From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada
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ABSTRACT |
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CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear. In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains. A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) was catalytically active and found to exist primarily as a dimer. In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) existed as a monomer and had no catalytic activity against any of the substrates tested. Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicated that rD1/D2 was 2-3-fold more catalytically active, was more thermostable, and existed primarily as a monomer. Limited trypsin digestion of rD1/D2 provided evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction. Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay. Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45. In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation.
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INTRODUCTION |
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CD45 is a transmembrane protein-tyrosine phosphatase (PTP)1 that is expressed on all nucleated hematopoietic cells. Its phosphatase activity is required for efficient signal transduction initiated by lymphocyte antigen receptors, which is essential for T cell development and T and B cell activation (reviewed in Refs. 1 and 2). In T cells, CD45 has been shown to dephosphorylate p56lck and p59fyn in vitro and to be responsible for their dephosphorylation in vivo (3-7). The role of CD45 in the dephosphorylation of p56lck and p59fyn is likely to be a necessary prerequisite step for their effective participation in T cell receptor-mediated signaling events. In vitro, CD45 can dephosphorylate several artificial and phosphopeptide substrates, yet only a limited number of substrates have been identified for CD45 in vivo. How substrate specificity is achieved in the cell is not yet understood. However, it is of interest to note that CD45 can associate with p56lck, a major substrate for CD45 in T cells, both in vitro (8, 9) and in T cells, albeit with low stoichiometry (10, 11).
It is also not known how the phosphatase activity of CD45 is regulated.
By analogy with receptors possessing tyrosine kinase activity, it is
possible that receptor tyrosine phosphatases may be regulated by
dimerization. It has been suggested by cross-linking studies in T cells
that CD45 may exist in dimeric form together with CD45AP, a 30-kDa
CD45-associated protein (12). Induced dimerization of an epidermal
growth factor receptor-CD45 chimera with epidermal growth factor has
been shown to inhibit the restoration of T cell signaling events (13),
suggesting the possibility that CD45 activity may be regulated by
dimerization. In addition, the first phosphatase domain (PTP-D1) of a
related phosphatase, RPTP, crystallized as a dimer, with the
membrane-proximal region of one PTP domain inserted into the catalytic
region of the second PTP domain. It was predicted that if such an
interaction occurred under more physiological conditions, phosphatase
activity would be inhibited (14). However, to date, no direct evidence
has been obtained for the formation of CD45 dimers.
CD45, like RPTP, belongs to the growing family of protein-tyrosine
phosphatases. For most members of the transmembrane, two-domain phosphatase family, the majority of catalytic activity has been shown
to reside in PTP-D1 and the function of the second phosphatase domain
(PTP-D2) is unclear. PTP-D2 of RPTP
has been shown to possess
catalytic activity, albeit at much lower levels than PTP-D1 (15). In
other transmembrane PTPs, no phosphatase activity has been detected for
PTP-D2, which in some instances lack key residues involved in catalysis
(Refs. 16-19 and reviewed in Refs. 20-22). In these cases, a
regulatory role for PTP-D2 has been suggested, and in some two-domain
PTPs, the presence of PTP-D2 has been shown to be required for optimal
PTP-D1 activity (15, 17, 19). For CD45, contradictory data exists as to
whether CD45 is an active phosphatase in the absence of PTP-D2 (17, 18,
23). Recent data shows that CD45 PTP-D1 is active when coupled to a
maltose-binding protein fusion partner but is not active when the
fusion partner is proteolytically removed (24). Likewise, conflicting
data exists as to whether CD45 PTP-D2 is an active phosphatase. No catalytic activity has been attributed to CD45 PTP-D2 when expressed in vitro as a recombinant protein (18) or after the
essential cysteine in PTP-D1 has been mutated to a serine and expressed in vitro (16-18). Mutation of the critical cysteine in
PTP-D1, but not in PTP-D2, prevented the restoration of CD45-mediated T
cell signaling events (25), indicating that phosphatase activity residing in PTP-D1, and not PTP-D2, was essential for this function. However, expression in eukaryotic cells of an active, truncated form of
CD45 lacking the catalytic cysteine present in PTP-D1 has led
researchers to suggest that PTP-D2 has phosphatase activity (26). Thus
the function of PTP-D2 of CD45 is unclear.
To clarify the function of the individual PTP domains of CD45 and to determine the role of these domains in the enzymatic function of CD45, recombinant CD45 cytoplasmic domain proteins were produced in Escherichia coli, purified, and analyzed. It was found that rD1 of CD45 was an active protein-tyrosine phosphatase in the absence of PTP-D2 and was present as a dimer. In contrast, no phosphatase activity was detected for rD2 of CD45. In the two-domain CD45 cytoplasmic domain protein, the presence of PTP-D2 enhanced the stability of the enzyme and was required for optimal enzymatic activity. Specific binding of rD1 to rD2 and the association of N- and C-terminal tryptic fragments of rD1/D2 provided evidence for an intramolecular interaction occurring in rD1/D2. In the absence of the C-terminal region including PTP-D2, intermolecular interactions occur, leading to the formation of rD1 dimers.
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EXPERIMENTAL PROCEDURES |
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Materials--
Nickel-NTA-agarose was from Qiagen Inc. (Santa
Clarita, CA.), Sephadex G-25M PD-10, MonoQ and Superose 12 columns were
from Amersham Pharmacia Biotech. p-Nitrophenyl phosphate
(pNPP) was from Pierce, tyrosine-phosphorylated peptides (CD3 pY83,
LGRREEpYDVLEKKRA; fyn pY531; TATEPQpYQPGENL; src pY416; LIEDNEpYTARQGA;
cdc2 pY15; KIGEGTpYGVVYKA; and PDGF-R pY1021; NEGDNDpYIIPLPD) were from
Dr. F. Jirik and Dr. I. Clark-Lewis (Biomedical Research Center,
University of British Columbia). GST-p56lck was
made and purified as described (9), and 4G10-antiphosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid,
N.Y.). Anti-His antibody was from Amersham, and sequencing grade
trypsin was from Promega (Madison, WI.). The GST-Crk-SH3 construct was
from Dr. M. Matsuda (International Medical Center of Japan, Tokyo,
Japan), and 6-His-glucosidase-cellulose binding domain fusion protein
was from G. Doheny (University of British Columbia).
Recombinant DNA Constructs--
Recombinant 6-His-tagged murine
CD45 cytoplasmic domain proteins, rD1/D2, containing residues 564-1268
(numbered as in Ref. 27) and the catalytically inactive protein, with a
mutation of cysteine 817 to serine (C817S), were as described (28). A truncation mutant (rD1) encoding residues 564 to 927, containing the
membrane-proximal region, PTP-D1, and the spacer region, was constructed previously (18) and subcloned into the pET-3D-6HisIEGR vector (28). Another truncation mutant (rD2) encoding residues 901-1268, which contains part of the spacer region, PTP-D2, and the
carboxy tail, was made by removing a DraIII fragment and
adding a linker (5'-AGATCTCCA-3') to introduce a BglII site.
A second construct encoding the membrane-proximal region, PTP-D2, and
carboxy tail (rD2.2) was made by deleting a
HincII-NdeI fragment containing residues 649 to
943 and adding a HincII-NdeI linker
(5'-TATGGAACAACATT-3'), restoring residues 940 to 943. BglII
fragments containing rD2 and rD2.2 in pBluescript SK (Stratagene, La
Jolla, CA) were subcloned into pET-3D-6HisIEGR (28), and
rD2 was also subcloned into pGEX-3X vector (Amersham).
Expression and Purification of Recombinant Proteins--
Vectors
were expressed in BL21(DE3) (29) or XL1-Blue E. coli
(Stratagene). rD2.2 was expressed in conjunction with a pT-Trx vector
(30). rD1/D2, the C817S mutant protein, rD1, and rD2 were purified
essentially as described (28) with the following modifications.
Cultures were induced at 26 °C, and all other steps were at 4 °C.
Cells were lysed in 100 µg/ml lysozyme, 0.5% Triton X-100, 20 mM Tris, pH 7.5, 150 mM NaCl, 20 mM
imidazole, pH 7.2, 0.025% -mercaptoethanol, 1 mM EDTA.
Proteins bound to Nickel-NTA-agarose were washed with lysis buffer plus
0.5 M NaCl and 1.0 M NaCl and eluted in 1 M imidazole, pH 7.2, 150 mM NaCl, 0.025%
-mercaptoethanol, and 0.1% Triton X-100. The eluent was passed
through a PD-10 column equilibrated in 90% buffer A (20 mM
Tris, pH 7.2, 0.1% Triton X-100, 0.025%
-mercaptoethanol), 10%
buffer B (1 M NaCl in buffer A) and eluted from a MonoQ
column using a linear gradient of 10 to 40% buffer B. rD2.2 was
purified as above except instead of separation on a MonoQ column, it
was loaded onto a Superose 12 column equilibrated in 20 mM
Tris, pH 7.2, 0.1% Triton X-100, 0.025%
-mercaptoethanol, 160 mM NaCl. All buffers used for protein purification
contained 0.2 mM phenylmethylsulfonyl fluoride and 1.0 µg/ml each of pepstatin, leupeptin, and aprotinin. After
purification, recombinant proteins were concentrated using the
Centricon 10 concentrator from Amicon (Beverly, MA.), glycerol was
added to 50%, and proteins were stored at
80 °C. GST and GST-rD2
were purified as per manufacturer's instructions and left bound to glutathione-Sepharose 4B beads (Amersham), glycerol was added to 50%,
and GST and GST-rD2 were stored at
80 °C.
Phosphatase Assays--
Assays using the phosphopeptides as
substrates were performed as described (31). Purified recombinant
proteins were diluted in 10 µl of phosphatase buffer (50 mM Tris, pH 7.2, 1 mM EDTA, 0.1%
-mercaptoethanol), and assayed at equal molar concentrations (23 nM final concentration) with 10 µl of phosphopeptide (15 µM to 2 mM final concentration). For assays
using pNPP, 5 µl of recombinant protein diluted in phosphatase buffer
(final concentration 7.5 nM) was added to 195 µl of 19 µM to 2.5 mM pNPP at 30 °C to start the
reaction. The increase in absorbance at 405 nm was monitored continuously using a SOFTmax PRO kinetic analysis program in a SpectraMax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). A molar extinction coefficient of 1.8 × 104
M
1 cm
1 was used to calculate
the concentration of p-nitrophenolate. pH optimum curves
were obtained by measuring phosphatase activity of equal molar
concentrations of rD1 and rD1/D2 (ranging from 22 to 50 nM
final concentrations), 13.5 mM pNPP as substrate in the
following buffers plus 1 mM EDTA, 0.1%
-mercaptoethanol, 50 mM sodium citrate for pH 4.3 to
6.0, 50 mM carbonate/bicarbonate for pH 6.8 and 10.1, and
50 mM Tris for pH 7.2 to 8.8. Thermostability experiments
were performed where 233 nM rD1/D2 and rD1 were incubated at temperatures ranging from 30 to 65 °C for 5 min before
phosphatase activity was measured at 30 °C by adding 5 µl of
enzyme to 195 µl of 2.5 mM pNPP.
Dephosphorylation of GST-p56lck-- This was performed at 30 °C as described (32). 5 µl of rD1 or rD1/D2 (60 nM) was added to 5 µl of autophosphorylated GST-p56lck (55 nM) to initiate the reaction. The reaction was stopped by immersion in a dry ice/ethanol bath, samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Immobilon P, Millipore Corp., Bedford MA.), and the amount of phosphorylated substrate remaining was determined by Western blot analysis with 4G10 antiphosphotyrosine antibody diluted 1/10,000 as the primary antibody and horseradish peroxidase-labeled goat anti-mouse antibody diluted 1/5000 (Southern Biotechnology Associates, Inc., Birmingham, AL) as the secondary antibody. The blot was developed using enhanced chemiluminescence (ECL) reagents (Amersham) and exposed to BioMax film from Eastman Kodak Co.
Limited Trypsin Digestion-- Trypsin was added to 1.5 µg of rD1/D2 in a total volume of 20 µl of substrate buffer (50 mM Tris, pH 7.5, 1 mM CaCl2) at ratios of 1:100, 1:50, 1:25, and 1:10 w/w. The digest was incubated at 37 °C for 1 to 4 h and terminated by the addition of 1 µl of 100 mM phenylmethylsulfonyl fluoride. 5 µl of each sample was run on a native polyacrylamide gel, and 15 µl was run on a 10% SDS-polyacrylamide gel under reducing conditions.
N-terminal Sequencing-- 12 µg of rD1/D2 was digested with trypsin for 1.5 h at 1:25 w/w rD1/D2:trypsin, reducing sample buffer was added at 37 °C for 15 min, and the sample was electrophoresed on a 12.5% SDS-polyacrylamide gel and transferred to Immobilon P (polyvinylidene difluoride) membrane. The membrane was stained with 0.025% Coomassie R-250, the 53-kDa and 27-kDa bands were excised, and the N-terminal sequence was obtained from the Nucleic Acid-Protein Service (NAPS) unit (University of British Columbia, Vancouver, B.C., Canada).
Gel Filtration--
A Superose 12 gel filtration column was
equilibrated with 50 mM Tris, pH7.5, 100 mM
NaCl, 0.025% -mercaptoethanol. The column was calibrated with
globular protein gel filtration standards from Bio-Rad (gamma globulin
(158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa)) and bovine albumin
fraction V (67 kDa) from Life Technologies, Inc. rD1, rD2, rD2.2, and
rD1/D2 were each applied separately to the column in concentrations
ranging from 1 to 100 µg/ml for rD1, rD2 at 5 to 20 µg/ml, rD2.2 at
100 µg/ml, and rD1/D2 at 5 µg/ml to 1 mg/ml.
Native Gel Electrophoresis-- 20% homogeneous native polyacrylamide gels were run on the Pharmacia PhastSystem according to manufacturer's instructions. Bovine serum albumin (67 kDa) and ovalbumin (44 kDa) were used for size reference.
Protein Binding Assay--
1 µg of GST fusion protein bound to
glutathione-Sepharose 4B beads was washed with binding buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet
P40, and 0.025% -mercaptoethanol), and 1 µg of soluble
6-His-tagged protein was added to the immobilized protein. Binding
buffer was added to a final volume of 50 µl, and the mixture was
incubated on ice, shaking for 2 h. The mixture was then washed 5 times with 1 ml of binding buffer and separated on a 10%
SDS-polyacrylamide gel, transferred to polyvinylidene difluoride
membrane, and immunoblotted with anti-His antibody diluted 1/1000 as
the primary and horseradish-peroxidase-labeled goat anti-mouse antibody
diluted 1/2500 as the secondary. Blots were developed as described
above. The membrane was then stained with Coomassie Blue to visualize
the immobilized proteins and subjected to densitometry scan
analysis.
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RESULTS |
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Catalytic Activity of the Individual Phosphatase Domains of
CD45--
Recombinant 6-His-tagged CD45 cytoplasmic domain proteins:
rD1, containing the membrane-proximal region, PTP-D1, and the spacer region, (residues 564-927); rD2, containing part of the spacer region,
PTP-D2, and the carboxy tail (residues 901-1268); a second PTP-D2
construct, rD2.2, containing the membrane-proximal region, PTP-D2, and
the carboxy tail (residues 649-939 deleted); the full-length cytoplasmic domain proteins (residues 564-1268), rD1/D2, and a catalytically inactive mutant, C817S, are represented schematically in
Fig. 1A. These proteins were
purified from E. coli (Fig. 1B). rD1 typically
purified as a doublet with both bands detected by anti-CD45 cytoplasmic
domain antisera (data not shown), suggesting that the lower band is a
proteolytic fragment. Equal molar amounts were assayed for phosphatase
activity, and rD1 was found to be catalytically active using the CD3
pY83 phosphopeptide as a substrate, although it was about 2-fold less
active than rD1/D2 (Fig. 2). Both rD2 and
C817S had no detectable activity against this substrate. Kinetic
analysis of rD1 and rD1/D2 activity using pNPP and other tyrosine-phosphorylated peptides as substrates indicated that the
two-domain phosphatase (rD1/D2) was a 2-3-fold more efficient enzyme
than rD1 (Table I). In contrast to this,
the rD2 proteins expressing either the membrane-proximal region or the
spacer region at the N terminus of the protein did not exhibit any
detectable phosphatase activity for any of the substrates listed in
Table I. Even when 100× more enzyme (2.6 µM) was assayed
for up to 4 h with CD3
pY83 or pNPP substrates, no detectable
activity was observed. The presence of PTP-D2 in the rD1/D2 protein did not affect the substrate specificity of CD45, at least when tested against the substrates indicated in Table I. rD1 and rD1/D2 were also
able to dephosphorylate autophosphorylated recombinant
GST-p56lck (Fig. 3). Comparison
of the initial rates of dephosphorylation (Fig. 3B)
indicated that the recombinant two domain phosphatase, rD1/D2, was
again approximately 2-fold more efficient than rD1.
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The Presence of Domain 2 Influences the Catalytic Activity and Enzyme Stability of CD45-- To further investigate the differences between the single (rD1) and two-domain CD45 phosphatase (rD1/D2), enzymatic activity was compared at different pHs (Fig. 4). Both rD1 and rD1/D2 showed optimal activity at pH 7.2 using pNPP as the substrate and displayed a similar pattern of activity in the basic pH range. However, in the acidic pH range, between pH 5.5-7.0, rD1/D2 consistently showed a higher level of activity than rD1 alone, indicating that the presence of PTP-D2 creates a more favorable environment for an ionizable group involved in the catalytic reaction. Thermostability of enzyme activity was also assessed by incubating rD1/D2 and rD1 at the indicated temperatures in Fig. 5 for 5 min then assaying equal molar concentrations at 30 °C using pNPP as a substrate. rD1 consistently lost phosphatase activity at lower temperatures than rD1/D2, indicating that it is less thermostable.
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rD1/D2 and rD2 Are Primarily Monomers, rD1 Is Primarily a Dimer, and rD2.2 Aggregates in Solution-- The molecular sizes of the native recombinant proteins were estimated by gel filtration (Fig. 6A). Interestingly rD1, but not rD2, eluted at a similar volume to rD1/D2, suggesting that the majority of rD1 was present in a dimeric form, whereas the elution volume of rD2 suggested that it was primarily present as a monomer. rD1 eluted primarily as a single peak at this volume over a concentration range of 1-100 µg/ml, indicating that rD1 is primarily present in a dimeric form, whereas rD1/D2 eluted as a monomer over a similar concentration range. Although rD2 was present as a monomer, rD2.2, containing the membrane-proximal region in place of the spacer region, was present in large aggregates and eluted in the void volume (data not shown).
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Evidence for an Intramolecular Interaction in rD1/D2-- The fact that rD1 forms a dimer in solution but rD1/D2 does not suggested that an intramolecular interaction may be occurring in the full-length cytoplasmic domain of CD45 between PTP-D1 and PTP-D2, thus preventing dimerization. This would also help to explain the effect of PTP-D2 on the catalytic activity and stability of PTP-D1 in the rD1/D2 protein. To try and establish whether intramolecular interactions were occurring in rD1/D2, limited trypsin digestion was performed, and the digested products were electrophoresed on both native and SDS-polyacrylamide gels. Fig. 7A shows that with increasing amounts of trypsin, rD1/D2 is digested primarily into two major fragments of approximately 27 and 53 kDa. Interestingly, these tryptic fragments electrophoresed as a single band on the native gel, indicating that both fragments associated with one another (Fig. 7B). Excision of this band from the native gel and subsequent electrophoresis on an SDS-polyacrylamide gel confirmed that both tryptic fragments were present (Fig. 7C). To further localize the regions involved in this interaction, N-terminal sequence was obtained for each tryptic fragment. From the N-terminal sequence, SSNLDE, the 27-kDa fragment was found to begin at residue 573, thus comprising the N-terminal portion of the CD45 cytoplasmic domain estimated to contain the majority of the membrane-proximal region and approximately two-thirds of the first phosphatase domain. The 53-kDa fragment was found to begin at residue 770 with the sequence ATGREV and thus can be estimated to consist of the last third of the first phosphatase domain (including the catalytic cysteine 817), the spacer region, the entire second phosphatase domain, and probably some of the carboxy tail. This indicates the presence of a noncovalent association between the N- and C-terminal regions of the cytoplasmic domain of CD45.
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DISCUSSION |
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This data establishes that PTP-D1 of CD45 does not require the presence of PTP-D2 to be catalytically active. This contradicts our previously published results, which did not detect any phosphatase activity from an in vitro translated and immunoprecipitated rD1 protein (18). Likewise, Streuli et al. (17) detected only 0.1% wild-type activity from a rD1 protein assayed from bacterial cell lysates, and Lorenzo et al. (24) recently found that a recombinant protein expressing CD45 PTP-D1 was not active when its maltose-binding protein fusion partner was cleaved (24). In this present study, several differences were evident, which may help explain why significant levels of activity were observed for the rD1 protein. First, the rD1 protein was produced in large amounts and was isolated and purified away from other potentially inhibitory compounds. Second, the isolation and purification procedure was optimized to minimize exposure to conditions that favor protein unfolding and degradation (see "Experimental Procedures"). Third, assays such as CD spectral analysis were performed, which helped to confirm the structural integrity of the protein (data not shown).
No catalytic activity was detected for rD2 or rD2.2. rD2 was found to be a monomer in solution, whereas rD2.2 was present as a large aggregate. rD2.2 contained the membrane-proximal region of CD45, which may have contributed to this aggregation. Alternatively, the splicing of this region to PTP-D2 may have produced an unstable or misfolded protein, although CD spectral analysis indicated that this molecule possessed a similar secondary structure to that observed in the rD1/D2 protein (data not shown). Under similar purification conditions that produced an active rD1, no detectable activity was observed for rD2, even when tested in a 100-fold excess for several h. Thus we conclude that PTP-D2 is not catalytically active against any of the substrates tested when expressed as a recombinant protein. This result supports previous data obtained from in vitro translated and immunoprecipitated recombinant PTP-D2-containing proteins (18). In addition, the sequence at the predicted catalytic region of CD45 PTP-D2 is lacking an arginine and aspartate residue, two residues that are thought to play a crucial role in the enzymatic reaction (reviewed in Refs. 20-22). Mutation of the presumed catalytic cysteine in PTP-D2 had no effect on the protein-tyrosine phosphatase activity of CD45 in recombinant systems and in the restoration of T cell function by a chimeric epidermal growth factor receptor/CD45 phosphatase (16, 18, 25), again implying that PTP-D2 is not an active phosphatase either as a recombinant protein or when expressed in T cells. However, one group isolated two truncated CD45 proteins lacking different regions of PTP-D1 from eukaryotic cells, which had catalytic activity, and this was attributed to an active PTP-D2 (26). In this study we have demonstrated that in the absence of any PTP-D1 sequence, two purified recombinant PTP-D2 proteins were not catalytically active against any of the substrates tested.
This present work establishes that CD45 is similar to other two domain
phosphatases such as LAR, RPTP, and RPTPµ, where PTP-D1 has been
shown to be independently catalytically active (15, 17, 19). With the
exception of LAR (17, 33), optimal catalytic activity was observed with
these phosphatases when both PTP-D1 and PTP-D2 were present. Like CD45,
no catalytic activity has been observed for the second domain of these
phosphatases, except for RPTP
, where the observed catalytic activity
for PTP-D2 was much lower than that observed with PTP-D1 (15). In other
two domain phosphatases, PTP-D2 lacks key residues thought to be
crucial for catalytic activity, making it very unlikely that these
domains would be active phosphatases (reviewed in Refs. 20-22). Thus
the second phosphatase domain in two-domain phosphatases has been shown
to have little or no activity in comparison to their first phosphatase
domain, and in many cases these domains are required for optimal
activity of the first domain.
It has been suggested that PTP-D2 or phosphorylation of PTP-D2 can alter the substrate specificity of CD45 against certain artificial substrates (17, 34). In this study, using pNPP, a variety of peptide substrates, and the protein substrate GST-p56lck, no evidence was obtained for a major role of PTP-D2 in influencing substrate specificity. Comparison of rD1 and rD1/D2 activities indicated that PTP-D2 is required for optimal enzymatic stability and optimal activity of the first phosphatase domain of CD45. The fact that the presence of PTP-D2 influences catalytic activity at acidic pH suggests that PTP-D2 can influence the catalytic environment of PTP-D1. Whether PTP-D2 is acting directly to modify the catalytic environment or indirectly by altering the conformation of PTP-D1 is not known. However, this and data indicating an interaction between rD1 and rD2 suggests that PTP-D1 and PTP-D2 may be in close proximity to one another.
Crystal structures of the first phosphatase domains of two
transmembrane two-domain tyrosine phosphatases, RPTP and RPTPµ, have been determined (14, 35). Although both crystallized as dimers, it
was suggested that dimerization of the RPTPµ rD1 was an artifact of
crystallization, as gel filtration indicated that the protein was a
monomer in solution up to concentrations of 7.5 mg/ml (35). Dimers of
RPTP
rD1 were formed by the membrane-proximal region of one rD1
molecule forming a "wedge" and binding to the active-site region of
the second, an interaction that was predicted to block phosphatase
activity. Dimers and higher oligomer formation occurred for RPTP
rD1
at concentrations between 0.1 and 5 mg/ml but did not occur for the rD2
protein (14). However, kinetic analysis of rD1 of RPTP
at lower
concentrations was consistent with it being active as a monomer in
solution (36). In this study, CD45 rD1 existed primarily as a dimer at
the concentrations tested (1-100 µg/ml), whereas the two-domain
phosphatase, rD1/D2, existed primarily as a monomer (5-1000 µg/ml).
This suggests that the presence of PTP-D2 in the molecule prevents its
dimerization. Contrary to the prediction from RPTP
studies (14),
dimer formation of CD45 rD1 did not result in an inactive phosphatase;
however, it was 2-3-fold less active than rD1/D2, raising the
possibility that dimer formation may down-regulate activity. However,
we cannot easily assess whether one or both of the phosphatase domains
are active in the rD1-rD1 dimer.
Attempts to define the regions responsible for a potential intramolecular interaction in rD1/D2 identified a 27-kDa fragment containing the N-terminal region (the membrane-proximal region and two-thirds of PTP-D1) interacting with a 53-kDa fragment containing the C-terminal region (the last third of PTP-D1, the spacer region, PTP-D2, and the carboxy tail) after limited trypsin digestion. This 53-kDa tryptic fragment is the same fragment identified and purified by Tan et al. (26) who concluded that the catalytic activity was derived from PTP-D2. However, in these studies, we found this tryptic fragment noncovalently associated with a 27-kDa fragment derived from PTP-D1 and, despite finding no activity associated with rD2 proteins, found this complex to be catalytically active (data not shown).
Evidence for a noncovalent intramolecular interaction from tryptic digestion studies and for a rD1-rD2 association suggests an interaction between the N-terminal part of the protein containing PTP-D1 and the C-terminal part containing PTP-D2. It is tempting to speculate that in the rD1/D2 protein, the membrane-proximal region interacts with PTP-D2 to promote the intramolecular interaction and prevent intermolecular dimer formation, resulting in rD1/D2 monomers. In the absence of PTP-D2, the membrane-proximal region of rD1 may interact with another rD1 molecule, thereby promoting dimer formation. Thus, PTP-D2 may act to prevent dimerization. Regulation of this membrane-proximal region-PTP-D2 interaction would result in the formation of intra- or intermolecular interactions, which may act to regulate CD45 function. Consistent with this model is data from CD45-negative T cells expressing an epidermal growth factor receptor-CD45 chimera. Here, epidermal growth factor addition and presumed dimerization of the epidermal growth factor receptor-CD45 chimera interferes with its function in T cell activation (13). Interestingly, recent work suggests that a specific residue within the membrane-proximal region is required to mediate this effect (37). Although this model is consistent with current data, it remains to be proven, and attempts to generate an rD1 protein lacking the membrane-proximal region to directly assess its role in dimerization have not yet generated a stable protein.
It is becoming evident that interactions between different regions within a single protein can occur as a form of regulation. Examples of this can be found in the src family protein-tyrosine kinases, where two recent crystal structures demonstrate complex intramolecular interactions between the SH3 domain and a linker region as well as the SH2 domain near the N terminus with a regulatory phosphotyrosine residue close to the C terminus (38, 39). Such an interaction results in a closed, inactive conformation that can be converted to an open, active conformation upon dephosphorylation of the regulatory tyrosine (reviewed in Ref. 40). Likewise, the recent determination of the crystal structure of SHP-2 confirms that an intramolecular interaction occurs between the N-terminal SH2 domain and the C-terminal PTP domain (41). Release of this intramolecular interaction is thought to occur upon occupation of the SH2 domains, leading to a more active, open conformation (42-44). Furthermore, intramolecular regulation may also occur for T cell-PTP, where the noncatalytic C-terminal domain of T cell-PTP has been shown to affect its phosphatase activity (45). We now provide initial evidence for intra- and intermolecular interactions occurring in the cytoplasmic domain of CD45, raising the possibility that CD45 activity may also be regulated by intramolecular interactions mediated by the C-terminal region containing the second phosphatase domain, PTP-D2.
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ACKNOWLEDGEMENTS |
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We thank Dr. F. Jirik and Dr. I. Clark-Lewis for providing the tyrosine-phosphorylated peptides, Dr. M. Matsuda and R. Ingham for GST-Crk-SH3 protein, G. Doheny for 6-His-glucosidase protein, and D. Ng for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by funds from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Natural Sciences and Engineering Research Council
of Canada Studentship.
§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 300-6174 University Blvd, Vancouver, B. C., V6T 1Z3, Canada. Tel.: 604-822-8980; Fax: 604-822-6041; E-mail: pauline{at}unixg.ubc.ca.
1 The abbreviations used are: PTP, protein-tyrosine phosphatase; GST, glutathione S-transferase; pNPP; para-nitrophenyl phosphate; rD1/D2, recombinant 6-His-tagged CD45 cytoplasmic domain protein; rD1, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the first phosphatase domain, and the spacer region; rD2, recombinant 6-His-tagged CD45 protein containing part of the spacer region, the second phosphatase domain, and the carboxy tail; rD2.2, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the second phosphatase domain, and the carboxy tail; PTP-D1, the first phosphatase domain; PTP-D2, the second phosphatase domain; RPTP, receptor-like protein-tyrosine phosphatase; CD, circular dichroism; NTA, nitrilotriacetic acid.
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REFERENCES |
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