From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of T lymphocytes to produce cytokines
is regulated by the counterbalance of protein-tyrosine kinases and
protein-tyrosine phosphatases, many of which have a high degree of
substrate specificity because of physical association with their
targets. Overexpression of hematopoietic protein-tyrosine phosphatase
(HePTP) results in suppression of T lymphocyte activation as measured
by T cell antigen receptor-induced activation of transcription factors
binding to the 5' promoter of the interleukin-2 gene. Efforts to
pinpoint the exact site of action and specificity of HePTP in the
signaling cascade revealed that HePTP acts directly on the
mitogen-activated protein (MAP) kinases Erk1 and 2 and consequently
reduces the magnitude and duration of their catalytic activation in
intact T cells. In contrast, HePTP had no effects on N-terminal c-Jun kinase or on events upstream of the MAP kinases. The specificity of
HePTP correlated with its physical association through its noncatalytic
N terminus with Erk and another MAP kinase, p38, but not Jnk or other
proteins. We propose that HePTP plays a negative role in antigen
receptor signaling by specifically regulating MAP kinases in the
cytosol and at early time points of T cell activation before the
activation-induced expression of nuclear dual-specific MAP kinase phosphatases.
Phosphorylation of proteins on tyrosyl residues is an important
mechanism for many signal transduction pathways controlling cell
growth, differentiation, and development (1-3). Although the
phosphotyrosine (Tyr(P)) content of cellular proteins is the net result
of the opposing effects of protein-tyrosine kinases and
protein-tyrosine phosphatases
(PTPases),1 most
investigators have concentrated on the protein-tyrosine kinases, and
considerably less is currently known about the PTPases. The
hematopoietic protein-tyrosine phosphatase (HePTP) was cloned from
human T lymphocytes (4, 5), and it is expressed in thymus, spleen, and
in most leukemic cell lines examined, including Jurkat T leukemia cells
(6). HePTP belongs to a subgroup of PTPases with two other members,
STEP (7) and PCPTP1 (8, 9). In contrast to HePTP, the other two enzymes
are not expressed in hematopoietic cells but mainly in the central
nervous system: STEP mainly in striatum (7), and PCPTP1 particularly in
cerebellum (8). Like PCPTP1 and the 46-kDa isoform of STEP, HePTP
consists of a single PTPase domain that occupies the C-terminal 3/4 of the enzyme and is preceded by an ~80-amino acid noncatalytic N terminus. As might be expected from the lack of putative transmembrane sequences or other recognizable targeting motifs, immunofluorescence microscopy indicates that HePTP is located exclusively in the cytosol
in RBL mast cells (10) and Jurkat T
cells.2
The biological function of HePTP has remained elusive. A potential role
in cell proliferation or differentiation was suggested by the finding
that the HePTP gene is located at 1q32.1 (11) on the long arm of
chromosome 1, which is often found in extra copies (trisomy) in bone
marrow cells from patients with myelodysplastic syndrome (12, 13), a
disease characterized by reduced hematopoiesis. In contrast, deletions
of 1q32 have been reported in non-Hodkin lymphomas and chronic
lymphoproliferative disorders (14). Thus, these findings suggest that
excess HePTP may correlate with reduced proliferation (in
myelodysplasia) and loss of HePTP with increased cell proliferation
and/or survival. Amplification and overexpression of HePTP has also
been reported in a case of myelogenous leukemia (11). A connection with
lymphoid proliferation is also supported by the finding that the HePTP
gene is transcriptionally activated in T cells treated with
interleukin-2 (15). Although mRNA levels increased severalfold upon
stimulation of normal mouse lymphocytes with phytohemagglutinin,
lipopolysaccharide, concanavalin A, or anti-CD3 (4), the HePTP protein
was present in resting cells, and its amount increased only moderately.
Finally, HePTP has been reported to become phosphorylated on tyrosine
in RBL-2H3 mast cells stimulated through their Fc We recently reported that transient expression of HePTP in T cells
caused a clear reduction in antigen receptor-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T
cells (NFAT)/activator protein-1 (AP-1) element taken from the interleukin-2 gene promoter (6). In contrast, a catalytically inactive
C270S mutant of HePTP had no effect, suggesting that the PTPase
activity of HePTP was required for inhibition. We have continued this
work and have found that HePTP inhibits NFAT/AP-1 activation, and
thereby the entire 5' interleukin-2 promoter, by dephosphorylating the
Erk MAP kinase. Specificity for this substrate is the result of a
physical association between HePTP and Erk mediated by the noncatalytic
N terminus of HePTP. This region also bound the p38 MAP kinase but not
the N-terminal c-Jun kinases Jnk1 and Jnk2 or other signaling proteins.
We also show that HePTP is a substrate for Erk and p38, and we identify
the sites of phosphorylation and effects of phosphorylation on the association between HePTP and these kinases.
Reagents--
Most antibodies and plasmids were as
described before (6). The HePTP fragments Site-directed Mutagenesis--
As described earlier (6) for the
catalytically inactive mutant of HePTP (C270S), the S72A and T45A
mutants were generated using the TransformerTM
site-directed mutagenesis kit as recommended by the manufacturer (CLONTECH, Palo Alto, CA). Mutations were verified
by nucleotide sequencing.
Cells, Transfections, and Luciferase Assays--
Jurkat T
leukemia cells and two variants of this cell line, JCaM1.6, which lacks
Lck (17), and P116, which lacks Zap-70 (Ref. 18; a kind gift from R. Abraham), were kept at logarithmic growth in RPMI 1640 medium with 10%
fetal calf serum, L-glutamine, and antibiotics. These cells
were transiently transfected with a total of 5-10 µg of DNA by
electroporation at 950 microfarads and 240 V as before. Empty vector
was added to control samples to make a constant amount of DNA in each
sample. Luciferase assays were done as described in detail before (6,
19, 20).
Immunoblots, Immunoprecipitation, and in Vitro Kinase
Assays--
Immunoprecipitation and in vitro kinase assays
were done as before (6, 20). All immunoblots were developed by the
enhanced chemiluminescence technique (ECL kit, Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
Phosphoamino Acid Analysis and Tryptic Peptide
Mapping--
GST-HePTP protein phosphorylated in vitro by
the bound kinase in the presence of [ HePTP Reduces AP-1-dependent Gene Activation--
A
crucial step in the initiation of an immune response is the production
of cytokines by T cells challenged with properly presented antigen
(22). T cell antigen receptor-induced activation of the interleukin-2
gene is the result of the coordinated action of several transcription
factors (23, 24), including a trimeric complex consisting of a NFAT
family protein (25) and an AP-1 dimer (26, 27) of Fos and Jun family
proteins, with some assistance from octamer-binding proteins (Oct) and
NF-
When HePTP was expressed in Jurkat T cells together with a luciferase
reporter gene under the control of the 5' interleukin-2 promoter, the
activation of this reporter was reduced to 58.1 ± 9.3%
(n = 9). In contrast, expression of the catalytically
inactive mutant HePTP-C270S or two other PTPases, SHP2 and TCPTP, did
not affect the activation of the reporter gene although being expressed at similar levels. The inhibition by HePTP was not as pronounced as its
effect on a reporter gene driven by a subregion of the interleukin-2
promoter, one of the NFAT/AP-1 response elements, which was inhibited
by more than 80% (6). This discrepancy is probably explained by the
minimal effects of HePTP on luciferase reporter constructs driven by
Oct or NF- HePTP Inhibits Erk but Not Jnk--
First, we measured the two
types of MAP kinases known to be involved in AP-1 activation in T
cells, Erk and Jnk (27, 28). Jurkat T cells were transiently
co-transfected with epitope-tagged Erk2 together with HePTP,
HePTP-C270S, TCPTP,or SHP2. After stimulation of the cells, the
catalytic activity of the kinases was measured. These experiments
consistently revealed that the antigen receptor-induced activation of
Erk was significantly reduced by HePTP. In cells stimulated for
different periods of time with anti-CD3
In agreement with the notion that HePTP inhibits the activation of the
interleukin-2 gene by reducing the magnitude and duration of Erk
activation, we observed that expression of Erk2 plus an activated
mutant (29) of Mek, the upstream activator of Erk, augmented
interleukin-2 promoter activation and increased its sensitivity to
HePTP (Fig. 1c). As a control, co-expression of activated
Mkk6 plus p38 kinases did not affect the interleukin-2 reporter or its
inhibition by HePTP (not shown). Furthermore, the activation of another
gene known to be up-regulated, in part, through Erk-mediated
phosphorylation of the Elk-1 transcription factor (30),
c-fos, was also reduced in T cells overexpressing HePTP but
not in cells expressing the inactive HePTP-C270S (not shown). Together,
all these results indicate that HePTP reduces the activation of Erk
in vivo. This notion is supported by the opposite effect of
catalytically inactive HePTP-C270S in the same assays.
HePTP Inhibits Endogenous Erk but Not Upstream Events--
To
ascertain that the observed inhibition by HePTP was not limited to
exogenous transfected MAP kinase, we next utilized the JCaM1 cell line,
which is unresponsive to T cell antigen receptor stimulation because of
lack of Lck kinase (17). Transient expression of Lck (or Syk) restores
responsiveness (20). JCaM1 cells were transfected with Lck together
with HePTP, HePTP-C270S, or control PTPases and used for analysis of
antigen receptor-induced appearance of activated and phosphorylated MAP
kinases by immunoblotting with activation-specific antibodies. Fig.
1d shows that HePTP reduced the appearance of phospho-Erk1
and -2, while at the same time and in the same cells, not having any
effects on activation of Mek or the receptor-induced tyrosine
phosphorylation of cellular proteins (Fig. 1e), including the HePTP Also Inhibits Phorbol Ester-induced Erk Activation--
The
MAP kinase pathway can also be efficiently activated by phorbol esters,
which bypass all receptor-induced proximal tyrosine phosphorylation
events by activating the Raf kinase through protein kinase C (31, 32).
When tagged Erk2 was expressed in JCaM1 cells together with HePTP and
the cells were stimulated with 20 nM phorbol myristate
acetate, the activation of the MAP kinase was profoundly inhibited
compared with cells expressing Erk2 alone (Fig. 1f). In
contrast to cells stimulated by anti-CD3 (lanes 2,
4, and 6), phorbol ester-induced MAP kinase
activation did not require expression of Lck. Control blots confirmed
that the immunoprecipitates contained equal amounts of Erk2 and that
equal amounts of HePTP and Lck were expressed in the transfectants
(Fig. 1f, lower panels). Because HePTP is
specific for Tyr(P), this result supports the conclusion that HePTP
does not block MAP kinase activation by dephosphorylating
receptor-proximal tyrosine phosphorylation events. Rather, these data
suggest a more direct effect on Erk.
Direct Effect of HePTP on Erk--
Having found that HePTP had no
effects on the phosphorylation of Mek in the same cells where Erk
phosphorylation was reduced, we asked if HePTP acts directly on
phospho-Erk. First, we phosphorylated recombinant kinase-inactive Erk1
at the activation loop threonine and tyrosine residues using active
recombinant Mek and treated the resulting phospho-Erk with recombinant
HePTP at 37 °C. As shown in Fig.
2a, the Tyr(P) content of Erk
decreased detectably within 10-30 s and was very low by 1-5 min.
Phosphoamino acid analysis of similarly treated Erk1 phosphorylated by
Mek in the presence of [ Physical Association between HePTP and MAP Kinase--
To directly
test the possibility that HePTP binds Erk, we used GST fusion proteins
of HePTP, HePTP-C270S, or SHP2 and found that both HePTP and
HePTP-C270S readily bound Erk (Fig.
3a) in lysates of resting T
cells or cells treated with pervanadate (to maximize tyrosine
phosphorylation (33)). The precipitates also reacted strongly with
antibodies to another MAP kinase, p38. In contrast, neither Jnk1 nor
Jnk2 bound, and control GST, GST-SHP2 (Fig. 3a), or
GST-TCPTP (not shown) did not precipitate any of these kinases.
Immunoblotting with antibodies to the 36-38-kDa LAT (34) or to other
signaling molecules gave negative results. The Erk and p38 that bound
active HePTP did not contain Tyr(P) and were catalytically inactive. In
contrast, Erk and p38 bound to inactive HePTP-C270S from
pervanadate-treated cells were phosphorylated on tyrosine and
enzymatically highly active against myelin basic protein or GST-ATF2, a
preferred substrate for p38 kinase (Fig. 3b). In these
reactions, GST-HePTP was also phosphorylated by the bound Erk and p38.
HePTP also co-immunoprecipitated with p38 from untreated or activated
Jurkat T cells (Fig. 3c). Co-immunoprecipitation of HePTP
and Erk was not as clean because of nonspecific binding of Erk to
Sepharose beads coated with irrelevant immunoglobulin (Fig.
3d). We conclude that HePTP specifically associates with Erk
and p38 MAP kinases.
HePTP Also Inhibits Activation of p38 Kinase but Not Activation of
Jnk--
The finding that HePTP associates with both Erk and p38, but
not with Jnk, prompted us to study the direct effects of HePTP on the
kinase activity of p38 in vitro and on the activation of p38
and Jnk in intact cells. First, we used 10 ng of recombinant active p38
kinase, added 100 ng of GST-HePTP, GST-HePTP-C270S, control GST, or
GST-SHP2, and incubated the samples with [ Mapping of the Binding Site in HePTP--
The region of HePTP that
binds Erk and p38 was first mapped to its noncatalytic N-terminal 92 amino acids. A GST fusion protein of HePTP lacking this region
(GST- Phosphorylation of the N terminus of HePTP by MAP Kinase--
This
region also contains two potential phosphorylation sites for a
proline-directed kinase (such as Erk), Ser-72, and Thr-45. Because
HePTP was readily phosphorylated on both serine and threonine by the
bound Erk and p38 (Fig. 3b) and by recombinant Erk2 (Fig. 6a) or p38 (not shown), we
mutated these residues to alanines and examined their phosphorylation
by tryptic peptide mapping, which revealed that both peptides
containing Ser(P) (peptides 1 and 2) contained Ser-72, whereas Thr-45
was the phosphorylated residue in peptides 3-5 (Fig. 6b). These
peptides were also seen in tryptic peptide maps of HePTP from
metabolically 32Pi-labeled T cells but were
missing in a HePTP-T45A/S72A mutant (not shown). Thus, the noncatalytic
N terminus of HePTP binds Erk and p38 and is phosphorylated at Ser-72
and Thr-45 by these kinases.
Possible Role of Phosphorylation at Thr-45 and Ser-72--
The
phosphorylation of the N terminus of HePTP by the Erk or p38 kinases
introduces the possibility of a regulatory role of this event.
Measurement of the catalytic activity of HePTP revealed a small
decrease in activity upon phosphorylation by Erk (not shown). This
decrease is presently of questionable significance. Instead, we
addressed the possibility that phosphorylation regulates the physical
association between HePTP and Erk or p38. Because a GST-N ter protein
having both the T45A and S72A mutations still bound Erk and p38 as
readily as the wild-type GST-N ter protein (Fig. 5b), it
seems that the hydroxyl groups of Thr-45 and Ser-72 or their
phosphorylation are not required for binding of Erk or p38. To test the
opposite, namely that phosphorylation is involved in dissociation of
the kinases, we incubated HePTP-Erk complexes in the presence of ATP
and Mg2+ at 37 °C and measured the release of HePTP.
These experiments utilized catalytically active GST-Erk2 adsorbed onto
glutathione-Sepharose with bound HA-tagged HePTP-C270S from transfected
Jurkat T cells and resulted in a time-dependent
dissociation of HePTP from the beads (Fig. 6c). Thus, the N
terminus of HePTP binds Erk and p38 but may release them upon
phosphorylation. Curiously, both phosphorylation sites are outside the
minimal necessary binding site for Erk and p38, but their
phosphorylation could influence the conformation of the binding site.
Elimination of the Phosphorylation Sites Augments Inhibition of MAP
Kinase by HePTP--
To study the impact of phosphorylation of HePTP
at Thr-45 and Ser-72 in intact cells, we generated a mutant in which
both residues were replaced by alanine residues. The mutant,
HePTP-T45A/S72A, was included in a co-transfection experiment similar
to that in Fig. 1f using JCaM1 cells. As shown in Fig.
6d, wild-type HePTP reduced the anti-CD3-induced activity of
Erk2 to about half, whereas HePTP-C270S augmented it. In contrast,
HePTP-T45A/S72A was more efficient than wild-type HePTP and essentially
eliminated any increase in MAP kinase activation in the anti-CD3
stimulated cells. Thus, it is clear that phosphorylation of HePTP at
Thr-45 and/or Ser-72 is not required for inhibition of MAP kinase.
Rather, it seems that phosphorylation has the opposite effect, namely
to lessen the inhibitory effect of HePTP. This fits our hypothesis that
phosphorylation causes a dissociation of Erk from HePTP; the T45A/S72A
mutant would not allow bound Erk to escape, and the inhibition of Erk
activation would be stronger.
Taken together, our findings show that HePTP, a strictly
Tyr(P)-specific protein phosphatase, forms a physical complex through a
small region in its unique N terminus with the Erk and p38 MAP kinases.
Because HePTP very efficiently dephosphorylates these kinases and
inactivates them in vitro, it seems that the physical association serves to position HePTP correctly for this catalysis. The
small amount of HePTP required for MAP kinase inactivation in
vitro and the reduction in Erk phosphorylation and activity in
intact T cells transfected with HePTP indicate that this function of
HePTP is physiologically significant. This conclusion is also supported
by the finding that HePTP is readily phosphorylated by Erk and p38 at
Thr-45 and Ser-72, both of which are phosphorylated in intact cells.
Finally, very similar conclusions were recently drawn using mast cells
from mice deficient in
HePTP.3 Together these
observations indicate that the role of HePTP is to negatively regulate
the Erk and p38 MAP kinases in hematopoietic cells. Whether the related
PTPases STEP and PCPTP1 carry out this task in other cell types remains
to be determined. Both share an N-terminal sequence with a high degree
of homology to the Erk/p38 binding region of HePTP.
It is well established that MAP kinases are the targets for
dual-specificity phosphatases (35-37) in T cells; particularly, the
Pac1 phosphatase (36). In contrast to HePTP (4, 6), however, these
enzymes are not present in resting T cells but are induced and
synthesized some 30-60 min after receptor ligation (35, 36). Thus,
they are unlikely to be responsible for suppression of MAP kinases in
resting T lymphocytes or during early time points of T cell activation.
It has been shown also in other cell types that the inactivation of MAP
kinases precedes the induction of dual-specificity phosphatases and
that a cytosolic PTPase is involved (38, 39). In the budding yeast,
Saccharomyces cerevisiae, the mating pheromone-induced
activation of the MAP kinases Fus3p and Kss1p is tightly regulated by
the concerted action of the dual-specificity protein phosphatase Msg5p
and the conventional PTPases Ptp2p and Ptp3p (40). In this system, the
latter are responsible for the basal suppression of the MAP kinases and
for terminating their ligand-induced activation. In contrast, the dual-specificity phosphatase is encoded by an inducible gene, and the
role of the enzyme is primarily to dephosphorylate the MAP kinases at
later time points ("recovery"). Our findings suggest that a
mammalian PTPase, HePTP, is a functional homologue of Ptp2p and Ptp3p
and is responsible for basal dephosphorylation and control at early
time points.
Lymphocyte activation is initiated by the action of several
protein-tyrosine kinases (41, 42) and is very likely to be negatively
regulated by a number of PTPases (43). Only one such PTPase is
currently known, namely SHP1 (44, 45), which dephosphorylates receptor-associated signaling molecules. Here we show that HePTP functions at a distinct step to negatively regulate lymphocyte activation. In contrast to SHP1, HePTP has a high degree of specificity for Erk and perhaps p38 MAP kinases and does not affect the tyrosine phosphorylation events upstream of these kinases. This specificity is
due, in part, to a strong physical association of HePTP with Erk and
p38, an interaction that is constitutive and occurs through the
unphosphorylated N terminus of HePTP. We suggest that upon activation
of Erk by Mek, the activated Erk either rapidly phosphorylates Thr-45
and Ser-72, dissociates (perhaps also because of other mechanisms), and
escapes into the nucleus to phosphorylate its nuclear substrates. A
fraction of the activated Erk molecules, however, are rapidly
inactivated by the bound HePTP. As determined by immunofluorescence,
HePTP appears to remain exclusively cytosolic (Ref. 10)2
and, thus, unable to inactivate MAP kinase molecules in the nucleus. Instead, these are later inactivated by the nuclear dual-specificity phosphatases, which are absent in resting T cells but are induced within 30-60 min of T cell activation. This sequential phosphatase model (Fig. 7) provides a flexible
mechanism for the regulation and fine-tuning of MAP kinases, which
function as crucial signal integration and decision points in T cell
activation as well as in growth and stress responses in many other cell
types. Our model predicts a potential role for HePTP in positive
selection of T cells in the thymus (46), cytokine production (27, 47), T cell proliferation (48), or anergy (49) and potentially in other MAP
kinase-dependent aspects of T cell differentiation and
activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RI (10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
N (amino acids 92-339), N
ter (amino acids 1-92), N1 (amino acids 1-40), N2 (amino acids
10-55), and N3 (amino acids 30-80) were amplified by polymerase chain
reaction using primers tailed with NheI and NdeI
sites and the HePTP cDNA as a template. The resulting fragments
were then subcloned into the pGEX-4T-3 vector and verified by
sequencing. The GST fusion proteins were expressed and purified with
glutathione-Sepharose using standard techniques as before (6). The
cDNA for activated Mek (S218D/S222D mutated) in the pUSE vector and
recombinant active Mek, kinase-inactive Erk1, active Erk2, and active
p38 were from Upstate Biotechnology Inc. (Lake Placid, NY). Luciferase
reporter constructs in the pGL2 promoter vector (Promega) containing
multiple copies of NF-
B (
211 to
192, 8 times), Oct (
97 to
64, 7 times), or the promoters of the c-fos and
c-jun genes were a kind gift from T. Kawakami (16).
Anti-phospho-Erk and anti-phospho-Mek were purchased from Promega and
New England Biolabs.
-32P]ATP was
resolved on 10% SDS gels and transferred onto a nitrocellulose filter,
and the phosphorylated band was excised and digested with TPCK-treated
trypsin as described in detail by Luo et al. (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B. Although these proteins operate in a synergistic manner, they
can be measured separately using their target DNA elements coupled to a
reporter gene.
B (not shown). Together, these results further support the
notion that HePTP may play a role in antigen-induced T cell activation
and suggests that HePTP dephosphorylates some signaling molecule in the
pathways that lead from the antigen receptor to the activation of the
NFAT/AP-1 response elements in the interleukin-2 gene. Therefore, we
decided to examine several receptor-proximal signaling steps upstream of this transcription factor complex.
(Fig. 1a), the inhibition by HePTP
was seen at all time points but most clearly at 2-5 min, coinciding
with the peak of Erk activity. Similar results were also obtained in
Zap-70-deficient P116 cells (18) co-transfected with Zap-70, except
that the effects of both wild-type HePTP and HePTP-C270S were even
stronger (Fig. 1b), presumably because of the lower levels
of endogenous HePTP in these cells.2 HePTP also
blocked interleukin-2 promoter activation more efficiently in these
cells (not shown). Thus, the effect of transfected HePTP correlates
inversely with the amount of endogenous HePTP, suggesting that the
observed inhibition of MAP kinase represents the normal function of
HePTP.
View larger version (59K):
[in a new window]
Fig. 1.
Inhibition of MAP kinase by HePTP.
a, time course of Erk2 activation in the absence ( ) or
presence (
) of a co-transfected HePTP. The data represent in
vitro kinase assay of myc-tagged Erk2 MAP kinase expressed in
Jurkat T cells alone or together with HePTP and treated with
anti-CD3
mAbs for the indicated times. After cell lysis, the tagged
Erk2 was immunoprecipitated with an anti-Myc mAb (9E10), and the kinase
activity was measured using myelin basic protein (MBP) as substrate.
The radioactivity in the MBP band was counted in a
-counter and is
presented as % of that in the 0-min sample. The shown data represent
the mean and S.D. (error bars) from three independent
experiments. Anti-Erk2 blots showed equal amounts of the kinase in each
sample, and the expression of HePTP was verified by anti-HA tag
immunoblotting of the lysates. b, activation of Erk2 in
Zap-deficient P116 Jurkat T cells transfected with Myc-tagged Erk2 plus
empty vector, Zap, Zap plus HePTP, or Zap plus HePTP-C270S (as
indicated). The assay was done as in a. The lower
panel shows the expression of both Zap and HePTP, both of which
are HA-tagged. wt, wild type. c, activation of a luciferase
reporter gene driven by the entire 5' interleukin-2 (IL-2)
promoter in Jurkat T cells transiently transfected with the indicated
plasmids. 48 h after transfection, the cells were stimulated by a
combination of anti-CD3 (OKT3) and anti-CD28 (9.3) mAbs plus a
secondary sheep anti-mouse Ig antibody for 8 h, lysed, and used
for measurement of luciferase activity. The luciferase activity was
normalized for protein concentration, and the values shown represent
the mean and S.D. from four separate determinations. The expression of
transfected PTPases was verified by immunoblotting. d,
anti-phospho-Erk immunoblot of lysates of Lck-negative JCaM1 Jurkat T
cells transfected with the indicated plasmids and left untreated or
stimulated for 5 min with anti-CD3
. The same lysates were blotted
with anti-Erk2 (lower half of upper panel), anti-HA-tag
(middle panel), and anti-Lck (lower panel).
e, immunoblot of the same lysates with anti-phospho-Mek and
anti-Mek (upper panel), anti-Tyr(P) (anti-PTyr,
lower panel). The lower half of the lower panel
represents a longer exposure of the same blot than the upper (to
optimize the visualization of bands). Similar results were obtained in
two other experiments. f, in vitro kinase assay
of myc-tagged Erk2 MAP kinase expressed in JCaM1 cells alone or
together with Lck or Lck plus HePTP (as indicated) and either left
unstimulated or treated with anti-CD3
mAbs for 5 min (lanes
2, 4, and 6) or 20 nM phorbol
myristate acetate (TPA) (lanes 8, 10,
and 12). The upper panel shows the autoradiogram
of the kinase assay with MBP as substrate. The amount of Erk2 in the
precipitates was visualized by immunoblotting (middle
panel) and the expression of HePTP was visualized by anti-HA
tag immunoblotting of the lysates (bottom panel), and
expression of Lck was visualized by anti-Lck blotting (fourth
panel). Note that the Mr of Lck changes
upon phorbol 12-myristate 13-acetate treatment. The same result was
obtained in another independent experiment.
chain
of the T cell antigen receptor and Zap-70 (verified by
immunoprecipitation). Additional immunoblots of the same samples showed
that the expression of endogenous Erk and Mek, as well as transfected
Lck and PTPases, was equal in all samples. On longer exposures, it was
also noted that the catalytically inactive HePTP-C270S elevated the
amount of phospho-Erk in the resting cells. Thus, HePTP readily
inhibits endogenous MAP kinase, and the catalytically inactive
HePTP-C270S acts as a dominant negative reducing the action of
endogenous HePTP.
-32P]ATP revealed that HePTP
caused a rapid loss of phosphate from tyrosine without hydrolyzing
phosphothreonine (Fig. 2b). Furthermore, a brief incubation
at 37 °C of active recombinant Erk with HePTP resulted in a total
loss of its kinase activity (Fig. 2c). In contrast, GST,
HePTP-C270S, or SHP2 had no effects. Using 10 ng (150 fmol) of
recombinant Erk, we found that 10 ng (150 fmol, 7.5 nM) or
more of HePTP caused a complete inactivation of Erk within the first
minute of the assay, whereas the addition of 1 ng (15 fmol) of HePTP
had insignificant (<10%) effects even during a 30-min assay (Fig.
2d). This result suggests that HePTP acts on Erk at a 1:1
stoichiometry, perhaps by binding and primarily dephosphorylating only
the bound kinase molecules.
View larger version (59K):
[in a new window]
Fig. 2.
Direct inhibition of Erk by HePTP.
a, anti-Tyr(P) (anti-PTyr) immunoblot of
kinase-inactive GST-Erk1 phosphorylated by active Mek in the presence
of 1 mM ATP for 10 min and then incubated with active HePTP
for the indicated times. PSer, Ser(P). b,
phosphoamino acid analysis (PAA) of kinase-inactive GST-Erk1
phosphorylated by Mek in the presence of [ -32P]ATP for
10 min and then incubated with active HePTP for the indicated times.
c, phosphorylation of MBP during a 30-min assay by 10 ng of
recombinant Erk2 in the presence of 100 ng of GST-HePTP, GST,
GST-HePTP-C270S, or GST-SHP2. d, similar assay in the
presence of the indicated amounts of GST-HePTP or controls. Similar
results were obtained in at least three independent experiments
each.
View larger version (55K):
[in a new window]
Fig. 3.
Physical association of HePTP with Erk and
p38 but not Jnk. a, anti-Tyr(P) (anti-PTyr,
upper panel), anti-Erk2 (second panel), anti-Jnk1
(third panel), and anti-p38 (fourth panel)
immunoblot of material bound to control GST, GST-HePTP,
GST-HePTP-C270S, or GST-SHP2 in lysates of untreated Jurkat T cells or
cells treated with 100 µM pervanadate for 5 min. The last
lane is cell lysate. The same result has been obtained in
two other experiments, and blotting with anti-Jnk2 was also negative.
wt, wild type. b, upper panel,
autoradiogram of a kinase reaction of the same samples in the presence
of [ -32P]ATP and MBP for 10 min. Lower
panel, autoradiogram of a kinase reaction of the same samples with
GST-ATF2 as a substrate. c, anti-p38 immunoblot of
immunoprecipitates obtained with serum from a rabbit before
(lanes 1 and 2) or after immunization with HePTP
(lanes 3 and 4) or from another rabbit before
(lanes 5 and 6) or after immunization with
GST-TCPTP (lanes 7 and 8). Lane 9 is
total cell lysate. d, anti-Erk immunoblot of the same
immunoprecipitates. Lane 1 is total cell lysate.
-32P]ATP for
30 min. As shown in Fig. 4a,
active HePTP inhibited p38 profoundly, whereas the inactive C270S
mutant, GST, and the control SHP2 PTPase lacked effects. Next, we
co-transfected HA-tagged p38 or Jnk2 with empty vector, with HePTP, or
with HePTP-C270S. Two days later, the cells were stimulated with a
combination of anti-CD3 and anti-CD28 mAbs (as neither mAb alone
activates these kinases). After 15-20 min at 37 °C, the cells were
lysed, the tagged kinases were immunoprecipitated, and their activity
was measured with GST-ATF2 or GST-c-Jun-N as substrates. These assays revealed that HePTP reduced both the basal activity of p38 and its
further activation (Fig. 4b) but not the activation of Jnk2 (Fig. 4c). The cpm in the GST-ATF2 band were reduced in
lane 5 compared with lane 3 by 55% and in
lane 6 compared with lane 4 by 78%. The
expression of p38 was difficult to evaluate due the similarity in size
with HePTP but at least did not appear to be any less in lanes
5 and 6. Jnk2 was expressed at relatively low levels
but equally in all samples. We conclude that HePTP inhibits p38, but
not Jnk, in intact cells.
View larger version (45K):
[in a new window]
Fig. 4.
Inhibition of p38, but not Jnk, by
HePTP. a, phosphorylation of MBP during a 30-min assay
by 10 ng of recombinant p38 in the presence of 100 ng of GST-HePTP,
GST, GST-HePTP-C270S, or GST-SHP2. wt, wild
type.b, in vitro kinase assay with GST-ATF2 as a
substrate of HA-tagged p38 kinase expressed in Jurkat T cells alone or
together with the indicated plasmids and either left unstimulated or
treated with anti-CD3 plus anti-CD28 mAbs for 20 min. The
upper panel shows the autoradiogram of the kinase assay. The
expression of p38 and HePTP was visualized by anti-HA tag
immunoblotting (lower panel). The same result was obtained
in another independent experiment. c, in vitro
kinase assay with GST-c-Jun-N as a substrate of HA-tagged Jnk2 kinase
expressed in Jurkat T cells alone or together with the indicated
plasmids and either left unstimulated or treated with anti-CD3
plus
anti-CD28 mAbs for 15 min. The upper panel shows the
autoradiogram of the kinase assay. The expression of Jnk2 and HePTP was
visualized by anti-HA tag immunoblotting (lower panel). The
same result was obtained in several independent experiments.
N) failed to bind any Tyr(P)-containing proteins, Erk, or p38
(Fig. 5a) in lysates of Jurkat
T cells. In contrast, a GST fusion protein containing only the
N-terminal 92 amino acids of HePTP (GST-N ter) bound both Erk and p38
(Fig. 5b). To determine the binding region more precisely,
we made three smaller constructs encompassing amino acids 1-40,
10-55, 30-80, respectively. These three GST fusion proteins were
incubated with cell lysates, washed extensively, and immunoblotted for
the presence of Erk and p38. As shown in Fig. 5c, the two
first fragments bound both Erk and p38 readily, whereas the third did
not. We conclude that the binding site must reside within amino acids
10-30, perhaps extending into the 30-40 region, which is not
sufficient for binding by itself.
View larger version (54K):
[in a new window]
Fig. 5.
Mapping of the binding site for Erk and p38
in HePTP. a, anti-Tyr(P) (anti-PTyr,
upper panel), anti-Erk2 (second panel), anti-p38
(third panel), and anti-GST (lowest
panel) immunoblot of material bound to control
GST, GST-HePTP, GST-HePTP- N, GST-HePTP-C270S, or
GST-HePTP-
N-C270S, as indicated, in lysates of untreated Jurkat T
cells or cells treated with 100 µM pervanadate for 5 min.
The last lane is cell lysate. wt, wild type.
b, anti-Tyr(P) (upper panel), anti-Erk2
(middle panel), and anti-p38 (lower panel)
immunoblot of material bound to control GST-HePTP-N ter,
GST-HePTP-
N, or GST-HePTP-N ter-T45A/S72A in a similar experiment.
c, similar experiment with the shorter N-terminal
constructs, GST-HePTP-N1, -N2, and -N3. The size and amount of the GST
fusion proteins used in panels b and c were
verified during their preparation.
View larger version (43K):
[in a new window]
Fig. 6.
Mapping of phosphorylation sites in N
terminus of HePTP. a, first panel,
autoradiogram of GST-HePTP-C270S incubated with a lysate from untreated
(lane 1) or pervanadate-treated cells (lane 2),
washed extensively, and then incubated with [ -32P]ATP
for 5 min. Second panel, tryptic peptide map of the band in
lane 2 of the first panel. Third
panel, phosphoamino acid analysis (PAA) of peptides 1, 2, 4, and 5 from the same tryptic peptide map. PSer, Ser(P);
PTyr, Tyr(P). b, tryptic peptide maps of
GST-HePTP, GST-HePTP-S72A, and GST-HePTP-T45A phosphorylated by
recombinant Erk2 in vitro in the presence of
[
-32P]ATP. wt, wild type. c,
anti-HA immunoblots of supernatants (upper panel;
soluble) or pellets (lower panel;
bound) of glutathione-Sepharose beads preadsorbed with 0.5 µg of active GST-Erk2 incubated in a lysate of T cells transfected
with HA-tagged HePTP-C270S, washed extensively, and then incubated with
10 µM ATP at 37 °C for the indicated time. Note the
increase in soluble HePTP and the decrease in bound HePTP. Similar
results were obtained in at least one other experiment for each panel.
d, in vitro kinase assay of myc-tagged Erk2 MAP
kinase expressed in JCaM1 cells alone or together with the indicated
plasmids and either left unstimulated or treated with anti-CD3
mAbs
for 5 min. The upper panel shows the autoradiogram of the
kinase assay. The amount of Erk2 in the precipitates was visualized by
immunoblotting (second panel) and the expression of HePTP by
anti-HA tag immunoblotting of the lysates (third panel). The
same result was obtained in another independent experiment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 7.
Proposed sequential phosphatase model of Erk
regulation. TCR, T cell antigen receptor.
Arrows refer to stimulation, and T-shaped lines
refer to inhibition. PTKs, protein-tyrosine kinases.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Brent Zanke for the kind gift of the HePTP cDNA and for valuable discussions, to Toshi Kawakami and Bob Abraham for reagents, and to Drs. Carl Ware and Douglas Green for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a fellowship from the Alfred Benzon Foundation (to J. B.) and Grants GM48960, AI35603, AI41481, and AI40552 from the National Institutes of Health (to T. M.).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.
Current address: Laboratory of Signal Transduction, Sidney Kimmel
Cancer Center, 10835 Altman Row, San Diego, CA 92121.
§ Current address: Institute of Medical Microbiology and Immunology, University of Copenhagen, 2200 Copenhagen, Denmark.
¶ To whom correspondence should be addressed: Laboratory of Signal Transduction, Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Tel.: 619-450-5990 (ext. 330); E-mail: tmustelin{at}skcc.org.
2 M. Saxena, S. Williams, and T. Mustelin, unpublished observation.
3 B. Zanke, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTPase, protein-tyrosine phosphatase; HePTP, hematopoietic protein-tyrosine phosphatase; NFAT, nuclear factor of activated T cells; AP-1, activator protein-1; MAP, mitogen-activated protein; GST, glutathione S-transferase; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; MBP, myelin basic protein; HA, hemagglutinin; mAb, monoclonal antibody.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|