(Received for publication, May 23, 1995)
From the
The activation of Janus protein-tyrosine kinases (Jaks)
and the subsequent phosphorylation and activation of latent signal
transducers and activators of transcription (Stats) are common elements
in signal transduction through the cytokine receptor superfamily. To
assess the role and specificity of Jaks in Stat activation, we have
utilized baculovirus expression systems to produce Stat1 and the Jaks.
Co-expression of Stat1 with Tyk2, Jak1, or Jak2 resulted in the
specific tyrosine phosphorylation of Stat1 at Tyr, the
residue phosphorylated in mammalian cells stimulated with interferon
. Alternatively, Stat1, purified to apparent homogeneity from
insect cell extracts, was phosphorylated at Tyr
in Jak
immune complex kinase reactions. Phosphorylation of purified Stat1 was
necessary and sufficient for the acquisition of DNA binding activity.
The specificity in both systems was indicated by the inability of a
Jak2 catalytically inactive mutant (Jak2-Glu
) or the Tec
protein-tyrosine kinase to phosphorylate Stat1. However, immune
complex-purified epidermal growth factor receptor was capable of
phosphorylating purified Stat1 at Tyr
and activating its
DNA binding activity in in vitro reactions.
Cellular functions are regulated through the interaction of
extracellular ligands with their cognate receptors. One group of
ligands, variably termed growth factors, cytokines, or
colony-stimulating factors, utilize structurally and functionally
similar receptors of the cytokine receptor superfamily(1) .
These receptors couple ligand binding to the activation of
protein-tyrosine phosphorylation through their association with members
of the Janus protein-tyrosine kinase (Jaks) family (reviewed
in (2) and (3) ). The Jak family currently consists of
Jak1, Jak2, Jak3, and Tyk2, which vary in size from 120-135 kDa.
One or more of the Jaks associate with one or more of the cytokine
receptor chains at a membrane proximal site of the cytoplasmic domain.
The association and activation of Jaks is critical for signaling
through these receptors as indicated by the inactivation of receptor
function by deletions or point mutations in the receptors that disrupt
their ability to associate with the Jaks. A critical role for Jaks in
interferon (IFN) ()signaling has also been established
through the identification of cell lines selected for the inability to
respond to IFN that lack individual Jaks. In these cells, introduction
of the appropriate Jak restores IFN signaling. Lastly, recent studies (4, 5) have shown that kinase inactive Jaks can
function as dominant negatives in cytokine signaling.
The activation
of Jaks is required for the subsequent tyrosine phosphorylation of a
variety of proteins that are involved in signal transduction. In
particular, analysis of receptor mutants has indicated that Jak
activation is required for the tyrosine phosphorylation of SHC, the p85
subunit of phosphatidylinositol 3-kinase, Vav, as well as members of
the Stat family of proteins. The Stats (signal transducers and
activators of transcription) were initially identified in IFN signaling
and have been shown to be essential for many of the responses to IFN
(reviewed in (6) ). Individual cytokines induce the tyrosine
phosphorylation of one or more of the seven known Stats (Stat1, Stat2,
Stat3, Stat4, Stat5a, Stat5b, and Stat6). In the response to
IFN/
, Jak1 and Tyk2 are activated, and Stat1 and Stat2 are
subsequently tyrosine-phosphorylated. Following phosphorylation, Stat1
and Stat2 form a complex with a DNA-binding protein, p48, and the
complex translocates to the nucleus, binds DNA, and is required for
activation of transcription of IFN responsive genes. The absence of any
of the components negates the response. In the response to IFN
,
Jak1 and Jak2 are activated, and Stat1 is tyrosine-phosphorylated and
forms a DNA binding homodimer that translocates to the nucleus and is
required for activation of the transcription of IFN
responsive
genes.
Although a strong correlation exists between Jak activation and the tyrosine phosphorylation of the Stats, there has not been direct evidence to support the hypothesis that the Stats are immediate substrates for the Jaks. Indeed, Stat activation has been seen with other receptor systems, including the epidermal growth factor receptor (EGFR)(7, 8, 9) . Nevertheless, the utilization of specific Jaks by individual cytokines and activation of specific Stats have suggested the possibility that individual Jaks may specifically phosphorylate Stats. The studies presented here were designed to address these questions. Using baculovirus expressions systems, we demonstrate that Stat1, either co-expressed with the Jaks or as a purified protein added to immune complex reactions, is a substrate for Jak1, Jak2, or Tyk2. Phosphorylation occurs at the single site previously identified as the in vivo site of tyrosine phosphorylation and as a consequence, Stat1 acquires DNA binding activity. Lastly we demonstrate that Stat1 is similarly a substrate for EGFR but not for the cytoplasmic protein-tyrosine kinase Tec.
For Stat1 kinase reactions, immune complex Jak kinases were mixed with purified Stat1 (8 µg of protein) in kinase buffer plus 5 mM ATP. Following 30 min of incubation at 25 °C, reactions were stopped by the addition of EDTA to a 15 mM final concentration. The products of these reactions were separated by centrifugation into soluble (Stat1) and insoluble (Jak kinase) fractions for use in SDS-PAGE and gel mobility shift assays.
EGFR immune complexes were prepared by adding 4 µl of anti-EGFR
serum to 1 ml of a centrifuged (12,000 g for 10 min)
10% homogenate of adult mouse liver prepared with a lysis buffer
containing 20 mM HEPES, pH 7.4, 1% Trition X-1000, 50 mM sodium
-glycerophosphate, 10 mM sodium fluoride, 100
µM sodium vanadate, 0.1 M sodium chloride. After
standing for 2 h at 0 °C, 100 µl of a 50% slurry of protein
A-Sepharose was added, and incubation, with mixing, was continued for
30 min. The suspension was centrifuged, washed three times with 1 ml of
homogenizing buffer and stored frozen at -70 °C. Before use,
the complex was washed once with 1 ml of 20 mM HEPES pH 7.4
buffer containing 1 mM dithiothreitol.
Cytokine-dependent activation of the Jaks is associated with
the phosphorylation and activation of one or more of the known Stats.
To initially explore the role of the Jaks in Stat phosphorylation, the
ability of various Jaks to phosphorylate Stat1 in insect cells was
examined. Baculovirus expression constructs were developed for Jak1,
Jak2, Tyk2, and a kinase inactive mutant Jak2-Glu as well
as for Stat1. An expression construct containing the Tec kinase (18) was also utilized to determine specificity relative to the
Jaks. Each of the wild type kinases was expressed at comparable levels
and was tyrosine-phosphorylated constitutively and catalytically active
in immune kinase reactions (data not shown). As illustrated in Fig. 1, when expressed alone, Stat1 was not detectably
tyrosine-phosphorylated. However co-expression of Stat1 with Jak1,
Jak2, or Tyk2 resulted in the tyrosine phosphorylation of Stat1.
Moreover, over several experiments, the levels of phosphorylation with
the various Jaks were comparable with the levels of Jak expression,
indicating that Stat1 was not preferentially phosphorylated by a
particular Jak. The specificity was demonstrated by a lack of Stat1
phosphorylation by the kinase inactive Jak2 mutant
(Jak2-Glu
) or by Tec.
Figure 1:
Phosphorylation of Stat1
by Jak kinases. SF9 cells were infected with baculovirus expression
vectors for Stat1, Tyk2, Jak1, Jak2, an inactive point mutant of Jak2 (Jak2-E), or Tec. Lysates from infected cells
were immunoprecipitated (IP) with Stat1 specific antisera (A), pooled antisera specific for Tyk2, Jak1, or Jak2 (B), or antisera specific for Tec (C).
Immunoprecipitated proteins were separated on 7% SDS-polyacrylamide
gels, transferred to nitrocellulose filters, and probed with the
anti-phosphotyrosine antibody PY20. Alternatively, filters were probed
with the same sera used for
immunoprecipitation.
In the cellular response to IFN,
tyrosine phosphorylation of Stat1 occurs on a single residue
(Tyr)(19) . To determine whether the Jaks
phosphorylate this site and/or additional sites in Stat1,
two-dimensional tryptic peptide analysis was done (Fig. 2).
Stat1 expressed alone contained a single phosphopeptide that based on
the above data is not a phosphotyrosine-containing peptide. When
expressed with Jak1, Jak2, or Tyk2, a single additional phosphopeptide
was detected. This phosphopeptide co-migrated with a synthetic tryptic
peptide containing phosphotyrosine at position Tyr
(data
not shown).
Figure 2:
Phosphopeptide mapping of Stat1. SF9 cells
were infected with Stat1 virus (A) or co-infected with virus
for Stat1 and Tyk2 (B), Stat1 and Jak1 (C), or Stat1
and Jak2 (D). 60 h after infection, cells were labeled with
[P]orthophosphate. Subsequently, Stat1 was
isolated by immunoprecipitation and SDS-PAGE separation. Tryptic
peptides of the isolated Stat1 were prepared and mixed with a synthetic
phosphopeptide corresponding to the predicted Tyr
peptide
of Stat1. Separation was performed in two dimensions, and labeled
phosphopeptides were visualized using a PhosphorImager. The positions
of the synthetic peptides were determined by ninhydrin staining and are
indicated by the broken circles. The origin of separation is
indicated by the arrowhead.
The tyrosine phosphorylation of Stat1 in mammalian cells
is associated with the acquisition of the ability to bind DNA. In the
response to IFN/
, Stat1 participates in a complex containing
an additional protein, p48, that is essential for DNA binding, whereas
in the response to IFN
p48 is not required for DNA binding. It was
therefore important to determine whether Stat1, phosphorylated in a
non-mammalian cell where associating proteins are less likely to be
present, would bind its cognate sequence. As illustrated in Fig. 3, expression of Stat1 alone resulted in the appearance of
no detectable GAS binding activity. However, co-expression of Stat1
with any of the wild type Jaks resulted in the appearance of DNA
binding activity. The contribution of Stat1 to this activity is evident
from the ability of antisera to Stat1 to supershift the complexes (Fig. 3B).
Figure 3: GAS binding activity of phosphorylated Stat1. A, the cell lysates prepared for Fig. 1were used directly for gel shift analysis using an oligonucleotide containing the core GAS element of the interferon regulatory factor 1 promotor. B, alternatively, lysates were mixed with normal rabbit serum (control antibody) or Stat1 specific serum prior to gel shift analysis.
To further establish the ability of Jaks
to directly phosphorylate Stat1, Jaks were purified by
immunoprecipitation and examined for their ability to phosphorylate
either a peptide containing Stat1 Tyr or purified Stat1.
As shown in Fig. 4A, Jak2, but not the kinase inactive
mutant Jak2-Glu
, phosphorylated the Stat1 peptide. The
specificity is indicated by the lack of phosphorylation of a peptide
containing Stat1 Tyr
, a tyrosine that is conserved in all
the Stats. Comparable results were obtained with Jak1 and Tyk2 (data
not shown). Jak2 was also able to phosphorylate peptides containing the
predicted phosphorylation sites of Stat2 and Stat4 (data not shown).
Figure 4:
Jak2 phosphorylation of a
Stat1-Tyr peptide. Jak2 immunoprecipitates were prepared
for SF9 cells infected with a control virus, Jak2 virus, or
Jak2-Glu
virus. A portion of each immunoprecipitation was
used for in vitro kinase reactions with the
Tyr
-peptide substrate (A) or the
Y
-peptide substrate (B). A portion of the
immunoprecipitation was also analyzed by SDS-PAGE and Western blotting
with Jak2 antisera (C).
The ability of Jaks to phosphorylate purified Stat1 is shown in Fig. 5. For these experiments, Stat1 was purified to apparent
homogeneity (Fig. 5A) by sequential ion exchange and
gel filtration chromatography as detailed under ``Materials and
Methods.'' The purified Stat1 was added to the immune complex
kinase reactions, and the products were resolved by SDS-PAGE and
examined by autoradiography (Fig. 5B). As indicated,
the reactions were separated into a fraction containing the immune
complexes (insoluble) and the supernatant fraction (soluble). Jak1 and
Jak2 autophosphorylations were seen in the immune complexes as
expected. In addition, a fraction of the phosphorylated Stat1 was
co-isolated with the immune complexes. However, phosphorylated Stat1
was also seen in the soluble fraction. The requirements for the Jaks is
indicated by the lack of Stat1 phosphorylation by the kinase inactive
Jak2-Glu. Tryptic peptide analysis of in vitro phosphorylated Stat1 demonstrated the phosphorylation of a single
tryptic fragment that co-migrated with the Tyr
tryptic
peptide (Fig. 5, C and D). Lastly, in
vitro phosphorylation of purified Stat1 activated its latent DNA
binding activity (Fig. 6A), and this activation was
dependent upon the addition of ATP (Fig. 6D).
Interestingly, two distinct GAS binding complexes were seen with in
vitro phosphorylated Stat1. The major, faster migrating complex
co-migrated with the complex seen in the co-expression studies above.
The slower migrating complex was more variably observed. The basis for
the second complex is unknown.
Figure 5:
In vitro phosphorylation of
Stat1. Stat1 expressed in SF9 cells was purified by sequential ion
exchange and gel filtration chromatography. Purified Stat1 was
separated by SDS-PAGE, and the gel was stained with Coomassie Blue (A). Immune complex kinase preparations were made for Jak1,
Jak2, Jak2-Glu, Tec, EGFR, or control (no kinase) as
described under ``Materials and Methods.'' Each kinase
preparation was reacted with purified Stat1 in the presence of
[
-
P]ATP. The reaction products were
separated by centrifugation into soluble (Stat1) and insoluble (kinase)
fractions. Each fraction was further separated by SDS-PAGE and
visualized by autoradiography (B). Stat1 phosphorylated by
Jak1 (C), Jak2 (D), or EGF-R (E) was then
excised from gel slices of the soluble reaction product and used for
tryptic phosphopeptide mapping as described in the legend to Fig. 2.
Figure 6: In vitro activation of Stat1. Immune complex kinase reactions were preformed in the presence or the absence of purified Stat1. Following the kinase reaction, the soluble (Stat1) and insoluble (kinase) components were separated by centrifugation. An aliquot of the soluble portion of each in vitro kinase reaction was assayed for GAS binding activity (A). Aliquots of the insoluble fractions were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies for phosphotyrosine (B) or with a pool of antisera specific for Tyk2, Jak1, Jak2, and Jak3 (C). Alternatively, Jak2 immune complex kinase reactions were performed with purified Stat1 in the presence or absence of ATP. Aliquots of the soluble fraction of each reaction were incubated with non-reactive rabbit serum (NRS) or anti-Stat1 serum prior to the gel mobility shift assay (D).
Previous studies (7, 8, 9) have shown that EGF stimulation,
both in tissue culture cell lines and in vivo, can induce the
tyrosine phosphorylation of Stat1 as well as Stat3. However, it was not
determined whether this occurred directly or through the activation of
a Jak by the EGFR. As illustrated in Fig. 5B, immune
complex purified EGFR readily phosphorylated purified Stat1 and this
phosphorylation occurred on a single tryptic peptide corresponding to
the peptide containing Tyr (Fig. 5E). The
specificity is indicated by the inability of Tec to phosphorylate
Stat1, although autophosphorylation was readily detectable in these
reactions (Fig. 6B). It should also be noted that the
immune complex purified Tec and EGFR contained no detectable Jak1 or
Jak2 by Western blotting of the preparations (Fig. 6, B and C). As with the Jaks, phosphorylation of Stat1 by
EGFR resulted in the activation of its latent DNA binding activity (Fig. 6A).
The Jaks are required for cytokine-stimulated signal transduction pathways leading to the tyrosine phosphorylation and activation of the Stats. However, whether the Stats are immediate substrates of the Jaks or substrates of a Jak-activated kinase has not been established. The studies described here address this question and the related question of the specificity of the kinases for Stats. Our results demonstrate that the Jaks are capable of mediating the specific tyrosine phosphorylation of Stat1 in vivo in insect cells and in in vitro reactions with purified proteins. Similar to Stat1, co-expression of Jak2 with Stat2, Stat3, Stat4, Stat5, or Stat6 resulted in their tyrosine phosphorylation (data not shown), although these results have not as yet been done with the respective purified proteins. Therefore Stats are potentially immediate substrates of the activated Jaks in vivo.
Strikingly, tyrosine
phosphorylation, both in insect cells and in vitro, occurs at
a single site, Tyr, although numerous tyrosines are
present in Stat1. This specificity is particularly noteworthy because
it occurs in the absence of any relevant receptor complex. The
specificity for Tyr
may be determined solely by the
immediate flanking sequences and/or other structural determinants of
Stat1 that contribute to the tertiary structure and position
Tyr
to be accessible for phosphorylation. The ability of
Jaks to phosphorylate a peptide containing Tyr
demonstrates the ability of the catalytic pocket to bind the site
of phosphorylation in the absence of other structural determinants. In
this regard it should be noted that considerable similarity exists at
comparable sites in the other Stats, as indicated in Table 1.
Among these peptides, those for Stat4 and Stat2 peptides are also
substrates of Jak2 (data not shown); the remainder have not been
examined.
In addition to the Jak-dependent tyrosine phosphorylation
of Tyr, a second site of phosphorylation was seen in
Stat1 produced in insect cells. This phosphorylation was seen without
co-expression of the Jaks, suggesting that an endogenous insect cell
kinase is involved. The lack of reactivity of Stat1 produced alone in
insect cells with antiphosphotyrosine reagents would suggest that this
second site of phosphorylation is either a serine or threonine residue.
The potential significance of this phosphorylation site is unclear.
Because the insect cell-produced Stat1 is constitutively phosphorylated
at this site, we cannot rule out a role in DNA binding. In this regard,
recent studies have indicated that phosphorylation of Stat1 S
may be required for maximal transcriptional activation (20) .
The above data are consistent with a relatively simple recognition of Stat1 by the Jaks. However, we have also begun to examine the ability of various mutants of Stat proteins to be phosphorylated when co-expressed with Jaks. Strikingly many mutations, deletions, or truncations eliminate the ability of Stats to be recognized and phosphorylated by the Jaks (data not shown). For example, relatively small deletions in the amino-terminal region of Stat4 eliminate its ability to be phosphorylated by Jak2. The basis for these effects are not known and are currently being examined, although they demonstrate that the interaction of Jaks and Stats may be more complex and may be dependent upon Stat tertiary structure.
The
complexity of Stat activation is further indicated by recent studies
that have demonstrated that Stat2 is required for IFN-induced
tyrosine phosphorylation of Stat1(21) . The basis for this
dependence is not known; however, it can be hypothesized that Stat2 is
required to recruit Stat1 to the receptor complex. Recruitment may rely
on the SH2 domain of Stat1 interacting with phosphorylated Stat2.
Alternatively, recruitment may rely on heterodimerization of the Stat1
and Stat2 through other domains that affect the ability of the complex
to associate with the activated Jaks.
When co-expressed in insect
cells, Jak1, Jak2, or Tyk2 were equally capable of phosphorylating and
activating Stat1. Therefore the specificity with which different
cytokines induce the tyrosine phosphorylation of specific Stat proteins in vivo cannot be ascribed to the Jaks. Rather, the
specificity must reside in the ability of individual receptor complexes
to recruit specific Stat proteins to the associated Jaks. Recent
studies have provided compelling evidence that this recruitment
involves the interaction of the SH2 domains of the Stats with sites of
tyrosine phosphorylation on the receptor. Specifically, a single
tyrosine in the IFN receptor
(R1) chain has been implicated
in recruiting Stat1(22) . Similarly, tyrosine residues within
gp130 or the IL-4 receptor
chain have been shown to be docking
sites for Stat3 (23) or Stat6(24, 25) ,
respectively. Lastly, exchanging SH2 domains has been shown to alter
the specificity with which Stat proteins associate with receptor
complexes(26) . It is important to note that in this model and
based on our results it is essential that the activated Jaks be
restricted to the activated receptor complex to maintain specificity.
Indeed, overexpression of Jaks in mammalian cells results in their
constitutive, ligand-independent activation and the concommitant
constitutive activation of the endogenous
Stats(27, 28) .
The phosphorylation of Stat1, either in vivo in insect cells or in vitro, resulted in the acquisition of GAS DNA binding activity. This result supports the hypothesis that unlike the ISGF3 complex, Stat1 is capable of binding DNA in the absence of any additional factors. Because of this result we have also examined the properties of Stat2 phosphorylated in insect cells by the Jaks (data not shown). Consistent with the current models, no DNA binding activity was detected against either various GAS sequences or the ISRE sequence.
The Stat proteins are utilized in
signaling pathways other than those activated by receptors of the
cytokine receptor superfamily. In particular, EGF has been shown to
induce the tyrosine phosphorylation of Stat1 as well as
Stat3(7, 8, 9) . Stat1 is hypothesized to be
a necessary component of an EGF induced DNA binding activity that
recognizes the SIE (c-sis-inducible element) in the c-fos gene promoter. From these studies, however, it was not clear
whether Stat1 phosphorylation was mediated by the EGF receptor or
through activation of a Jak. We therefore examined the ability of
immuno-purified EGFR to phosphorylate purified Stat1. As demonstrated,
EGFR catalyzed the tyrosine phosphorylation of Stat1 at
Tyr. Conversely, Stat1 was not detectably
tyrosine-phosphorylated when co-expressed with the Tec protein-tyrosine
kinase. Based on these results it will be of some interest to define
the spectrum of kinases that are capable of phosphorylating the various
Stats.