From the
To determine whether the receptor-like protein-tyrosine
phosphatase, RPTP
Signal transduction by protein-tyrosine kinases is relatively
well understood, but rather little is known about their enzymatic
counterpart, the protein-tyrosine phosphatases (PTPs).
RPTP
Relatively little is known about regulation of PTP activity, but
there are indications that both serine and tyrosine phosphorylation may
play an important role. Treatment of T cells with the calcium ionophore
ionomycin leads to a decrease in CD45 activity concomitant with a
decrease in CD45 serine phosphorylation
(15) . Serine
phosphorylation of CD45 and of the cytoplasmically localized PTP1B is
enhanced in response to phorbol ester treatment
(16, 17) . However, these phosphorylation events do not
significantly affect the activity of either PTP. In contrast, phorbol
ester treatment of HL-60 cells has been found to increase the
expression, activity and serine phosphorylation of PTP1C and its
translocation to the plasma membrane
(18) , but it is not clear
that this phosphorylation is directly due to PKC nor whether it affects
PTP1C activity indirectly. TPA treatment also leads to a small increase
in RPTP
There are also a
number of reports of tyrosine phosphorylation of PTPs. The
SH2-containing nonreceptor PTP, PTP1D, is phosphorylated on tyrosine in
response to activation of the EGF and platelet-derived growth factor
receptor-like protein-tyrosine kinases
(22, 23) , but
whether tyrosine phosphorylation affects PTP1D activity remains to be
established definitively. CSF-1 treatment of mouse macrophages induces
tyrosine phosphorylation of the related SH2-containing PTP, PTP1C
(24) . We have found that RPTP
To begin to characterize RPTP
To confirm the identification, Ser-204 was
mutated to alanine, and the S204A mutant bPTP
In this study, we have shown that RPTP
Our results
indicate that Ser-180 and Ser-204 are directly phosphorylated by PKC
in vivo. Ser-180 is in a conventional PKC consensus sequence,
and this, coupled with its location 14 residues from the inner face of
the plasma membrane, makes it an ideal target for PKC following its
translocation to the plasma membrane in response to increased levels of
diacylglycerol. Ser-204, on the other hand, is not in a perfect PKC
consensus sequence because the Arg on the C-terminal side is at the
+3 position rather than at +2
(40) . However, the Arg
at -3 on the N-terminal side of Ser-204, coupled with its
location close to the plasma membrane, may compensate for this.
Although RPTP
There are many precedents for membrane-associated
proteins that are phosphorylated and regulated by PKC. For instance,
the EGF receptor is phosphorylated by PKC at Thr-654, which is in the
juxtamembrane domain in an exactly analogous location to
Ser-180/Ser-204
(41) , and this reduces EGF-dependent activation
of the EGF receptor protein-tyrosine kinase
(43) . Another
membrane protein, CD4, has been shown to be phosphorylated in a similar
location in response to TPA. The phosphorylation of CD4 by PKC also
appears to be functionally important
(44, 45) .
Does
PKC phosphorylation of RPTP
Two other PTPs are known to be
phosphorylated by PKC. In vitro PKC phosphorylates PTP1B at
Ser-378, and phosphorylation of Ser-378 is stimulated in TPA-treated
cells
(17) . However, phosphorylation of Ser-378 does not appear
to affect PTP1B activity. PTP-PEST is phosphorylated in vitro by PKC and the cAMP-dependent protein kinase at Ser-39 and
Ser-435, and the same sites are phosphorylated in TPA- or
forskolin-treated HeLa cells
(20) . In this case,
phosphorylation at Ser39 decreases PTP-PEST activity by increasing its
K
Many PTPs are
discretely localized in the cell
(46) , and the subcellular
localization of PTPs presumably dictates access to substrate. Given its
primary structure and the fact that it is a heavily glycosylated
protein, one might expect RPTP
We are grateful to Ushio Kikkawa for supplying
purified protein kinase C, to Carl Hoeger and Jean Rivier for synthesis
of the C-terminal RPTP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which is widely expressed in both the
developing and adult mouse, is regulated by phosphorylation, we raised
antiserum against a C-terminal peptide. This antiserum precipitated a
140-kDa protein from metabolically
S-labeled NIH3T3 cells.
Using this antiserum, we showed that endogenous RPTP
is
constitutively phosphorylated in NIH3T3 cells, predominantly on two
serines, which we identified as Ser-180 and Ser-204, lying in the
juxtamembrane domain. 12- O-tetradecanoylphorbol-13-acetate
(TPA) stimulation of quiescent NIH3T3 cells rapidly increased
phosphorylation of Ser-180 and Ser-204. Purified protein kinase C (PKC)
phosphorylated bacterially expressed RPTP
at Ser-180 and Ser-204.
When wild type and S180A/S204A double mutant RPTP
s were
transiently expressed in 293 human embryonic kidney cells, TPA
stimulated phosphorylation of wild type but not of double mutant
RPTP
. PKC down-regulation following prolonged exposure to TPA
diminished TPA-stimulated RPTP
phosphorylation. Taken together,
these results indicate that RPTP
is a direct substrate for (PKC).
Examination of 293 cells expressing exogenous RPTP
using
immunofluorescence confocal microscopy showed that RPTP
exists
predominantly in two subcellular compartments: in dense intracellular
granules or dispersed within the plasma membrane. TPA treatment caused
redistribution of some intracellular RPTP
to the cell surface, but
this did not require direct phosphorylation of RPTP
at
Ser-180/Ser-204. Our results suggest that activation of PKC by
cytokines modulates RPTP
function in several different ways.
(
)
Over the past few years, a large number of PTPs have been
cloned, based on sequence homology within the catalytic domains (for
reviews, see Refs. 1-4). Broadly speaking, there are two types of
PTP: membrane-spanning receptor-like PTPs and nonreceptor PTPs. Most
but not all receptor-like PTPs (RPTPs) contain two cytoplasmic
catalytic PTP domains. The domain closest to the membrane exhibits the
majority of the PTP activity, but in some cases the second domain also
possesses detectable but low PTP activity
(5, 6) . RPTP
extracellular domains range in size from over 1600 to 27 residues.
Whether the extracellular domain of RPTPs can bind ligands and, if they
do, whether this modulates PTP activity remains to be determined.
(also known as HRPTP
(7) , HPTP
(8) , LRP (LCA-related phosphatase)
(9) , HLRP
(10) , and R-PTP-
(11) ) is a RPTP, with a rather
short extracellular domain lacking any obvious structural motifs or
cysteines
(1, 2) . Mammalian RPTP
contains 793
amino acids, including a 19-residue signal sequence, a 123-amino acid
extracellular domain, a transmembrane domain, two catalytic domains,
and a short C-terminal tail. Both catalytic domains have been shown to
have in vitro PTP activity when expressed individually in
bacteria
(5) or collectively when expressed in insect cells
(12) . RPTP
is most highly expressed in brain, both in
embryonic development and in the adult
(11) . The fact that
RPTP
mRNA expression is enhanced during differentiation of
neuronally derived cell lines suggests that RPTP
may be involved
in neuronal differentiation. This idea is reinforced by the finding
that overexpression of RPTP
in pluripotent P19 embryonal carcinoma
cells alters the differentiation fate of these cells in favor of
neuronal differentiation
(13) . The nonreceptor protein-tyrosine
kinase c-Src is activated in the RPTP
-overexpressing P19 cells
(13) and in RPTP
-overexpressing Rat-2 cells
(14) ,
possibly as a result of direct dephosphorylation of the c-Src
inhibitory tyrosine phosphorylation site (Tyr-527) by RPTP
.
activity, concomitant with an increase in serine
phosphorylation, and in this case phosphorylation has been shown to
decrease the K
for phosphopeptide
substrate
(19) . PTP-PEST is phosphorylated in vitro and in vivo by PKC, but in this case its activity is
decreased due to an increase in its K
for
substrate
(20) . It has been shown that MAP kinase
phosphorylates and inactivates PTP2C (also known as PTP1D, Syp, and
SH-PTP2) in vitro, and this phosphorylation may be responsible
for the inhibition of PTP2C observed in EGF-treated PC12 cells during
the time when MAP kinase is activated
(21) .
is constitutively
phosphorylated on Tyr-789 in fibroblasts, and that protein Tyr-789 acts
as a binding site for the adaptor protein Grb2
(25) . Su et
al.(26) have also shown that RPTP
is tyrosine
phosphorylated and associates with Grb2. RPTP CD45 is phosphorylated on
tyrosine in vivo following treatment of cells with the PTP
inhibitor phenylarsene oxide
(27) , but whether treatment
affects activity in vivo is unclear. The same group has
recently reported that the PTP activity of RPTP CD45 is increased
in vitro by sequential phosphorylation first by a
protein-tyrosine kinase and second by a protein-serine kinase
(28) . The order of phosphorylation is critical, and the
increase in activity is also specific for the RCML substrate.
Proteolytic removal of the first catalytic domain produces a similar
activation
(6) , implying that the increase in RCML-specific PTP
activity is due to the second catalytic domain, consistent with the
sites of phosphorylation being located within the N-terminal portion of
the second catalytic domain.
function, we asked whether it is phosphorylated in vivo and
whether its phosphorylation can be modulated in response to growth
factors. We have shown that RPTP
is constitutively phosphorylated
predominantly on serine and have located the two serine residues close
to the inner face of the plasma membrane as major sites of
phosphorylation. Phorbol ester treatment increases phosphorylation at
these sites, and purified PKC phosphorylates these same serines in
vitro.
Cloning PTP
Sequences for the PTPs PTP1B
(29) , CD45
(30) , and LAR
(31) had been reported at the onset of
this investigation. These sequences were aligned, and regions conserved
in the three PTPs were used to design oligonucleotide primers specific
for the catalytic domain. polymerase chain reactions were performed
using RNA from NIH3T3 cells as a template. The product of this reaction
was used to probe an NIH3T3 cDNA library. A clone encoding a protein
with a structure typical of a CD45-like phosphatase was isolated. This
clone, pPTP and Construction of Recombinant
bPTP
, was used in all subsequent experiments. The sequence
and predicted translation product of pPTP
are essentially
identical to other reported mouse RPTP
clones
(9, 11) . Recombinant PTP
was generated by fusing
the cytoplasmic domain of PTP
beginning at Phe-168 to the
glutathione S-transferase gene of the expression plasmid
pGEXKG (pGEXPTP
). The fusion protein was purified on
glutathione-Sepharose beads and cleaved with thrombin generating
bPTP
.
Cell Culture, Labeling, and Transient
Transfection
For P labeling, NIH3T3 cells were
grown to
75% confluence in Dulbecco's modified Eagle's
medium (DMEM) containing 10% calf serum (CS) and then shifted to DMEM
containing 0.5% CS. After 24 h, the cells were labeled in
phosphate-free DMEM containing 0.5% dialyzed CS and 2 mCi/ml
[
P]orthophosphate (ICN) for 16 h at 37 °C.
Cells were stimulated by the addition of 50 ng/ml TPA (Sigma) for 15
min at 37 °C. For
S labeling,
2
10
NIH3T3 cells grown under the same conditions were labeled with
100 µCi/ml [
S]methionine/cysteine (Express,
DuPont NEN) for 20 h at 37 °C in methionine-free DMEM containing 4%
dialyzed CS. For transfection experiments, human embryonic kidney 293
cells were seeded on plates coated with 0.1% gelatin in DMEM containing
10% calf serum. 24 h later, cells were transfected with 20 µg of
Sg5 vector, which utilizes a SV40 promoter to drive expression,
containing either full-length wild type
(13) or S180A/S204A
mutant RPTP
. Transfections were done using the calcium phosphate
method as previously described
(25) . 8 h after transfection,
the medium was changed to either methionine-free DMEM containing 1%
dialyzed CS or phosphate-free DMEM containing 1% dialyzed CS and
incubated an additional 16 h at 37 °C. Cells were stimulated by the
addition of 100 ng/ml TPA for 10 min at 37 °C (initial time course
experiments had shown that the shorter time was optimal for the
transfected cells).
Antiserum and
Immunoprecipitation
Polyclonal rabbit antiserum, 5201, was
raised against the peptide CYKVVQEYIDAFSDYANFK corresponding to the
C-terminal 19 residues of RPTP. The peptide was coupled to KLH
(Calbiochem) and used to immunize rabbits
(32) . Polyclonal
rabbit antiserum, 5478, was raised against purified bPTP
protein
and purified on a GST-bPTP
affinity column
(25) .
S- or
P-labeled cells were lysed in
SDS-boiling lysis buffer (10 mM sodium phosphate, pH 7.2, 0.5%
SDS, 1% aprotinin, 1 mM EDTA, 1 mM dithiothreitol
(DTT)), boiled, and diluted with 4 volumes of RIPA buffer (10
mM sodium phosphate, pH 7.0, 150 mM NaCl, 2
mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS)
containing 1 mM DTT but lacking SDS. Lysates were prepared and
proteins were immunoprecipitated with 8 µl of preimmune serum, 8
µl of immune serum, or 8 µl of immune serum preincubated with
10 µg of immunizing peptide. The immunoprecipitates were collected
on protein A-Sepharose and washed four times in RIPA buffer without
DTT. The labeled proteins were resolved on a 7.5% SDS-polyacrylamide
gel and transferred electrophoretically to Immobilon-P or
nitrocellulose membranes.
Expression, Purification, and in Vitro
Phosphorylation of Recombinant bPTP
Expression,
purification, and thrombin cleavage of bPTP were performed as
described
(33) except that cells were lysed by sonication in
PBS containing 1% Triton X-100 (Sigma), 1% aprotinin, and 1 mg/ml
lysozyme. Purified rat brain PKC was the gift of Ushio Kikkawa and was
prepared as described
(34) . bPTP
was phosphorylated in
vitro by purified PKC under the following reaction conditions: 20
mM Hepes, pH 7.4, 10 mM MgCl
, 0.5
mM CaCl
, 5 mM DTT, 8 µg/ml
phosphatidylserine (Sigma), 0.8 µg/ml diolein (Avanti), 100 ng of
purified PKC, and 25 µCi of [
-
P]ATP
(3000 Ci/mmol, Amersham Corp.). The reaction was incubated for 15 min
at 30 °C and was terminated by the addition of EDTA to a final
concentration of 5 mM. Sample buffer was added, and the
samples were then boiled and resolved on a 10% SDS-polyacrylamide gel
and transferred to nitrocellulose.
Phosphoamino Acid Analysis and Two-dimensional
Phosphotryptic Mapping
Following transfer to Immobilon, the
phosphoamino acid content of RPTP was analyzed as described
(35) . Phosphopeptide maps of in vivo and in vitro
P-labeled proteins were prepared from proteins
blotted to nitrocellulose membranes as described
(36) . The
liberated peptides were dissolved in pH 1.9 buffer, applied to
100-µm thin-layer cellulose plates, and separated by
electrophoresis at pH 1.9 for 30 min at 1.5 kV, followed by ascending
chromatography in phosphochromotography buffer
(37) .
Site-directed
Mutagenesis
Oligonucleotides AGGCGGAAAGCGTTGGAATGA (Ser-180
to Ala) and CCTGTTGGTGGCCGGGGACCT (Ser-204 to Ala) were synthesized on
a Cyclone DNA synthesizer (Biosearch). Mutagenesis was carried out
using the Amersham oligonucleotide-directed in vitro mutagenesis system according to the manufacturer's manual as
described
(38) . The S180A and S204A single mutations and the
double mutation were introduced into pGEXKGPTP and verified by
sequencing.
Immunofluorescence Confocal
Microscopy
Human 293 cells were trypsinized 24 h following
transfection and seeded onto Lab-Tek-chambered glass slides (Nunc) that
had been treated with 20 µg/ml poly-L-lysine. The cells
were grown in DMEM containing 0.5% CS for 24 h and treated with 100
ng/ml TPA or left untreated for controls. Cells were fixed in freshly
made 4% paraformaldehyde/PBS at room temperature for 15 min, rinsed
three times for 10 min in 0.5 mM glycine/PBS, permeabilized in
0.5% Triton X-100, 0.5 mM glycine/PBS for 10 min, rinsed three
times for 10 min in 0.5 mM glycine/PBS, and blocked in 2%
normal goat serum, 0.5 mM glycine/PBS for 20 min. Primary
anti-RPTP antibody 5478 was then added to a final dilution of
1:500 and incubated for 1 h in a humidified chamber. The cells were
then washed three times for 10 min in 2% normal goat serum, 0.5
mM glycine/PBS, and secondary fluorescein-conjugated goat
anti-rabbit Ig (Cappel Labs) was added at a final dilution of 1:50 in
2% normal goat serum, 0.5 mM glycine/PBS and incubated for 20
min. The cells were then washed three times for 10 min in 2% normal
goat serum, 0.5 mM glycine/PBS, coverslips were applied to the
slides, and the cells were viewed on an Noran Odyssey laser confocal
microscope.
RPTP
To
study RPTP Is a 140-kDa Phosphoprotein
Phosphorylated on Serine and Tyrosine Residues, and the Level of Serine
Phosphorylation Increases following TPA Stimulation
in vivo, we raised an antiserum against the
predicted C-terminal 19 residues of RPTP
(Fig. 1 A)
and additional antisera against bacterially expressed GST fusion
proteins containing either the first catalytic domain or the whole
cytoplasmic domain of RPTP
. The anti-peptide serum precipitated a
140-kDa protein from
[
S]methionine/cysteine-labeled NIH3T3 cells
(Fig. 1 A). Inclusion of the immunizing peptide
specifically blocked precipitation of p140. The anti-bacterial fusion
protein sera also precipitated p140 (data not shown). The major
[
S]methionine/cysteine-labeled tryptic peptides
of immunoprecipitated p140 were also present in the 100-kDa product of
an in vitro translation reaction of synthetic RPTP
mRNA
(data not shown), thus demonstrating that p140 is RPTP
. The size
discrepancy is presumably due to glycosylation of p140; there are 8
predicted N-linked glycosylation sites in the extracellular
domain of RPTP
. Treatment of NIH3T3 cells with tunicamycin reduced
its size from 130 to 110 kDa (data not shown), indicating that
RPTP
is N-glycosylated. The residual size difference is
presumably due to O-glycosylation events
(39) .
Pulse-chase analysis indicated that RPTP
maturation proceeds
through a
90-kDa precursor form (data not shown). Daum et al.(39) have reached similar conclusions.
Figure 1:
RPTP is a 140-kDa phosphoprotein
phosphorylated predominantly on serine residues. A, an
antiserum raised against the C-terminal 19 residues of RPTP
precipitated a 140-kDa protein from metabolically
S-labeled NIH3T3 cells (preimmune, lane1; immune, lane2). Precipitation was
blocked by the addition of peptide (immune + peptide, lane3). Proteins were analyzed on a 7.5% SDS-polyacrylamide
gel. The gel was impregnated with 2,5-diphenyloxazole and exposed to
Kodak XAR film for 6 days at -70 °C. B,
immunoprecipitation of RPTP
from in vivo
P-labeled resting (control, lane1)
and TPA-stimulated ( TPA, lane2) NIH3T3
cells. Lanes1 and 2 are nonadjacent lanes
from the same gel. Immunoprecipitated RPTP
was resolved on a 7.5%
SDS-polyacrylamide gel. Proteins were transferred to Immobilon and
exposed to Kodak XAR film for 16 h at -70 °C with an
intensifier screen. C-F, phosphoamino acid analysis of
P-labeled RPTP
from resting ( C and E) and
TPA-stimulated NIH3T3 cells ( D and F). E and F are 10-day exposures illustrating the presence of phosphothreonine
and phosphotyrosine, while C and D are 3-day
exposures illustrating the increase in phosphoserine. The
horizontalarrowhead indicates the position of
phosphotyrosine. The schematic indicates the relative positions of the
ninhydrin-stained phosphoamino acid markers.
To determine
whether RPTP is phosphorylated, we immunoprecipitated RPTP
from
P-labeled NIH3T3 cells. RPTP
was found to be
phosphorylated in cells growth arrested in low serum
(Fig. 1 B). Several mitogens were tested to determine
whether exogenous stimulation of NIH3T3 cells altered RPTP
phosphorylation. The level of phosphorylation increased modestly
following stimulation with TPA (Fig. 1 B),
platelet-derived growth factor (data not shown), and serum. In
different experiments, the level of RPTP
phosphorylation increased
30-50% following TPA treatment (Fig. 1 B), and the
increase was maximal by 15 min following TPA stimulation (data not
shown). Phosphoamino acid analysis of RPTP
showed it to be
phosphorylated predominantly on serine and to a lesser extent on
threonine and tyrosine (Fig. 1, C and E). The
increase in phosphorylation detected after TPA treatment was reflected
in an increase in phosphoserine (Fig. 1, D and
F).
PKC Phosphorylates Bacterially Expressed RPTP
To determine if the increase in phosphorylation of
RPTP in
Vitro at the Same Sites as the Major in Vivo Sites (Ser-180 and
Ser-204)
after TPA treatment was due to direct phosphorylation by PKC,
the entire cytoplasmic domain of RPTP
(residues 168-793) was
expressed in bacteria as a GST fusion protein, purified, and
phosphorylated in vitro by PKC purified from rat brain
(Fig. 2 A). Two-dimensional phosphotryptic mapping of
in vivo
P-labeled RPTP
revealed three major
phosphopeptides and several minor ones (Fig. 2 B). The
pattern of phosphopeptides was unchanged after TPA treatment, but the
intensity of the major phosphopeptides increased. Comparison of this
pattern with the phosphotryptic map from in vitro phosphorylated bPTP
showed that three of the peptides found
in in vivo
P-labeled RPTP
were generated
upon PKC phosphorylation. This was confirmed by the comigration of the
in vivo and in vitro generated phosphopeptides
(Fig. 2 B).
Figure 2:
Bacterially expressed RPTP is
phosphorylated in vitro by purified PKC at the same sites as
endogenous RPTP
. A, the cytoplasmic domain of RPTP
was fused to the glutathione S-transferase gene of the
expression plasmid pGEXKG (pGEXPTP
). The fusion protein was
purified on glutathione-Sepharose beads and cleaved with thrombin
generating bPTP
. The products of an in vitro phosphorylation reaction containing PKC and 0, 5, 25, or 50 ng of
bPTP
( lanes1-4) were resolved on a 10%
SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed to
Kodak XAR film for 15 min. Positions of PKC and bPTP
are
indicated. Molecular weight standards are Rainbow markers (Amersham).
B, phosphotryptic peptide maps of in vivo
P-labeled RPTP
and cytoplasmic bPTP
phosphorylated in vitro by PKC. Approximately 100 cpm were
analyzed in each case. Arrows indicate the origin.
Electrophoresis was in the horizontal dimension with the cathode on the
right. Panel1, control; panel2, TPA-stimulated NIH3T3 cells labeled with
[
P]orthophosphate as in Fig. 1 B;
panel3, in vitro phosphorylated bPTP
;
panel4, schematic diagram indicating the position of
the major phosphopeptides (phosphopeptides found in vivo and
in vitro ( A-C) and phosphopeptides found only
in vivo (1, 2)); panel5, mix of 50 cpm of
control and 50 cpm of in vitro phosphorylated bPTP
;
panel6, mix of 50 cpm of TPA-stimulated and 50 cpm
of in vitro phosphorylated bPTP
. Plates were dried and
exposed for 6 days at -70 °C with an intensifier
screen.
The sequence of RPTP was then
examined for tryptic peptides containing possible PKC phosphorylation
sites (Ser/Thr-hydrophobic-Arg)
(40) . Due to its neighboring
basic residues and position near the transmembrane domain,
characteristics similar to previously described PKC phosphorylation
sites
(41) , Ser-180 was chosen as a likely candidate for a PKC
site. Ser-180 was mutated to Ala, and the S180A mutant RPTP
was
expressed in bacteria and phosphorylated in vitro by PKC. The
phosphotryptic map of the S180A mutant bPTP
revealed the loss of
peptides B and C, but peptide A remained (Fig. 3 B),
indicating that peptides B and C contain Ser-180. The predicted
mobility of the tryptic peptide containing Ser-180 is approximately
that of peptide B. The N-terminal residue of this peptide is Gln, and
cyclization of this residue to pyrocarboxylic acid during the tryptic
mapping procedure would yield a more negatively charged, less
hydrophilic species at pH 1.9 corresponding to peptide C; a similar
situation exists for the major phosphopeptide derived from the
autophosphorylated v-Fps protein
(42) . Therefore, we believe
that peptide C is derived from peptide B during the generation of the
tryptic digest.
Figure 3:
Mutation of Ser-180 and Ser-204 abolishes
phosphorylation of bPTP by PKC in vitro. A,
in vitro phosphorylation of wild type ( WT) and mutant
bPTP
by purified PKC was performed as in Fig. 2 A. The
products of an in vitro phosphorylation reaction containing
either no added protein ( lane1), 50 ng of purified
GST protein from bacteria expressing the vector alone ( lane2), 50 ng of wild type bPTP
( lane3), 50 ng of S204A bPTP
( lane4),
or 50 ng of S180A/S204A bPTP
( lane5) were
analyzed on a 10% SDS-polyacrylamide gel, dried, and autoradiographed.
The positions of PKC and bPTP
are indicated. B,
phosphotryptic maps of wild type and the Ser-180 to Ala mutant (S180A)
and Ser-204 to Ala mutant (S204A) cytoplasmic domains expressed in
bacteria and phosphorylated in vitro by PKC. Panel1, wild type bPTP
(2000 cpm); panel2, Ser-180 to Ala mutant (S180A) bPTP
(2,000 cpm);
panel3, mix of 1000 cpm each of wild type and S180A
bPTP
; panel4, wild type bPTP
(2000 cpm);
panel5, Ser-204 to Ala mutant (S204A) bPTP
(2000 cpm); panel6, mix of 1000 cpm each of wild
type and S204A bPTP
. Because the experiment shown in panels1-3 was carried out on a different occasion to that
in panels4-6, two wild type controls are
shown. Plates were exposed to Kodak XAR film for 16 h at 23 °C.
C, schematic showing the position of PKC phosphorylation sites
in RPTP
. The inset depicts the sequence of the predicted
CNBr fragment from bPTP
(the upstream methionine is provided by
GST) and shows Ser-180 and Ser-204. The sequence begins one residue
inside the transmembrane domain as indicated by the horizontalarrow. Arrowheads indicate predicted sites of
trypsin cleavage.
To narrow the list of possible tryptic peptides
responsible for the remaining peptide A, manual Edman degradation was
performed. Free phosphate was released at the third cycle of
degradation, indicating that the phosphoserine was the third residue
from the N terminus of the peptide (data not shown). Two RPTP
peptides met this criterion. The tryptic peptide containing Ser-204 was
considered the most likely candidate due to its relatively hydrophilic
nature. It was also noted that Ser-204 is located in the same cyanogen
bromide (CNBr) fragment as Ser-180. If Ser-204 is the second site of
PKC phosphorylation, then in vitro phosphorylation of
bPTP
should generate a labeled CNBr fragment of
7 kDa, and
trypsin digestion of this fragment should yield all three major
phosphopeptides present in the intact protein. In addition, the S180A
mutant bPTP
should generate the same CNBr fragment containing only
peptide A. To test this hypothesis, in vitro phosphorylated
wild type and S180A bPTP
were isolated from an SDS-polyacrylamide
gel, excised, cleaved with CNBr, and run on a 20% SDS-polyacrylamide
gel. As predicted, a
7-kDa
P-labeled band was
obtained in both cases. The isolated bands were subjected to tryptic
digestion and peptide mapping, and the phosphopeptides were shown to be
identical to those obtained following phosphorylation of the intact
protein (data not shown).
was expressed in
bacteria, purified, and phosphorylated in vitro. the
S180A/S204A double mutant bPTP
failed to be phosphorylated in
vitro by PKC (Fig. 3 A). Moreover, the major
phosphopeptide, peptide A, was missing from the peptide map of S204A
mutant bPTP
while peptides B and C were still present
(Fig. 3 B). Therefore, we conclude that Ser-204 is the
other site of PKC phosphorylation in RPTP
. The remaining band in
the S180A/S204A lane of approximately the same mobility as bPTP
was shown to be identical to a background band in the GST control lane
by tryptic peptide mapping (data not shown). Fig. 3 C illustrates the position of the two phosphorylation sites relative
to the membrane spanning domain and the two catalytic domains of
RPTP
.
Transient Expression of Wild Type and S180A/S204A
RPTP
To examine the effect of the
phosphorylation site mutations in vivo, sequences encoding
either the entire wild type or the S180A/S204A RPTP in 293 Cells
were expressed
from the Sg5 vector by transient transfection of 293 cells, a human
embryonic kidney cell line immortalized with adenovirus E1A
(Fig. 4 A). As determined by immunoprecipitation of
S-labeled cells, the level of RPTP
expression in
transfected cells was greater than 20 times that in the
vector-transfected controls (Fig. 4 A). The protein
migrating at
87 kDa in transfected cells was shown to have an
identical [
S]methionine/cysteine-labeled tryptic
map to RPTP
(data not shown) and is likely to be the
unglycosylated RPTP
precursor
(39) .
(
)
Treatment of
P-labeled wild type
RPTP
-expressing 293 cells with TPA resulted in a large increase in
phosphorylation of RPTP
(Fig. 4 B). In contrast, TPA
treatment of cells expressing the S180A/S204A mutant RPTP
resulted
in only a very slight increase in
P incorporation. Tryptic
peptide mapping revealed that in resting cells, wild type RPTP
was
phosphorylated on the same three major peptides that were
phosphorylated in vitro by PKC (data not shown). Peptide maps
of S180A/S204A mutant RPTP
in resting cells contained a single
phosphopeptide that comigrated with the phosphopeptide that contains
Ser-204. This peptide contained only phosphoserine (data not shown).
TPA treatment of S180A/S204A RPTP
-expressing cells did not
increase the
P content of this major peptide but rather at
a large number of sites as demonstrated by the presence of a number of
new phosphopeptides. Since PKC did not phosphorylate the S180A/S204A
mutant RPTP
in vitro (Fig. 3 B), 293 cells
may contain a protein kinase distinct from PKC that phosphorylates
RPTP
at Ser-202, which is present in the same tryptic peptide as
Ser-204.
Figure 4:
Transient expression of wild type and
S180A/S204A mutant RPTP in human embryonic kidney (293) cells. 293
cells were transiently transfected overnight with the Sg5 expression
vector or Sg5 encoding either full-length wild type ( WT) or
S180A/S204A RPTP
. The following morning, the medium was changed
from DMEM containing 10% calf serum to DMEM containing 1% serum. After
8 h, the cells were metabolically labeled with either
[
S]methionine/cysteine ( A) or
[
P]orthophosphate ( B), and after an
additional 16 h, the cells were either treated with 100 ng/ml TPA for
10 min(+) or left untreated (-). RPTP
was then
immunoprecipitated with anti C-terminal peptide antiserum and analyzed
on a 7.5% SDS-polyacrylamide gel.
Down-regulation of PKC Results in Reduced
TPA-stimulated Phosphorylation of RPTP
It has been
well documented that long term exposure to TPA leads to down-regulation
of some isoforms of PKC. Reduced phosphorylation of RPTP following
preincubation with TPA would be taken as evidence that PKC was in fact
the protein kinase responsible for the in vivo phosphorylation. To test this experimentally, 293 cells were
transiently transfected with the expression vectors for wild type
RPTP
or the S180A/S204A mutant RPTP
, serum-starved, and
labeled with [
S]methionine/cysteine or
[
P]orthophosphate, and, where indicated, cells
were pretreated with 100 ng/ml TPA for 24 h to down-regulate PKC
(Fig. 5). In cells expressing wild type RPTP
, the increase
in phosphorylation seen following TPA stimulation was reduced when the
cells were preincubated with TPA. TPA-stimulated phosphorylation was
also reduced in cells expressing the S180A/S204A mutant RPTP
,
indicating that the sites that are phosphorylated in the absence of the
major PKC sites are also due to PKC activation and presumably result
from phosphorylation by a PKC-activated protein kinase. It is
interesting to note that there was a small (
50%) but reproducible
increase in the apparent amount of
S-labeled RPTP
present in immunoprecipitates from RPTP
-expressing transfected
cells that had been treated with TPA (Fig. 5). The increase was
also apparent in immunoblots of total cell lysates and in RPTP
immunoprecipitates from cells treated with TPA in the presence of
cycloheximide (data not shown). This result indicates that the apparent
increase in RPTP
was not the result of new protein synthesis,
which was to be expected given the short (10 min) TPA treatment. The
reported values for the increase in phosphorylation have been
calculated, taking the increase in the apparent amount of RPTP
protein into account.
Figure 5:
Down-regulation of PKC results in reduced
TPA-stimulated phosphorylation of RPTP. Human 293 cells were
transiently transfected, serum-starved, in vivo labeled,
immunoprecipitated, and analyzed as in Fig. 4 except that, where
indicated, cells were pretreated with 100 ng/ml TPA for the 24 h that
the cells were incubated in low serum. Upper left,
P-labeled wild type-transfected 293 cells. Upper
right,
P-labeled S180A/S204A-transfected 293 cells.
Lower left,
S-labeled wild type-transfected 293
cells. Lower right,
S-labeled
S180A/S204A-transfected 293 cells.
Immunofluorescence Staining of 293 Cells Transiently
Expressing RPTP
The puzzling observation that the
amount of RPTP protein apparently increased upon TPA treatment
prompted us to examine the subcellular location of RPTP
in
transfected 293 cells using immunofluorescence confocal microscopy.
RPTP
-expressing cells could readily be detected by this method and
represented 30-50% of the total population, depending on the
experiment (Fig. 6). The subcellular distributions of both wild
type and S180A/S204A mutant RPTP
were identical (data not shown).
In the majority of the transfected cells, RPTP
was present at the
cell surface as expected (Fig. 6, D-H). However,
in
30-40% of the positive cells, RPTP
staining appeared
in dense intracellular granules that were clearly distinct from the
Golgi apparatus (Fig. 6 B). Stimulation cells with TPA
resulted in a transient apparent increase in the percentage of cells
expressing exogenous RPTP
at the cell surface
(Fig. 6 D). The TPA-induced redistribution of RPTP
may reflect not only translocation but also maturation of the protein
into the 140-kDa form, thus accounting for the apparent protein
synthesis-independent increase in RPTP
in TPA-treated cells.
Figure 6:
Immunofluorescence staining of RPTP
in transiently transfected 293 cells. 293 cells were trypsinized 24 h
following transfection with Sg5 wild type RPTP
expressing and
seeded on to chambered glass slides. The cells were grown in DMEM
containing 0.5% serum for 24 h and treated with 100 ng/ml TPA for 10
min or left untreated for control. The cells were stained with
affinity-purified antibodies raised against bPTP
(5478).
A, phase contrast of a field of control cells. B, the
same field of cells viewed with fluorescence. A positive staining cell
is in the center of the field. Almost all of the stain is localized
within intracellular granules; very little or none was visible at the
cell surface. C, phase contrast of a field of TPA-treated
cells. D-E, the same field of cells viewed with
fluorescence and focused in D at the level of the substratum
and in E on the top of the cell. The majority of the staining
can be seen at the cell surface. F-H, other TPA-treated
cells exhibiting either entirely surface-associated staining ( F and G) or localized to both the cell surface and
intracellular granules ( H).
is a
phosphoprotein. In fibroblasts, RPTP
is phosphorylated on serine
at two major sites, Ser-180 and Ser-202, and as we have reported
elsewhere
(25) , on Tyr-789. The phosphorylation of Ser-180 and
Ser-202 is stimulated by TPA treatment, and, since these sites are
phosphorylated in vitro by PKC, we conclude that they are
phosphorylated by PKC in vivo. In NIH3T3 cells, the
stimulation by TPA was only modest but much more dramatic when
exogenous RPTP
was expressed in 293 cells. RPTP
is not an
abundant protein in fibroblasts, and we suspect that basal PKC activity
is sufficient to phosphorylate Ser-180 and Ser-204 even under
growth-arrested conditions. We have not determined the exact
stoichiometry of phosphorylation at these sites, but based on our
estimate of 1.2 mol of phosphate per mol of RPTP
in NIH3T3 cells
(25) , and the relative intensities of the Ser-180/Ser-202
peptides and the Tyr-789 peptide, the stoichiometry of phosphorylation
of Ser-180 and Ser-202 is probably close to 50% in NIH3T3 cells. Thus,
a 2-fold stimulation of phosphorylation is the maximum that can be
achieved. In 293 cells, where exogenous RPTP
is overexpressed
20-fold, the basal level of phosphorylation is much lower, and
PKC-dependent phosphorylation is much more dramatic.
and RPTP
are closely related proteins,
RPTP
lacks a Ser at the position equivalent to Ser-180. RPTP
does have a Ser at the position equivalent to Ser-204, and although the
surrounding sequence is not identical, the two basic residues are
conserved, and this Ser could be a substrate for PKC. Ser-202, which
may be phosphorylated in the S204A mutant RPTP
, is followed by a
proline and could be phosphorylated by a proline-directed protein
kinase such as a MAP kinase. We note that several other RPTPs,
including LAR, RPTP
, and RPTP
, have potential PKC
phosphorylation sites within 20 residues of the inner face of the
plasma membrane.
in the juxtamembrane domain regulate
its activity? den Hertog and colleagues
(19) have shown that
TPA stimulation of 293 or P19 cells expressing exogenous RPTP
increases the in vitro PTP activity of RPTP
2-3-fold, due to a decreased K
for
phosphorylated myelin basic protein substrate rather than an increase
in V
. The increased activity is reduced by
phosphatase treatment of RPTP
, probably as a result of
dephosphorylation of Ser-180/Ser-204, which are phosphorylated in
response to TPA in these cells but possibly of other sites as well. To
determine whether phosphorylation Ser-180/Ser-204 directly affects
RPTP
activity, we phosphorylated bPTP
with purified PKC.
Phosphorylation did not affect its activity in vitro (data not
shown), but the maximal phosphorylation stoichiometry we could achieve
was 10%, which would have prevented us from detecting a small change in
bPTP
activity. We have also been unable to find a conclusive
difference in activity between wild type and S180A/S204A mutant
RPTP
expressed in 293 cells upon TPA treatment (data not shown),
but this analysis is complicated by the TPA-induced increase in the
mature form of RPTP
(Fig. 5). It is also possible that
phosphorylation at other sites affects RPTP
activity. At present,
it is an open question whether a small decrease in
K
will significantly affect RPTP
function in vivo, but if RPTP
activity is stimulated via
PKC-mediated phosphorylation in vivo, then this could provide
a mechanism for down-regulating the activity of ligand-activated
receptor-like protein-tyrosine kinases, such as the platelet-derived
growth factor receptor, that stimulate phosphoinositide turnover,
either by dephosphorylating the receptor itself or substrates
phosphorylated by the receptor.
for substrate.
to be found on the cell surface. We
attempted to determine the subcellular distribution of RPTP
by
immunofluorescence staining, but we were unable to obtain convincing
staining of endogenous RPTP
in either NIH3T3 cells or in
untransfected 293 cells due to the low level of protein. Therefore, we
resorted to the use of transiently transfected 293 cells to localize
RPTP
, but it should be kept in mind that RPTP
is highly
overexpressed in this system, and this may affect its subcellular
localization. Nevertheless, with this reservation we found that most
cells displayed surface staining as expected if RPTP
is localized
to the plasma membrane. However, as assessed by immunofluorescence
confocal microscopy, the subcellular distribution of RPTP
varied
from entirely intracellular to entirely at the cell surface. The nature
of the dense intracellular granules that stained in
30-40%
of the RPTP
positive cells is unknown, but they were clearly
distinct from the Golgi apparatus. TPA stimulation resulted in a
transient increase in the fraction of cells expressing exogenous
RPTP
at the cell surface. This translocation did not require
direct phosphorylation of RPTP
by PKC because the S180A/S204A
double mutant RPTP
behaved in a similar fashion (data not shown).
TPA also rapidly increased the amount of the 140-kDa form of RPTP
in transiently transfected 293 cells, suggesting that a PKC-mediated
process accelerates the maturation of RPTP
. This effect of TPA was
also detected in NIH3T3 cells, suggesting that it is a normal cellular
response. The relocalization of RPTP
may modulate its access to
specific substrates. Thus, activation of PKC by cytokines can in
principle affect the amount of mature RPTP
, its localization, and
activity, and all of these may regulate RPTP
function.
, bacterial protein-tyrosine phosphatase
; CS, calf
serum.
peptide, and to Jeroen den Hertog for
communicating unpublished results and providing affinity-purified
anti-bPTP
antibodies. We thank Dan Chin for carrying out the
confocal microscopy.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.