(Received for publication, June 2, 1995; and in revised form, September 1, 1995)
From the
CD45 is a transmembrane protein-tyrosine phosphatase required
for antigen receptor signaling in lymphocytes. CD45 activates the Src
family protein-tyrosine kinases, p56and
p59
, by dephosphorylating a negative regulatory
tyrosine in the carboxyl terminus. Immunoprecipitation of CD45
precipitates p56
and CD45AP. Although the
function of CD45AP is unknown, it has been proposed to be an adapter
between p56
and CD45. To assess the ability of
CD45AP to function as an adapter, we determined the regions required
for the interaction with CD45 by expressing chimeric proteins in HeLa
cells. CD45AP has a region similar to a potential protein-protein
interaction domain, the WW domain. Surprisingly, this domain was not
necessary for the association with CD45. Rather, a 40-amino acid
sequence encompassing the putative transmembrane domain of CD45AP was
sufficient to mediate binding to CD45. Similarly, a 39-amino acid
sequence encompassing the CD45 transmembrane region was sufficient to
direct the interaction with CD45AP. Expression of p56
with CD45AP resulted in an interaction that could only be
detected by in vitro kinase reaction, suggesting that the
association of p56
and CD45AP is weak. These
data support a model in which CD45AP links CD45 with other proteins but
not necessarily p56
.
Engagement of the antigen receptor on T cells results in the
activation of protein-tyrosine kinases coupling antigen recognition to
subsequent signaling events. Two members of the Src family of
protein-tyrosine kinases, p56 and
p59
, have been implicated in T cell receptor
(TcR) (
)signaling. Therefore, protein-tyrosine phosphatases
are predicted to be antagonistic to receptor-signaling events. However,
CD45, the major transmembrane protein-tyrosine phosphatase expressed by
leukocytes, is required for signaling in T cells. Both CD4
and CD8
CD45-deficient T cell clones are
impaired in the ability to proliferate to
antigen(1, 2) . Furthermore, CD45-deficient T cell
leukemic lines fail to induce tyrosine phosphorylation in response to
CD3 cross-linking(3) . Thus, CD45 is required to initiate the
earliest steps of T cell receptor signaling.
p56 and p59
from CD45-deficient cells are
increased in tyrosine phosphorylation, suggesting that both enzymes are
substrates of CD45. All Src family members are negatively regulated by
tyrosine phosphorylation(4, 5, 6) . Peptide
mapping indicates that the site of increased phosphorylation is the
negative regulatory site. Consequently, both kinases from
CD45-deficient cells are decreased in kinase activity(6) . The
difference in tyrosine phosphorylation and kinase activity is observed
when the T cell clones are extensively rested, suggesting that the
requirement for CD45 is prior to antigen receptor
engagement(6) . Therefore, it is likely that the defect in
CD45-deficient cells is the failure to activate the Src family kinases
prior to antigen receptor engagement.
Comparison of p56 and p59
from CD45-deficient and
-expressing L3 T cells demonstrates an 8- and 2-fold increase in
tyrosine phosphorylation, respectively(6) . In the L3 T cell
clone, p59
is expressed at nearly twice the
level of p56
, (
)yet both are
comparable substrates for CD45 in vitro. Therefore, it appears
that CD45 preferentially interacts with p56
.
Furthermore, a chimeric protein consisting of the amino-terminal unique
domain of p59
and the remaining
carboxyl-terminal portion of p56
is
phosphorylated at the negative regulatory site in a manner similar to
p59
(7) . Interestingly,
p60
does not demonstrate increased
phosphorylation at the negative regulatory site if transfected into
either CD45-expressing or -deficient cells(5) . Therefore, the
interaction of CD45 with distinct Src family members appears to be
regulated. It is possible additional proteins, which interact with CD45
may modulate substrate accessibility.
A direct interaction between
p56 and CD45 has been demonstrated upon
phosphorylation of CD45 by p50
in
vitro(8) . Purified p56
SH2 domains
added in excess can inhibit the binding of p56
to CD45. However, it is unclear as to whether this is the
mechanism of interaction in vivo.
In vitro kinase
reactions of CD45 immunoprecipitates demonstrate the presence of
p56 and an associated protein of M
36,000, CD45AP, also termed lymphocyte
phosphatase-associated protein(9, 10, 11) .
Although CD45 is expressed by all nucleated cells of hematopoietic
origin, CD45AP expression is restricted to lymphocytes(11) .
The molecular mechanism by which CD45 and CD45AP associate are unknown.
Furthermore, it is unknown whether CD45AP is required for the
association of CD45 and p56
.
Sequence
analysis of the mouse and human CD45AP cDNAs predicts a mature protein
of 198 amino acids containing a 20-amino acid leader sequence, a
7-amino acid extracellular domain, a membrane-spanning region of 21
amino acids, and a 150-amino acid cytoplasmic domain (10, 11, 12) . Interestingly, the first 50
amino acids of the cytoplasmic domain of CD45AP have significant
sequence similarity to a potential protein interaction domain, the WW
domain (Fig. 1). The signature residues of a WW domain are
highly conserved tryptophan residues. WW domains are found in proteins
with diverse functions (13, 14) including dystrophin,
utrophin, Ess 1, a protein required for cell division in yeast, and
Yap, a protein that associates with p60. It is
possible WW domains may function in a manner similar to SH2, SH3, or PH
domains(14) .
Figure 1: Comparison of a potential WW domain within CD45AP with other putative WW domains. Sequences are derived from (14) . Identical residues and conservative replacements found in the majority of the sequences are indicated by boldface.
To define the sites by which CD45 and CD45AP
interact, we expressed chimeric proteins between CD45 and CD8 or CD45AP
and p60. These studies demonstrate
that CD45 and CD45 interact through their respective transmembrane
regions. Furthermore, co-immunoprecipitations studies suggest that the
association between CD45 and CD45AP is much stronger than the
association between CD45AP and p56
.
Figure 2:
Schematic diagram of the chimeric proteins
expressed in HeLa cells. Numbers above the bars indicate the position of the amino acids derived from each
protein. Portions derived from each protein are indicated as follows:
CD45, open bar; CD8, black bar; CD45AP, stippled
bar; the epitope-tag derived from c-myc, cross-hatched bar;
p60, checkered bar. The
dark shading at the amino terminus of CD45AP
indicates the
12 potential amino acids encoded by the possible alternatively spliced
isoform (see ``Experimental
Procedures'').
CD45 and
CD45AP were expressed in HeLa cells either individually or
together (Fig. 3A). In this system, neither protein required
the presence of the other for expression. This is in contrast to a
previous report that indicated that CD45AP was undetectable in
CD45-deficient Jurkat cells, suggesting that CD45AP required the
presence of CD45(11) . The difference may be due to the
transient, high level expression obtained with the vaccinia/HeLa
system.
Figure 3:
CD45 interacts with CD45AP through the
transmembrane region. A, CD45 associates with CD45AP in HeLa
cells. CD45 (lanes 3-6) and/or CD45AP (lanes 1, 2, 5, and 6) were expressed in HeLa cells.
Cells were lysed and immunoprecipitated with either anti-c-myc monoclonal antibody, 9E10 (lanes 1, 3, and 5) or anti-CD45 monoclonal antibody, I3/2.3 (lanes 2, 4, and 6). Immunoprecipitates were resolved on an
SDS-polyacrylamide gel and Western blotted with either anti-CD45
antiserum (top panel) or 9E10 (bottom panel). B, identification of CD45 sequences required for the
association with CD45AP. CD45AP (lanes 1-10)
and chimeric proteins CD8/8/45 (lanes 3 and 4),
CD8/45/45 (lanes 5 and 6), CD8/8/* (lanes 7 and 8), and CD8/45/* (lanes 9 and 10)
were expressed in HeLa cells. Cells were lysed and immunoprecipitated
with either 9E10 (lanes 1, 3, 5, 7,
and 9) or anti-CD8 monoclonal antibody 53-6.72 (lanes 2, 4, 6, 8, and 10). Immunoprecipitates were resolved on an SDS-polyacrylamide
gel and Western blotted with 9E10. C, cell surface expression
of the chimeric proteins expressed in B. Cells were stained
with directly fluorescein-conjugated anti-CD8 and analyzed on a
FACScan.
Immunoprecipitation of CD45 co-expressed with CD45AP demonstrated that the two proteins associate in HeLa cells (Fig. 3A). Likewise, CD45 will immunoprecipitate with
CD45AP
using the anti-c-myc monoclonal antibody.
Thus, we conclude that the association of CD45 with CD45AP is direct
and does not require the presence of other lymphoid-specific proteins.
Figure 4:
The
transmembrane region of CD45AP is required for the interaction with
CD45. A, identification of CD45AP sequences required to
interact with CD45. CD8/45/* (lanes 1, 2, 7, 8, 9, and 10) p60 (lanes 3, 4, 7, and 8),
and APsrc (lanes 5, 6, 9, and 10)
were expressed in HeLa cells. Cells were lysed and immunoprecipitated
with either anti-p60
antibody 327 (lanes 1, 3, 5, 7, and 9),
or anti-CD8 monoclonal antibody, 53-6.72 (lanes 2, 4, 6, 8, and 10).
Immunoprecipitates were subjected to a kinase reaction in the presence
of [
-
P]ATP, resolved on an
SDS-polyacrylamide gel and proteins visualized by autoradiography. B, cell surface expression of the chimeric proteins expressed
in A. Cells were stained with directly fluorescein-conjugated
anti-CD8 and analyzed on a FACScan.
Figure 5:
Weak interaction between CD45AP and
p56. CD45AP
(lanes 1, 2, 5, and 6) and p56
(lanes 3-6) were expressed in HeLa cells. The
cells were lysed and immunoprecipitated with either
anti-c-myc, 9E10 (lanes 1, 3, and 5) or anti-p56
antiserum (lanes
2, 4, and 6). Half of the immunoprecipitate was
resolved on an SDS-polyacrylamide gels and Western blotted with either
anti-p56
antibody, 3A5 (top panel) or
9E10 (middle panel). The remaining portion of the
immunoprecipitate was subjected to a kinase reaction in the presence of
[
-
P]ATP resolved on an SDS-polyacrylamide
gel, and proteins were visualized by autoradiography. B,
CD45AP
and p56
were expressed in
HeLa cells. Anti-c-myc (lane 1),
anti-p56
(lane 2), and anti-ICAM-1 (lane 3) immunoprecipitates were subjected to kinase reactions
as in A. Western blots were performed in parallel and were
similar to the results of A (data not
shown).
These studies demonstrate that CD45 and CD45AP associate
through a region encompassing their respective transmembrane regions.
The transmembrane regions of both CD45 and CD45AP are highly conserved.
Human and shark CD45 are 70% identical in the region we identified as
sufficient for interaction with CD45AP. ()Similarly, human
and mouse CD45AP are 79% identical in the region that can direct
binding to CD45. The high degree of conservation in these regions
suggests that the association of CD45 and CD45AP is of functional
importance. One potential function is that CD45AP may link CD45 to
other molecules. The identification of a potential WW domain within
CD45AP provides a possible mechanism by which CD45AP could function as
an adapter protein. It will be of much interest to determine whether
this region mediates interactions with other proteins. It is important
to note that WW domains are found in another potential adapter
molecule, Yap. Yap interacts with the p60
SH3 domain
through a proline-rich sequence carboxyl-terminal to the WW
domain(17) .
Chimeric proteins utilizing only the cytoplasmic domain of CD45 expressed in CD45-deficient cells are sufficient to rescue inositol trisphosphate turn over and intracellular calcium fluxes in response to TcR stimulation(18, 19, 20) . This implies that CD45AP is not required for the regulation of Src family member kinases, tyrosine phosphorylation, or second messenger production. It is possible that the association of CD45 with CD45AP is necessary to couple CD45 to responses not measured by these assays.
The
transmembrane association of CD45 with CD45AP is significant.
Association through the transmembrane regions has been demonstrated for
receptor complexes, most notably the T cell and B cell antigen
receptors. In the case of the TcR, the - and
-chains
associate with the CD3 complex and
-chain homodimers through
transmembrane regions. Transport to the plasma membrane requires the
association of the
- or
-chains(21) . CD45AP is
expressed only by lymphocytes and, therefore, unless there is an
analogous molecule in other hematopoietic-derived cells, CD45AP is not
required for the transport of CD45. However, it appears that CD45
expression increases the half-life of CD45AP, possibly by affecting
transport(11) .
Oligomerization of receptor components is required for the initiation of many signal transduction cascades. The transmembrane regions of receptor tyrosine kinases is required for dimerization and autophosphorylation. An oncogenic form of the neu receptor tyrosine kinase has a point mutation in the transmembrane domain that stabilizes dimer formation and causes constitutive signal transduction (22) . Therefore, the ability of CD45 to associate with CD45AP through transmembrane regions may affect the signal transduction properties.
A chimeric protein consisting of the extracellular and transmembrane regions of the epidermal growth factor receptor and the cytoplasmic domain of CD45 will rescue TcR-mediated signal transduction in CD45-deficient cells(18) . Addition of epidermal growth factor results in the dimerization of the chimeric receptor and blocks TcR-mediated signal transduction. It is possible that dimerization of CD45 will affect the ability of the TcR to function. Furthermore, CD45AP may mediate that effect, although it appears CD45AP can associate with both monomers and dimers of CD45 isolated by sucrose gradients(23) .
Expression of
p56 and CD45AP in HeLa results in a small proportion of
p56
associated with CD45AP. Significantly, the proportion
of p56
associated with CD45AP could only be detected by in vitro kinase reaction. This suggests that the direct
interaction of p56
with CD45AP is weak. The weak
interaction of p56
with CD45AP in HeLa cells may be the
result of a detergent effect or a co-localization to the caveoli, which
are found in HeLa cells but not in lymphocytes(24) .
p56
is modified in the amino-terminal region by both
myristylation and palmitylation and will associate with
glycophosphoinositol-linked (GPI-linked) proteins in
detergent-insoluble immunoprecipitates. This is dependent on the
palmitylation of the amino-terminal cysteines (25, 26, 27) .
p60
, which is not palmitylated, does not
associate with GPI-linked proteins(25, 28) .
Preliminary data indicates that the palmitylation of p56
is required to detect an interaction between CD45AP and
p56
in HeLa cells. A chimera of the first 10 amino acids
of p60
replacing the first 10 amino acids of
p56
did not co-immunoprecipitate with CD45AP in an in
vitro kinase reaction (data not shown). The association of
p56
with GPI-linked proteins may represent a
co-localization to a microdomain or an association driven by the use of
detergents since co-capping studies do not demonstrate co-localization
of a GPI-linked protein, Thy-1, with the detergent-insoluble glycolipid
GM
(24) . Therefore, the weak interaction of
p56
with CD45AP in HeLa cells may result from an
association in a microdomain or the dissolution of the membrane in
detergents. Consequently, it is important that a functional
relationship be established between CD45AP and p56
, since
the direct interaction is extremely weak. However, CD45AP may function
as an adapter for CD45 if additional lymphocyte specific proteins are
required as linkers between CD45AP and p56
.
It will be important to determine whether CD45AP affects CD45 function. The demonstration that CD45 and CD45AP interact through their respective transmembrane regions suggests a possible mechanism to block CD45 and CD45AP interaction and examine the effects on transport, dimerization, phosphatase activity, and signal transduction.