(Received for publication, April 12, 1995; and in revised form, June 6, 1995)
From the
The tyrosine kinase Tyk-2 is physically associated with the Type
I interferon (IFN) receptor complex and is rapidly activated during
IFN
Type I IFNs (
We initially sought to determine whether the SH2-containing
phosphotyrosine phosphatase HCP is a substrate for Type I IFN-dependent
tyrosine kinase activity. Molt-16 or Molt-4 cells were treated for
different time periods with IFN
Figure 1:
Tyrosine phosphorylation of a 135-kDa
HCP-associated protein during IFN
Fig. 2, A and B, shows anti-Tyk-2
immunoblots of cell lysates from Molt-16 or KG1A cells
immunoprecipitated with the
Figure 2:
Association of Tyk-2 with HCP(SHPTP-1). A, anti-Tyk-2 immunoblot. Serum-starved Molt-16 cells were
incubated for 5 min in the presence or absence of IFN
Figure 3:
Detection of in vitro kinase
activity in
We next
sought to determine whether inhibition of phosphatase activity can
alter the phosphorylation status of Tyk-2. Studies were performed in
which the phosphorylation status of Tyk-2 in response to treatment of
cells with the phosphatase inhibitor sodium orthovanadate was assessed. Fig. 4, A and B, shows that treatment of cells
with sodium orthovanadate induces intense tyrosine phosphorylation of a
Tyk-2-associated protein with an approximate molecular mass of 115 kDa
(p115) and weaker phosphorylation of Tyk-2 itself, suggesting that
inhibition of the associated HCP phosphatase activity results in
tyrosine phosphorylation of Tyk-2 and the associated p115 protein. To
determine whether Tyk-2 is a substrate for the phosphatase activity of
HCP in vitro, we performed phosphatase assays on
Figure 4:
Effect of the phosphatase inhibitor sodium
orthovanadate on the phosphorylation status of Tyk-2. A,
Molt-4 cells were incubated for 2 h at 37 °C in the presence or
absence of 1 mM sodium orthovanadate (VAN) as
indicated, and cell lysates were immunoprecipitated with
Figure 5:
In vitro dephosphorylation of
Tyk-2 and Jak-1 by an HCP fusion protein. Molt-4 (A) or HSB-2 (B) cells were stimulated with IFN
The phosphotyrosine phosphatase HCP contains two SH2 domains
in its amino terminus, a protein tyrosine phosphatase catalytic domain
in its carboxyl terminus, and is predominantly expressed in cells of
hematopoietic
origin(17, 20, 21, 22, 23) .
HCP has been implicated in the negative regulation of hematopoietic
cell growth(20, 21, 22, 23) ,
suggesting that its phosphatase activity may be inhibitory for
signaling pathways of cytokines that promote cell growth or necessary
for the activation of pathways of cytokines that inhibit cell growth.
Mice genetically deficient in HCP die shortly after birth due to
overexpansion of their macrophages and their accumulation in their
lungs(24, 25, 26) . These mice also have
significant increases in a variety of other hematopoietic cell
lineages(24, 25, 26) , underscoring the
regulatory role of HCP on hematopoietic cell proliferation. HCP has
been shown to interact with the c-kit receptor(17) , the
Although the exact
regulatory function of HCP in IFN
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
stimulation. We report that Tyk-2 forms stable complexes with
the SH2-containing hematopoietic cell phosphatase (HCP) in several
hematopoietic cell lines in vivo, and that the
IFN
-induced tyrosine-phosphorylated form of Tyk-2 is a substrate
for the phosphatase activity of HCP in in vitro assays.
Furthermore, treatment of cells with the phosphatase inhibitor sodium
orthovanadate induces tyrosine phosphorylation of Tyk-2 and an
associated 115-kDa protein. Altogether, these data suggest that HCP
regulates tyrosine phosphorylation of the Tyk-2 kinase, and thus its
function may be important in the transmission of signals generated at
the Type I IFN receptor level.
)exhibit multiple biological effects on
cells and tissues, including antiproliferative, antiviral, and
immunomodulatory activities(1) . Two kinases of the Janus
family, Tyk-2 and Jak-1, are physically associated with components of
the Type I IFN receptor complex(2, 3) . During
IFN
stimulation, the
and
subunits of the receptor
become phosphorylated on tyrosine(4, 5) , and the
tyrosine kinases Tyk-2 and Jak-1 are
activated(2, 6, 7, 8, 9) .
Although no definitive evidence exists at this time, activation of
these receptor-associated tyrosine kinases is presumed to regulate the
tyrosine phosphorylation of downstream IFN signaling elements, which
include the Stat 2, Stat 1
, and Stat 1
components of the
transcriptional activator
ISGF3
(10, 11, 12, 13) , the vav proto-oncogene product(14) , and the insulin
receptor substrate-1(15) . The exact sequence of events,
however, that lead to activation of Tyk-2 and Jak-1 after IFN
binding to its receptor are not well defined. Recent studies have
provided evidence for a phosphatase involvement in the early steps of
Type I IFN signaling, as suggested by experiments demonstrating that a
phosphatase inhibitor can block the formation of the active ISGF3
complex(16) . The authors of this report have proposed a model
in which a tyrosine phosphatase associates with an IFN-dependent
tyrosine kinase and regulates its activation(16) . In the
present study we demonstrate that the phosphotyrosine phosphatase HCP
(also named SHPTP-1, PTP1C, or SHP) forms a stable complex with the
active form of the tyrosine kinase Tyk-2 in intact cells, raising the
possibility that HCP is the phosphatase that regulates IFN-dependent
formation of the active ISGF3
complex.
Cells and Reagents
The human Molt-4 (acute
T-cell leukemia), Molt-16 (acute T-cell leukemia), HSB-2 (acute T-cell
leukemia), KG1A (acute myelogenous leukemia), and Daudi
(lymphoblastoid) cell lines were grown in RPMI 1640 (Life Technologies
Inc.) supplemented with 10% (v/v) defined calf serum (Hyclone
Laboratories) and antibiotics. Human recombinant IFN was provided
by Hoffmann-La Roche. The antiphosphotyrosine monoclonal antibody
(4G-10) and the rabbit polyclonal antibodies against the tyrosine
kinases Jak-1 and Jak-2 were obtained from Upstate Biotechnology (Lake
Placid, NY). A polyclonal antibody against the phosphotyrosine
phosphatase HCP was obtained from Santa Cruz Biotechnology. A GST-HCP
fusion protein has been described(17) . A rabbit polyclonal
antibody raised against a synthetic peptide corresponding to the
carboxyl-terminal 15 amino acids of Tyk-2 (5) was used for
immunoprecipitations. A monoclonal antibody against Tyk-2 was purchased
from Transduction Laboratories and used for immunoblotting. A
polyclonal antibody against Stat-2 (p113, 186-199) has been
described elsewhere(18) . Sodium orthovanadate and protease
inhibitors were obtained from Sigma.
Immunoprecipitations and Immunoblotting
Cells were
incubated in the presence or absence of 10 units/ml of
IFN
for the indicated periods of time. In some experiments, the
cells were serum-starved for 90-180 min prior to IFN
stimulation. After IFN
stimulation, the cells were rapidly
centrifuged and lysed in a phosphorylation lysis buffer (0.5% Triton
X-100 or Nonidet P-40, 150 mM NaCl, 200 µM sodium
orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5
mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). Cell
lysates were immunoprecipitated with the indicated antibodies, and,
after five washes with phosphorylation lysis buffer containing 0.1%
Triton X-100 or Nonidet P-40, were analyzed by SDS-PAGE. The proteins
were transferred onto polyvinylidene difluoride filters (Immobilon),
and the residual binding sites on the filters were blocked by
incubating with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), 10% bovine serum albumin for 1-3 h at
room temperature or overnight at 4 °C. The filters were
subsequently incubated with the indicated antibodies and developed
using an enhanced chemiluminescence (ECL) kit following the
manufacturer's recommended procedure (Amersham).
In Vitro Kinase Assays
In vitro kinase
assays were performed as described previously(19) . Briefly,
cells were treated with IFN for different time periods, the cells
were lysed in phosphorylation lysis buffer, and cell lysates were
immunoprecipitated with the polyclonal antibody against HCP.
Immunoprecipitates were washed three times with phosphorylation lysis
buffer and twice with in vitro kinase buffer (12 mM
MgCl
, 50 mM Tris-HCl, pH 7.4, 5 mM sodium
orthovanadate, 150 mM NaCl). The immunocomplex-protein
G-Sepharose beads were resuspended in 30 µl of in vitro kinase buffer, in which MnCl
at a final concentration
of 2.5 mM and 20 µCi of
[
-
P]ATP were added. The beads were
incubated for 30 min at room temperature, and the reaction was
terminated by the addition of loading buffer.
In Vitro Phosphatase Assays
In vitro phosphatase assays were performed as described previously with
minor modifications (17) . Briefly, Tyk-2 and Jak-1 proteins
were immunoprecipitated from IFN-stimulated cells and washed four
times with phosphorylation lysis buffer and once in phosphatase buffer
(25 mM PIPES, 5 mM EDTA, 10 mM dithiothreitol). The beads were incubated in 50 µl of
phosphatase buffer in the presence or absence of 1 µg of GST-HCP
fusion protein or 1 µg of GST alone for 10 min at 37 °C. The
reactions were terminated by adding loading buffer. The phosphotyrosine
content of Tyk-2 and Jak-1 was determined by resolving the proteins by
SDS-PAGE, transferring the proteins to Immobilon membranes, and probing
the blots with antiphosphotyrosine.
, and cell lysates were
immunoprecipitated with an antibody against HCP, followed by SDS-PAGE
analysis and immunoblotting with antiphosphotyrosine. Fig. 1shows that a tyrosine-phosphorylated protein corresponding
to HCP(SHPTP-1) was detectable in these blots, but no change in its
phosphorylation status was seen during IFN
stimulation. However, a
135-kDa protein (p135) was coimmunoprecipitated by the
HCP(SHPTP-1) antibody and was transiently phosphorylated on
tyrosine during IFN
stimulation. A 135-kDa HCP-associated
phosphoprotein was also noticeable in experiments in which Molt-4 cells
were metabolically labeled with
[
P]orthophosphoric acid, and cell lysates were
immunoprecipitated with the
HCP antibody (data not shown). As the M
of this protein was similar to the M
of the IFN-dependent tyrosine kinase Tyk-2, we
sought to determine whether p135 corresponds to Tyk-2.
stimulation. Antiphosphotyrosine
immunoblots are shown. Molt-16 (serum-starved, 6.8
10
/lane) or Molt-4 (2.1
10
/lane) cells
were stimulated with 10
units/ml IFN
for the indicated
times. Molt-16 cell lysates were immunoprecipitated with either an
antibody against HCP(SHPTP-1) (lanes 1-4) or with
control rabbit immunoglobulin (lane 5). Molt-4 cell lysates
were immunoprecipitated with an antibody against HCP(SHPTP-1) (lanes 1 and 2).
HCP antibody. Tyk-2 was clearly
detectable in the
HCP immunoprecipitates from both cell extracts,
establishing that p135 is indeed Tyk-2. Furthermore, when
immunoprecipitates obtained from Molt-16 cell lysates with polyclonal
antibodies against Tyk-2 or Stat-2 were immunoblotted with
HCP,
HCP was detectable in
Tyk-2 but not in
Stat-2
immunoprecipitates (Fig. 2C). A decrease in the amount
of HCP coprecipitated by
Tyk-2 after IFN
stimulation in this
experiment was not seen consistently. In experiments with Daudi cells,
HCP was also detectable in
Tyk-2 immunoprecipitates, but not in
immunoprecipitates obtained with antibodies against the related Janus
family kinases Jak-1 and Jak-2 (Fig. 2D). We cannot,
however, exclude the possibility that the inability to detect HCP in
association with Jak-1 or Jak-2 is due to low stoichiometries of such
associations in these cells. Altogether, these studies strongly
suggested that Tyk-2 and HCP form a complex in intact cells. In
experiments in which HCP was immunoprecipitated from cell lysates of
IFN
-treated or untreated cells and in vitro kinase assays
were performed on such immunoprecipitates, we found that HCP was
phosphorylated and a 135-kDa phosphoprotein was also present in
HCP immunoprecipitates (Fig. 3A), suggesting that
HCP associates with the active form of Tyk-2. Phosphoamino acid
analysis of the bands corresponding to HCP showed that HCP is
phosphorylated on tyrosine, but such phosphorylation was not increased
in response to IFN
treatment (Fig. 3B).
as
indicated, and cell lysates were immunoprecipitated with
HCP(SHPTP-1) or
Tyk-2 or control RIgG as indicated. B, anti-Tyk-2 immunoblot. Serum-starved KG1A cells were
incubated for 15 min in the absence or presence of IFN
as
indicated, and cell lysates were immunoprecipitated with the indicated
antibodies. C,
HCP(SHPTP-1) immunoblot. Molt-16 cells
were incubated for 15 min in the absence or presence of IFN
as
indicated, and cell lysates were immunoprecipitated with the indicated
antibodies. D, Daudi cell lysates were immunoprecipitated with
the indicated antibodies and immunoblotted with an antibody against
HCP(SHPTP-1).
HCP(SHPTP-1) immunoprecipitates. A, Molt-16
cells were incubated in the absence or presence of IFN
at 37
°C, and cell lysates were immunoprecipitated with an
HCP(SHPTP-1) antibody, subjected to an in vitro kinase
assay, and analyzed by SDS-PAGE. Proteins were transferred to
Immobilon, and the membrane was treated with 1 M KOH for 2 h
to select for tyrosine-phosphorylated proteins prior to
autoradiography. B, phosphoamino acid analysis of HCP prior to
and after IFN
stimulation.
Tyk-2
immunoprecipitates from IFN
-stimulated cells. Fig. 5A shows that the IFN
-induced phosphorylated form of Tyk-2 and
the associated p115 is dephosphorylated by a GST-HCP fusion protein,
but not GST alone, establishing that Tyk-2 acts as a substrate for this
phosphatase in vitro. The GST-HCP fusion protein also
dephosphorylated the IFN
-induced phosphorylated form of Jak-1 (Fig. 5B). It remains to be determined whether the
dephosphorylation of Jak-1 results from a direct effect of HCP on this
kinase or the effect is indirect resulting from dephosphorylation of
Tyk-2, as previous studies have established that the activation of
these Janus kinases is interdependent(6) .
Tyk-2 or
nonimmune rabbit serum as indicated, analyzed by SDS-PAGE, and
immunoblotted with antiphosphotyrosine. B, Molt-16 cells were
incubated for 2 h at 37 °C in the presence or absence of 1.5 mM sodium orthovanadate (VAN) as indicated. Cells were
stimulated for 5 min with IFN
as indicated, and cell lysates were
immunoprecipitated with the indicated antibodies and immunoblotted with
antiphosphotyrosine.
for 5 min as
indicated, and cell lysates were immunoprecipitated with polyclonal
antibodies against Tyk-2 or Jak-1 or with nonimmune rabbit serum (NRS) as indicated. Immunoprecipitates were incubated in the
presence or absence of an HCP(SHPTP-1) fusion protein or GST alone for
10 min at 37 °C as indicated, analyzed by SDS-PAGE, and
immunoblotted with antiphosphotyrosine.
-subunit of the interleukin-3 receptor(27) , the insulin
receptor tyrosine kinase(28) , and the erythropoietin
receptor(29) . Although the consequences of such associations
are not well understood, it has been hypothesized that they lead to
inhibition of the kinase activities associated with these receptor
complexes and negative regulation of downstream signaling pathways
promoting cell growth. Such a hypothesis is supported by a recent study
that demonstrated that cells expressing mutant erythropoietin (Epo)
receptors for the HCP binding site exhibit prolonged Epo-induced Jak-2
autophosphorylation and are hypersensitive to Epo(30) . The
role of HCP in the signaling pathways of cytokines that inhibit cell
proliferation is not known. Type I IFNs are strong inhibitors of
hematopoietic cell proliferation. These cytokines transduce signals by
activating the receptor-associated Tyk-2 and Jak-1 tyrosine
kinases(2, 6, 7, 8, 9) ,
and by tyrosine phosphorylation of the transcription factors Stat-2,
Stat-1
, and Stat-1
, which are the proteins that form the
ISGF3
complex(10, 11, 12, 13) .
The formation of the ISGF3
complex in response to IFN
stimulation is inhibited by the phosphatase inhibitor sodium
orthovanadate(16) , suggesting that a tyrosine phosphatase may
be associated with the Type I IFN receptor complex and, upon IFN
binding, may regulate IFN
-dependent tyrosine kinases. We now
report that the phosphotyrosine phosphatase HCP forms a stable complex
with the IFN
-dependent tyrosine kinase Tyk-2 in several cell lines
of diverse hematopoietic origin, suggesting that HCP is involved in the
regulation of activation of this kinase.
signaling remains unknown at
this time, our studies suggest that inhibition of the Tyk-2-associated
HCP phosphatase activity leads to enhanced tyrosine phosphorylation of
Tyk-2 and an associated p115 protein. In addition, Tyk-2 is a substrate
for the phosphatase activity of HCP in in vitro phosphatase
reactions. One interpretation of our findings is that HCP
dephosphorylates a region of Tyk-2 that controls its enzymatic
activity. Such dephosphorylation may be necessary for activation of
Tyk-2 and generation of downstream signals. Such a functional role for
the HCP-Tyk-2 complex would also be consistent with the phenotype of
mice genetically deficient in HCP. If activation of Tyk-2 is impaired
due to lack of HCP in these mice, this may lead to inhibition of
IFN
signals that regulate hematopoietic cell proliferation and
lead to overexpansion of certain hematopoietic lineages. An alternative
interpretation of our data, however, is that the phosphatase activity
of HCP may exhibit a negative regulatory effect on the activation of
Tyk-2. Future studies to identify the tyrosine residues of Tyk-2 that
are targets for the phosphatase activity of HCP should provide
important information on the functional role of this phosphatase in
Type I IFN signaling. There is also evidence that Tyk-2 is involved in
cell signaling for a group of cytokines that utilize gp130, which
includes interleukin-6, oncostatin M, leukemia inhibitory factor, and
ciliary neurotropic factor(31) . The finding of HCP association
with this kinase suggests a mechanism through which HCP may regulate
the signaling pathways of these cytokines as well.
We thank Dr. Michael Brunda (Hoffmann-La Roche) for
providing us with IFN.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.