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J Biol Chem, Vol. 275, Issue 20, 15350-15356, May 19, 2000
From the Division of Cellular Biochemistry, The Netherlands Cancer
Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
RPTPµ is a receptor-like protein-tyrosine
phosphatase (RPTP) whose ectodomain mediates homotypic cell-cell
interactions. The intracellular part of RPTPµ contains a relatively
long juxtamembrane domain (158 amino acids; aa) and two conserved
phosphatase domains (C1 and C2). The membrane-proximal C1 domain is
responsible for the catalytic activity of RPTPµ, whereas the
membrane-distal C2 domain serves an unknown function. The regulation of
RPTP activity remains poorly understood, although dimerization has been
proposed as a general mechanism of inactivation. Using the yeast
two-hybrid system, we find that the C1 domain binds to an N-terminal
noncatalytic region in RPTPµ, termed JM (aa 803-955), consisting of
a large part of the juxtamembrane domain (120 aa) and a small part of the C1 domain (33 aa). When co-expressed in COS cells, the JM polypeptide binds to both the C1 and the C2 domain. Strikingly, the
isolated JM polypeptide fails to interact with either full-length RPTPµ or with truncated versions of RPTPµ that contain the JM region, consistent with the JM-C1 and JM-C2 interactions being intramolecular rather than intermolecular. Furthermore, we find that
large part of the juxtamembrane domain (aa 814-922) is essential for
C1 to be catalytically active. Our findings suggest a model in which
RPTPµ activity is regulated by the juxtamembrane domain undergoing
intramolecular interactions with both the C1 and C2 domain.
Protein-tyrosine phosphatases
(PTPs)1 play important roles
in signal transduction pathways regulated by tyrosine phosphorylation. Members of the superfamily of PTPs use the same catalytic mechanism and
are broadly classified into transmembrane or receptor-like PTPs (RPTPs)
and intracellular, nonreceptor PTPs (reviewed in Refs. 1 and 2).
Members of the RPTP subfamily are type I membrane proteins consisting
of a variable ectodomain, a single membrane-spanning region, and in
most cases, two conserved intracellular phosphatase domains. The RPTPs
are further classified according to the structure of their ectodomains
(reviewed in Refs. 3 and 4). The large variety in ectodomain structure
suggests the existence of an equal number of putative ligands, yet in
most cases the corresponding ligands have not been identified.
RPTPµ is the prototype member of a subfamily of RPTPs that mediate
homophilic cell-cell interactions via their ectodomains and, hence, are
thought to play a role in cell adhesion-mediated processes (5-8). The
ectodomain of RPTPµ shows similarities with that of cell-cell
adhesion molecules and consists of an N-terminal "MAM" domain,
which is critical for mediating cell-cell adhesion (9), followed by an
Ig-like domain and four fibronectin type III repeats (10). Its
intracellular part consists of a juxtamembrane domain of 158 amino
acids (aa), which is relatively long compared with that in other RPTPs,
and two tandem phosphatase domains referred to as C1 and C2. As in most
other RPTPs, the membrane-proximal C1 domain of RPTPµ is
catalytically active, whereas the membrane-distal C2 domain shows no
activity, at least in vitro (11). The C2 domains of most
RPTPs have been proposed to play a regulatory role (12), but how it
might contribute to RPTP activity is not known.
One major unresolved question is how ligand binding may influence the
catalytic activity of RPTPs to affect signal transduction events. A
recently proposed model involves dimerization, as inferred from the
crystal structure of RPTP Here we present evidence for a new type of interdomain interaction
involved in the regulation of RPTPµ activity. In a search for
potential binding partners of the C1 domain using the yeast two-hybrid
system, we isolated a cDNA clone encoding part of RPTPµ itself,
consisting of a large part of the juxtamembrane domain and a small part
of the C1 domain. We show that this "JM" segment can interact with
both the C1 and C2 domain and present evidence suggesting that this
interaction is intramolecular rather than intermolecular. We further
show that the juxtamembrane domain is essential for catalytic activity
of the C1 domain. Based on these findings, we propose a model in which
the juxtamembrane domain may contribute to the regulation of RPTPµ activity.
Cells, Transfections, and Antibodies--
COS-7 cells were
cultured in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with antibiotics and 8% fetal calf serum. Transient
transfections of COS-7 cells were performed by the DEAE-dextran method
as described in Ref. 21. Antibodies against the HA tag (12CA5) and
Myc-tag (9E10) were obtained from hybridoma supernatants. Biotinylated
anti-HA antibody and anti-FLAG tag monoclonal antibody M2 were
purchased from Roche Molecular Biochemicals and Eastman Kodak Co.,
respectively. Monoclonal antibody 3D7 directed against the
extracellular domain of RPTPµ has been described previously (22).
Yeast Two-hybrid Library Screen--
For use as a bait in the
two-hybrid screen, the first catalytic domain of RPTPµ (RPTPµC1)
was polymerase chain reaction-amplified using primers
5'-TATGTCGACAACAGAATGAAGAACAGATACG and 5'-CCGGAATTCCTCTTTAATCTG. RPTPµC1 was fused to the Gal4 DNA binding domain by
SalI-EcoRI subcloning into pMD4 (23) containing a
trp1 marker for selection. pMD4-RPTPµC1 was co-transfected
into the lacZ and his3 containing yeast strain
Y190, together with a pVP16-based (24) human testis cDNA library
(kindly provided by R. Bernards) that carries the leu2
marker. Yeast transformants expressing the reporter genes were selected
on medium lacking histidine and supplemented with 25 mM
3-amino-1,2,4-triazole. Positive colonies were identified by
cDNA Constructs--
Plasmid pMT2-HA-cl.2 was constructed by
subcloning the insert from pVP16-clone 2 into a modified pMT2 vector
containing an HA tag. pMT2-FLAG-RPTPµC1, C1M, and C2 plasmids were
constructed by polymerase chain reaction amplification and standard
cloning procedures. The wild type and mutant first PTP domain
were amplified by sense (5'-TATGTCGACAACAGAATGAAGAACAGATACG) and
antisense (5'-GCGTCTAGAATTCCTCTTTAATCTG) primers using hFL and hFLm
constructs as template (5), respectively. The second PTP domain was
amplified using sense (5'-ATTACTCGAGCGGACGCTAAACATGGTGAC) and antisense
(5'-TCATTCTAGAACACCATCAGCCAGAATTCA) primers and hFL as template. All
polymerase chain reaction products were verified by sequencing.
pMT2-FLAG-RPTPµC1C2 was constructed by inserting an EcoRI
fragment containing the second PTP domain into plasmid pMT2-FLAG-RPTPµC1. HA- and Myc-tagged constructs encoding the juxtamembrane region and first catalytic domain (RPTPµJC1) were generated using primers 5'-TATGTCGACCTGAATGGGAGATCTGTGTC and
5'-TATGAATTCCTCATCTTTCTTAGCCGAGT. Amplified product was subcloned into
pMT2-SM-HA and pMT2-SM-Myc and verified by sequencing. pMT2-hFL
containing full-length RPTPµ cDNA has previously been described
(11). The pMT2-HA-cl.2/E896R and pMT2-HA-RPTPµJC1/E896R plasmids
(mutated glutamate 896 to arginine) were generated by site-directed
mutagenesis (Promega) using primers 5'-GATGAAGTGTGCGCGGGGCTACGGCTTC)
and 5'-GAAGCCGTAGCCCCGCGCACACTTCATC.
Protein Analysis and Phosphatase Assays--
Cells were washed
once with ice-cold phosphate-buffered saline and lysed on ice in 1 ml
(per 10 cm plate) of Nonidet P-40 lysis buffer (50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 1.5 mM EDTA, 10% glycerol, 1% Nonidet P-40) supplemented with 5 µg/ml leupeptin, 2.5 µg/ml aprotinin, and PefablocSC (Roche Molecular Biochemicals). After centrifugation, 200 µl of supernatant (for immunoblot analysis) or 1 ml of supernatant (for phosphatase assays) was incubated for
2 h with protein A-Sepharose beads (Amersham Pharmacia Biotech) precoupled to specific antibodies. Immunoprecipitates were washed three
times with lysis buffer and analyzed by Western blotting or assayed for
phosphatase activity. For expression controls, 10 µl of total lysate
was analyzed by Western blotting. Tyrosine phosphatase activity of
immunoprecipitates was measured using a nonradioactive protein-tyrosine
phosphatase assay kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Signals on Western blots were detected by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
C1 Interdomain Interaction in the Yeast Two-hybrid System--
In
an attempt to identify proteins that interact with the C1 domain of
RPTPµ, we used this domain as bait (RPTPµC1, aa 923-1190) in a
yeast two-hybrid screen of a human testis cDNA library. Two positive colonies were identified that contained identical
testis-derived cDNA clones, termed clone 1 and clone 2. Strikingly,
both clones encode a membrane-proximal region of RPTPµ (aa 803-955)
consisting of a large part of the juxtamembrane domain (120 aa) and a
small part of the C1 domain (33 aa) (Fig.
1A), which we refer to as either cl.2 or the JM region. As shown in Fig. 1B,
co-expression of RPTPµC1 and cl.2/JM in yeast results in the
activation of the lacZ reporter gene. These results strongly
indicate that the C1 domain undergoes either intermolecular or
intramolecular interaction with the JM region.
The Membrane-proximal JM Region Interacts with Both Catalytic
Domains of RPTPµ in COS Cells: Evidence for Intramolecular
Interactions--
To confirm the observed C1-JM interdomain
interaction in mammalian cells, HA-tagged clone 2 (HA-cl.2) encoding
the JM polypeptide was transiently expressed together with
epitope-tagged C1 (FLAG-RPTPµC1) in COS cells (Fig.
2A). When both proteins were
co-expressed, C1 was co-precipitated with the anti-HA monoclonal
antibody (not shown), whereas HA-cl.2 was co-precipitated with
anti-FLAG monoclonal antibody. Thus, the C1-JM interdomain interaction
occurs in both yeast and mammalian cells. Given the sequence
similarities between the C1 and C2 domains, we tested whether the C2
domain might also interact with JM. As shown in Fig. 2A,
this is indeed the case; when HA-cl.2 was co-expressed with the
isolated C2 domain (FLAG-RPTPµC2; aa 1191-1452) or with both C1 and
C2 domains in tandem (FLAG-RPTPµC1C2; aa 923-1452), HA-cl.2 was
co-precipitated with anti-FLAG monoclonal antibody. It thus appears
that the JM polypeptide does not discriminate between the C1 and C2
domain for binding in COS cells. It is of note that the RPTPµC1C2
construct, containing both phosphatase domains, yields a stronger
binding signal than either C1 or C2 (Fig. 2A, last
lane), as one would expect if each catalytic domain binds one
HA-cl.2 molecule (JM polypeptide).
We next examined whether the nature of the JM-C1 and JM-C2 interactions
is intramolecular or intermolecular. To this end, COS cells were
transfected with various epitope-tagged RPTPµ constructs and then
subjected to immunoprecipitation and blotting assays. Strikingly,
whereas HA-cl.2 (JM polypeptide) co-precipitates with the individual
phosphatase domains as well as the tandem C1C2 domain (Fig.
2A), HA-cl.2 fails to interact with longer versions of
RPTPµ: either a Myc-tagged polypeptide consisting of a large part of
the juxtamembrane domain and the C1 domain (JC1, aa 814-1190) (not
shown) or full-length RPTPµ (Fig. 2C). Furthermore, we
find that HA-tagged JC1 does not co-precipitate with Myc-tagged JC1 (Fig. 2B) nor with full-length RPTPµ (Fig. 2C).
The results of the co-immunoprecipitation analysis are summarized in
Fig. 2D. From these findings we conclude that the observed
JM-C1/C2 interactions do not occur between different RPTPµ molecules.
Thus, our results can only be explained by the JM-C1/C2 interactions
being intramolecular rather than intermolecular.
Mutational Analysis: Effects of Point Mutations on the JM-C1/C2
Interaction--
To determine which residues are involved in the
interaction between the JM and C1 domains, we transfected RPTPµ
constructs in which the following critical residues were mutated:
cysteine 1095 to a serine (C1095S) and glutamate 896 to an arginine
(E896R). The conserved cysteine 1095 is essential for catalytic
activity of the C1 domain of RPTPµ. Mutation of cysteine 1095 to a
serine (C1095S) was shown to completely abolish phosphatase activity (11). Glutamate 896 is analogous to aspartate 228 of RPTP The Juxtamembrane Domain Is Essential for Catalytic Activity of the
C1 Domain--
To examine how the distinct domains of RPTPµ
contribute to catalytic activity, we determined tyrosine phosphatase
activity in immune complexes using a nonradioactive tyrosine
phosphatase assay (see "Experimental Procedures"). We measured the
activity of both full-length RPTPµ and different epitope-tagged
constructs of RPTPµ (Fig. 4,
A and B) expressed in COS cells. We found that the isolated C1 and C2 domains as well as C1C2 are inactive (Fig. 4B). In marked contrast, however, N-terminal extension of
the C1 domain leads to phosphatase activity as inferred from the JC1 polypeptide being active in the assay. In other words, the
juxtamembrane domain is required for activity of the C1 domain. This is
consistent with reports on LAR and RPTP In the present study, we have shown that the juxtamembrane domain
of RPTPµ can bind to both the first and the second phosphatase domain
(C1 and C2) and that this interaction is likely to be intramolecular rather than intermolecular. Furthermore, we have presented evidence that the juxtamembrane domain is required for the C1 domain to become
fully active.
Through yeast two-hybrid analysis, we found that the RPTPµC1 domain
binds to an RPTPµ segment, termed JM, consisting of a large part of
the juxtamembrane domain and a small part of the C1 domain. Our COS
cell experiments revealed that the JM segment interacts not only with
C1 but also with the C2 domain of RPTPµ. These results would be
consistent with RPTPµ forming dimers, in which the JM region of one
molecule interacts with the juxtaposed C1 and/or C2 domains in the
partner RPTPµ molecule, analogous to what has been proposed for the
C1 domain of RPTP In a recent crystallographic study on the RPTPµC1 domain (residues
874-1168), it was concluded that the protein behaves as a monomer in
solution and that C1-C1 dimerization is most likely a consequence of
crystallization (18). The C1 crystal structure revealed that the
catalytic site is unhindered and adopts an open conformation. Caution
is needed, however, to extrapolate findings obtained with RPTPµC1 to
the full-length molecule, particularly because the juxtamembrane domain
was excluded from crystallographic analysis and, hence, any JM-C1
interaction would go undetected. The N terminus of the C1 domain used
for crystallization starts at the helix-turn-helix segment (at the
membrane-distal end) very close to the boundary of the C1 domain. This
would imply that a more membrane-proximal part of the juxtamembrane
domain (immediately N-terminal to the helix-turn-helix structure) is
involved in the observed JM-C1/C2 interaction. Further crystallization
studies using N-terminally extended versions of the C1 domain are
required to clarify this point.
It seems likely that the interdomain interactions found in RPTPµ also
occur in other members of the RPTP family. It is of note that the
juxtamembrane domain of RPTPµ, in common with the other MAM domain
containing RPTPs, is about 70 residues longer than that in all other
RPTPs (10); the significance of this extension remains unknown. It will
be interesting to see whether JM-C1/C2 interactions are a specific
feature of the MAM domain-containing subfamily of RPTPs. Our results
support the view that dimerization is not involved in the regulation of
RPTPµ activity, in contrast to what has been proposed for the
regulation of RPTP We also have shown that the juxtamembrane region of RPTPµ is required
for the C1 domain to be catalytically active, consistent with an
intramolecular JM-C1 interaction regulating catalytic activity.
Previous reports have shown that LAR and RPTP Based on our findings, we propose a model explaining how the observed
interdomain interactions may contribute to the regulation of catalytic
activity (Fig. 5). In this model, RPTPµ
can adopt two different conformations. In one conformation, the
juxtamembrane domain interacts with the regulatory, catalytically
inactive C2 domain. In this way, the C1 domain lacks interaction with
the juxtamembrane domain and thereby remains inactive. When a proper tyrosine-phosphorylated substrate is presented, RPTPµ adopts a new
conformation, in which the JM-C2 domain interaction dissociates to
promote the formation of a JM-C1 intramolecular complex thereby stimulating catalytic activity and allowing substrate
dephosphorylation. The precise function of the juxtamembrane domain
remains to be elucidated. It could be important for proper folding of
the C1 domain, but it might also be involved in substrate recognition and/or binding. Similarly, the role of the C2 domain remains poorly understood. Interactions between the C2 domain and other signaling molecules might be involved but still remain to be established. Recent
mutational analysis has raised the intriguing possibility that C2 might
in fact be an active PTPase domain in the correct cellular context
(28). The role of the juxtamembrane domain and the C2 domain are key
issues that need to be addressed for better understanding of the
regulation of RPTPµ activity. In conclusion, our findings reveal the
occurrence of interdomain interactions in RPTPµ, and given the lack
of any indication for intermolecular interactions, they support the
view that the dimerization model might not be applicable for the
regulation of RPTP activity in general and that of RPTPµ in
particular.
*
This work was supported by the Dutch Cancer Society.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.
§
To whom correspondence should be addressed. Tel.: 31-20-512-1971;
Fax: 31-20-512-1989; E-mail: wmoolen@nki.nl.
The abbreviations used are:
PTP, protein-tyrosine phosphatase;
RPTP, receptor protein-tyrosine
phosphatase;
aa, amino acid(s);
JM, juxtamembrane region (aa 803-955);
C1 and C2, phosphatase domains;
HA, hemagglutinin.
Intramolecular Interactions between the Juxtamembrane Domain
and Phosphatase Domains of Receptor Protein-tyrosine Phosphatase
RPTPµ
REGULATION OF CATALYTIC ACTIVITY*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(13). This model suggests that ligand
binding induces the formation of a symmetrical dimer in which the
catalytic site of one molecule is blocked by specific interactions with
a helix-turn-helix segment (termed the "wedge") in the
juxtamembrane domain of the other (13). There is no wedge-like region
present directly upstream of the C2 domain, suggesting a fundamental
difference between the C1 and C2 domains. Based on these structural
studies, dimerization has been proposed to be a universal mechanism of
inactivation of RPTPs (reviewed in Ref. 14). Consistent with this,
earlier studies had already indicated that the leukocyte-specific RPTP
CD45 can form homodimers (15) and that artificial induction of CD45
dimerization may lead to loss of function (16). Using a epidermal
growth factor receptor-CD45 chimera, a part of CD45 homologous to the
inhibitory helix-turn-helix wedge in RPTP was recently shown to
inhibit CD45 function after ligation by epidermal growth factor (17), in support of the dimerization model. On the other hand, however, the
crystal structure of RPTPµ does not reveal such intermolecular interactions between a wedge region and the C1 domain (18). It seems
that the catalytic site of RPTPµC1 is unhindered and adopts an open
conformation similar to what is observed in the cytosolic PTP, PTP1B
(19). It was suggested that the RPTPµ dimer may be the consequence of
crystallization, because dimers were not found in solution.
Furthermore, some residues important for the proposed dimerization
mechanism are less conserved in RPTPµ (18, 20), suggesting that
RPTPµ may not be regulated by dimerization (reviewed in Refs. 14 and
20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase filter assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Yeast two-hybrid screen for
RPTPµ-interacting proteins. A,
schematic representation of bait (RPTPµC1) and prey JM (two-hybrid
clone 1 and 2). The numbers correspond to the residues of
full-length RPTPµ. B, staining for -galactosidase
activity. The yeast colonies tested express RPTPµC1, clone 1 with or
without RPTPµC1, or clone 2 with or without RPTPµC1.
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Fig. 2.
Association of the membrane-proximal JM
region with both catalytic domains of RPTPµ in
COS cells: evidence for intramolecular interactions. A,
immunoblot analysis (lower panel) of anti-FLAG
immunoprecipitates from lysates of COS-7 cells transfected with empty
vector, pMT2-FLAG-RPTPµC1, pMT2-FLAG-RPTPµC2, or
pMT2-FLAG-RPTPµC1C2 without (lanes 1, 3-5) or with
pMT2-HA-cl.2 (lanes 2, 6-8). The blot was probed with
anti-HA antibody. The upper and middle
panels show expression controls. Molecular mass standards
(kDa) are indicated on the left. B, immunoblot
analysis (lower panel) of anti-HA
immunoprecipitates from lysates of COS-7 cells transfected with empty
vector (lane 1), pMT2-HA-JC1 (lane 2),
pMT2-Myc-JC1 (lane 3), or with both pMT2-HA-JC1 and
pMT2-Myc-JC1 (lane 4). The blot was probed with anti-Myc
antibody. The upper and middle panels
show expression controls. C, immunoblot analysis
(lower panel) of anti-HA immunoprecipitates from
lysates of COS-7 cells transfected with empty vector (lane
1), pMT2-HA-cl.2 (lane 2), pMT2-HA-JC1 (lane
3), pMT2-hFL (lane 4), or with pMT2-HA-cl.2 or
pMT2-HA-JC1 in combination with pMT2-hFL (lane 5 and
6). The blot was probed with anti-RPTPµ antibody 3D7
directed against the extracellular domain of RPTPµ. The
upper and middle panels show
expression controls. D, overview of co-immunoprecipitation
analysis with HA-tagged clone 2 or HA-tagged RPTPµJC1 and different
parts of RPTPµ. Experiments were carried out as described above.
HA-cl.2 or HA-RPTPµJC1 (as shown on the left) was
co-expressed with different parts of RPTPµ (shown on the
right). Plus (+) signs indicate that co-precipitation was
detected; minus ( 
) signs indicate no detectable co-precipitation
under the same conditions. IP, immunoprecipitate.
. In the
RPTP dimer, this residue is located in the N-terminal wedge that
inserts into the catalytic pocket of the C1 domain of the juxta-posed
RPTP molecule and thereby may block its activity (13). Glutamate 896 of RPTPµ is also analogous to glutamate 624 in CD45; mutation of this
residue was shown to abolish the inhibitory effect on T-cell receptor
signaling caused by CD45 dimerization (17). We find, however, that the
mutation C1095S in FLAG-RPTPµC1 did not affect association with
HA-cl.2 (Fig. 3A). We also
find that the mutation E896R in HA-cl.2 does not affect the association
with the C1 domain (Fig. 3B). Taken together, catalytic
activity and glutamate 896 are not essential for the association
between the juxtamembrane and the C1 domain of RPTPµ.
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Fig. 3.
Co-immunoprecipitation analysis of catalytic
inactive RPTPµC1 and mutant of clone 2. A, co-precipitation of HA-tagged clone 2 and catalytic
inactive FLAG-tagged RPTPµC1. Immunoblot analysis (lower
panel) of anti-HA immunoprecipitates from lysates of COS-7
cells transfected with pMT2-FLAG-RPTPµC1 (lane 1),
pMT2-FLAG-RPTPµC1M (lane 2), empty vector (lane
3), or with pMT2-FLAG-RPTPµC1 or pMT2-FLAG-RPTPµC1M in
combination with pMT2-HA-cl.2 (lanes 4 and 5).
The upper and middle panels show
expression controls. The blot was probed with anti-FLAG antibody.
B, co-immunoprecipitation of FLAG-tagged RPTPµC1 and
HA-tagged mutant of clone 2. Immunoblot analysis (lower
panel) of anti-HA immunoprecipitates from lysates of COS-7
cells transfected with empty expression vector (lane 1),
pMT2-HA-cl.2 (lane 2), pMT2-HA-cl.2/E896R (lane
3), pMT2-FLAG-RPTPµC1 (lane 4), or with pMT2-HA-cl.2
or pMT2-HA-cl.2/E896R in combination with pMT2-FLAGA-RPTPµC1
(lanes 5 and 6). The blot was probed with
anti-FLAG antibody. The upper panel shows
expression controls of the HA-tagged proteins. Molecular mass standards
in kDa are shown on the left of the immunoblots in
A and B. IP, immunoprecipitates.
, which show that the
isolated C1 domains require at least part of the juxtamembrane domain
for activity in vitro (12, 25). The present data also
indicate that the C2 domain is not required for activity of the C1
domain, although we cannot exclude the possibility that the C2 domain may somehow contribute to the activity of C1. Finally, we found that
the E896R mutation in the juxtamembrane domain of HA-RPTPµJC1 does
not affect phosphatase activity when compared with the wild-type JC1
polypeptide (Fig. 4B), indicating that glutamate 896 is not essential for activity of the C1 domain.
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Fig. 4.
Analysis of tyrosine phosphatase
activity. A, phosphatase activity of full-length
RPTPµ using a nonradioactive tyrosine phosphatase assay kit (Roche
Molecular Biochemicals). Anti-RPTPµ (3D7) immunoprecipitates of COS-7
cells transfected with empty expression vector (control) or with
pMT2-hFL were used in the nonradioactive phosphatase assay. The
absorbance at 405 nm is a reciprocal measure for phosphatase activity.
Absorbance at 490 nm is the reference wavelength. B,
analysis of phosphatase activity of different parts of RPTPµ, using
anti-FLAG immunoprecipitates from lysates of COS-7 cells transfected
with empty expression vector (control 1), pMT2-FLAG-RPTPµC1,
pMT2-FLAG-RPTPµ-C2, or pMT2-FLAG-RPTPµC1C2, and anti-HA
immunoprecipitates from lysates of COS-7 cells transfected with empty
vector (control 2) and pMT2-HA-RPTPµJC1 or
pMT2-HA-RPTPµJC1/E896R.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(13). Inconsistent with a homodimerization model,
however, is our finding that the JM segment fails to interact with
extended, JM-containing versions of RPTPµC1. JM also fails to
interact with full-length RPTPµ. We also did not detect any
interdomain interactions between versions of RPTPµ that comprise both
JM and C1. These results are most readily explained by a model in which
JM-C1/C2 binding represents an intramolecular interaction within one
single RPTPµ molecule. Mutational analysis indicates that the
interaction is independent of RPTPµ catalytic activity and of
glutamate 896 in the helix-turn-helix segment, which is analogous to
that in the corresponding motif of CD45, where it has been implicated
in dimerization-dependent inhibition of CD45 activity
caused by dimerization (17).
and CD45 (14, 20). In fact, there is no direct
evidence that RPTP dimers are catalytically inactive. Parts of
RPTP, containing the inhibitory wedge and the C1 domain, are
catalytically active and probably act as active monomers in solution
(25, 26). The RPTP dimerization concept has become even more complex
since the C1 domain of RPTP
was reported to interact with the C2
domain of RPTP
but not the RPTPC2 with RPTPC1 (27). This
apparent C1-C2 heterodimerization requires the wedge region of RPTP,
which was thought to bind the "pseudo-active" site in the
juxtaposed RPTPC2 domain (27). Although the precise cellular role of
the C2 domain remains unknown, the latter result does suggest that the
C2 domain is involved in a variety of protein-protein interactions. Very recently, structural studies on the tandem phosphatase domains of
RPTP LAR revealed a monomeric configuration without any indication of
dimer formation either in the crystal structure or in solution (28).
The LAR crystal structure further reveals that the N-terminal helix
wedge is not involved in any intermolecular interaction and that the
catalytic sites of both C1 and C2 are accessible, a configuration that
is in direct contrast to the previous model of dimeric-blocked
orientation based on the crystal structure of the RPTPC1 domain
alone (28).
similarly need the
juxtamembrane domain for full catalytic activity (12, 25), suggesting a
general regulatory mechanism of the juxtamembrane domain among RPTPs.
View larger version (12K):
[in a new window]
Fig. 5.
Proposed model of intramolecular interactions
regulating RPTPµ activity. Two possible
conformations of RPTPµ are schematically drawn. One conformation
represents the inactive state of the molecule, in which the second
catalytic domain (C2) of RPTPµ interacts with the juxtamembrane
domain thereby inhibiting the first catalytic domain (C1). In the other
conformation, C1 interacts with the juxtamembrane domain rendering an
active RPTPµ molecule as C1 is active in this conformation. For
details see "Discussion."
FOOTNOTES
Present address: Laboratory of Medical Oncology, Dept. of Internal
Medicine, University Medical Center, Heidelberglaan 100, 3584 CX
Utrecht, The Netherlands.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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