associate with the immunoprecipitating antibodies or
the Sepharose beads. The association is most likely not due to
cross-reactivity of the antibody because p116 is not immunoprecipitated
from lysates of CD45 cells. Identical results have
also been obtained using sets of CD45
and CD45
cell lines derived
from NZB.1, BW5147, and Yac-1 T-lymphomas (data not
shown).
Figure
2:CD45 immunoprecipitates from various cell
types. A, CD45 (I3/2) or control (M1/42)
immunoprecipitates prepared from freshly isolated C57Bl/6 thymocytes and
from cytotoxic T-cell line 2C were detected by India ink staining after
transfer of SDS-PAGE-separated proteins to Immobilon-P. B, India ink
stain of I3/2 immunoprecipitates prepared from CD45 and CD45
SAKRTLS 12.1 as
well as
2 cells expressing RO (
2-CD45/O)
or RB (
2-CD45/BC) isoforms of
CD45. Immunoprecipitates were also prepared from
2 control cells. All
immunoprecipitates in B were washed 5 times in lysis buffer
containing 0.5 M NaCl. Arrows denote the position of the 116-kDa
protein.
We have obtained 2 cells
that express various isoforms of CD45 that were generated by infection with
recombinant retrovirus containing various isoforms of CD45(28) . As expected, we were unable to detect either CD45
or p116 in I3/2 immunoprecipitates from control
2 cells. Interestingly, we have
found that a 116-kDa protein co-immunoprecipitates with two different
isoforms of CD45 expressed in
2 cells (Fig. 2B). These results raise the
possibility that p116 may be ubiquitously expressed, even in cells that do
not normally express CD45. Furthermore, the association between CD45 and
p116 appears to be relatively strong, in that it is stable in 0.5 M NaCl
in 0.1-1% Nonidet P-40 or digitonin (Fig. 2B and data not
shown).
Figure
3:CD45 immunoprecipitated from a cell line
lacking most of the cytoplasmic domain. India ink staining of I3/2
immunoprecipitates prepared from the BW5147 parent (BW), a CD45 variant
(BW/CD45
) and a revertant
(BW/rev) expressing a truncated cytoplasmic domain. The arrow
indicates the position of p116.
Figure
4:CD45 immunoprecipitated from cell lines
expressing various isoforms of CD45. I3/2 immunoprecipitates of SAKRTLS
12.1 CD45 and CD45
cells, control
2 cells,
and
2 cells expressing CD45/C
(RC), CD45/BC (RB), or CD45/ABC (RA). The
immunoprecipitates were washed in lysis buffer and mixed with lysates from
CD45
SAKRTLS 12.1 cells that had been metabolically labeled with [
S]methionine. After a
60-min incubation at 4 °C, the immunoprecipitates were washed and
subjected to SDS-PAGE followed by autoradiography. An arrow
indicates the position of the 116-kDa protein.
Figure
5:CD45 associates with a 116-kDa protein
found in membranes isolated from CD45 cells. The cytosolic fraction
(lanes3 and 4), Nonidet P-40-solubilized membrane
fraction (lanes5 and 6), and Nonidet P-40-solubilized
membrane fraction depleted of detergent-insoluble material
(lanes7 and 8) prepared from CD45
SAKRTLS 12.1 cells
were mixed with I3/2 beads in the presence (+) or absence (-) of
purified CD45. Immune complexes were washed as described under ``Materials
and Methods.'' As a control, beads and purified CD45 were incubated with
lysis buffer rather than cell extracts (lanes1 and
2). I3/2 immunoprecipitates from 4
10
CD45
(+) and CD45
(-) SAKRTLS 12.1 cells are
also shown (lanes9 and 10). The arrow marks
the position of p116.
To determine if the 116-kDa
protein contains an external domain, we cell-surface biotinylated CD45 and
CD45
SAKRTLS 12.1 cells and immunoprecipitated CD45 under conditions that
maintain the association with p116. It is clear from Fig. 6A that p116 can be biotinylated,
suggesting that it does have an external domain accessible to the
biotinylating agent. Control immunoprecipitates containing p56
or actin
demonstrate that intracellular molecules are not biotinylated (data not
shown). Protein staining of parallel immunoprecipitates run on the same gel
demonstrate that the biotinylated protein at 116 kDa comigrates with p116
as revealed by India ink staining and that both are found only in
immunoprecipitates from CD45
cells (Fig. 6A). Interestingly, we also observe
a 30-kDa biotinylated protein in immunoprecipitates prepared from
CD45
cells but not CD45
cells. It is possible that this is the previously described 30-kDa
adapter protein, which is predicted to have a very small external
domain(26) . Surface iodination
experiments have also been done, and p116 does become iodinated albeit to a
very low level (data not shown). These results, in conjunction with the
data presented in Fig. 5, suggest
that p116 is a membrane-bound protein with an external
domain.
Figure
6:p116 is a tyrosine-phosphorylated
transmembrane glycoprotein. A, I3/2 immunoprecipitates were prepared
in triplicate from CD45 (+) and CD45
(-) SAKRTLS 12.1
cells following biotinylation of cell-surface proteins. Samples were loaded
onto a single gel for SDS-PAGE and transferred to Immobilon-P for detection
by India ink staining or blotting with either Streptavidin or
anti-phosphotyrosine. An arrow indicates the position of the
116-kDa protein. B, I3/2 immunoprecipitates prepared from [
S]methionine-labeled CD45
(lane1) or CD45
(lanes2 and
3) SAKRTLS 12.1 cells were treated with N-glycosidase F (+)
or mock-treated(-) overnight at 37 °C. The immunoprecipitates were
then washed and subjected to SDS-PAGE followed by fluorography. The
arrowheads indicate the position of p116 with or without
N-glycosidase F (EndoF) treatment.
Since CD45 is a protein-tyrosine phosphatase, the determination of the phosphorylation status of p116 is significant. We found, by anti-phosphotyrosine immunoblotting, that a CD45-associated 116-kDa protein is tyrosine phosphorylated (Fig. 6A). The immunoprecipitates used for immunoblotting were run on the same gel with a parallel immunoprecipitate that was stained with India ink to reveal p116. It is clear that the tyrosine-phosphorylated protein comigrates with p116 (Fig. 6A). Identical results were obtained with two different rabbit anti-phosphotyrosine antisera, and antibody binding to the 116-kDa protein could be inhibited with phenylphosphate or phosphotyrosine (data not shown). Furthermore, very little tyrosine phosphorylation is detected when sodium vanadate is omitted from the lysis buffer, suggesting that the phosphorylation of p116 is regulated by a tyrosine phosphatase (data not shown). Taken together, these results support the conclusion that p116 is a tyrosine-phosphorylated protein.
To determine if p116 is a glycoprotein, we incubated immunoprecipitates containing CD45 and p116 with endoglycosidase F/N-glycosidase F, which removes high mannose and complex N-glycans. As shown in Fig. 6B, there is a dramatic shift in the mobility of CD45, which serves as our internal control, and a slight shift in the mobility of p116, suggesting that p116 is a glycoprotein. When these immunoprecipitates are immunoblotted with anti-phosphotyrosine antibodies, there is an identical shift in the tyrosine phosphorylated protein, further supporting the conclusion that p116 is a tyrosine-phosphorylated and glycosylated protein (data not shown). It is also interesting to note that the association between CD45 and p116 is not dependent on N-linked glycosylation of either CD45 or p116 since the association is stable after N-glycosidase F treatment. These results taken together with the membrane reconstitution and surface biotinylation experiments suggest that p116 is a transmembrane glycoprotein.
We have found that a 116-kDa protein specifically associates with CD45 in all CD45-expressing cells examined. This molecule appears to be a tyrosine-phosphorylated transmembrane glycoprotein. The interaction between CD45 and this 116-kDa protein is relatively stable and is easily reconstituted. Chemical cross-linkers are not required to detect the association, which is stable in buffer containing Nonidet P-40 and 0.5 M NaCl. The association is stable overnight at 37 °C and does not require the presence of N-linked glycosylation (Fig. 6B).
A number of studies have been done to identify molecules that are associated with CD45 on the T-cell surface. It has been shown to be associated with Thy-1, the T-cell antigen receptor, CD2, CD4, and CD8 (20, 21, 22, 23) . The expression of these cell-surface molecules is restricted to the T-cell lineage and therefore does not permit generalizations about the role of CD45 in the large number of different cell types in which it is expressed. We have found that p116 is associated with CD45 in every hematopoietic cell type examined, including non-lymphoid cells. Intriguingly, p116 was also detected in cells that do not normally express CD45, suggesting that p116 is ubiquitously expressed.
It has been demonstrated that CD45 is found in CD26 (dipeptidyl peptidase IV) immunoprecipitates prepared from digitonin, but not Nonidet P-40, extracts of human T-lymphocytes(24) . This molecule is about 110 kDa and has a wide tissue distribution raising the possibility that it might be the equivalent of the 116-kDa protein described herein. However we find this unlikely for three reasons. 1) CD26 is efficiently iodinated, whereas p116 is minimally iodinated. 2) CD26 has a different mobility than p116 by SDS-PAGE. 3) Mouse CD26 has no tyrosine in the cytoplasmic domain for phosphorylation, and 4) antibodies to CD26 do not react with p116 either by immunoprecipitation or immunoblotting (data not shown).
A number of observations have allowed us to
eliminate the possibility that p116 is a degradation product of
CD45. First, neither antiserum to the cytoplasmic domain of CD45 nor
monoclonal antibodies to the external domain react with p116 by Western
blotting (data not shown). Second, CD45 cells express p116 as
demonstrated by the labeled reconstitution experiments where labeled p116
from CD45
cells is able to bind to
unlabeled CD45 (Fig. 4). Third,
two-dimensional peptide mapping of CD45 versus p116 from [
S]methioninelabeled cells gives
very distinct patterns (data not shown). These observations, taken
together, suggest that p116 is not a degradation product of
CD45.
The identification of p116 will likely yield important
functional information about CD45. We have worked out a purification scheme
for p116 and are now trying to obtain sequence information. We have also
immunized rabbits with the purified protein to obtain antiserum that will
be useful for further characterization of the interaction. At present, one
can only speculate about the potential functions for such a molecule. One
possibility is that p116 regulates CD45 enzymatic activity either directly
or by allowing or preventing interactions with its substrate(s). Since p116
is tyrosine phosphorylated, it could be a substrate of CD45 and therefore a
downstream mediator of CD45-triggered signaling events. It is also possible
that p116 may be a linker molecule that connects CD45 to additional
signaling pathways similar to the -chain of the T-cell receptor
complex(33, 34)
. Consistent with this, p116 has limited glycosylation and is not readily
iodinated, suggesting that it may have a small external domain as does the
-chain(33) . Preliminary studies suggest that the
phosphorylation of p116 does not change upon T-cell activation; however,
this can be addressed more directly once we have antibodies to the
protein. Further characterization of p116 will provide insight into the
role of CD45 in signal transduction in lymphocytes and other hematopoietic
cells.