(Received for publication, September 1, 1995; and in revised form, December 1, 1995)
From the
A variety of cytokines and growth factors act through an
induction of gene expression mediated by a family of latent
transcription factors called STAT (signal transducers and activators of
transcription) proteins. Ligand-induced tyrosine phosphorylation of the
STATs promotes their homodimer and heterodimer formation and subsequent
nuclear translocation. We demonstrate here that STAT protein
heterocomplexes exist prior to cytokine treatment. When unstimulated
HeLa cells are ruptured in hypotonic buffer without salt or detergent,
immunoadsorption of either STAT1 or STAT2 from the resulting cytosol
yields coimmunoadsorption of the other STAT protein. Similarly,
STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic
cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte
lysate spontaneously form heterocomplexes when the translation lysates
are mixed at 0 °C. Our data suggest that
interferon-/
-induced tyrosine phosphorylation increases the
stability of a preexisting, latent, STAT1-STAT2 signaling complex.
Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3,
but we show that STAT2 and STAT3 exist in separate heterocomplexes with
STAT1, consistent with a model in which STAT1 contains a common binding
site for other STAT proteins.
It is now appreciated that a large number of cytokines, growth
factors, and hormones act through a sequence of steps that includes
binding to receptors in the cytokine receptor superfamily followed by
activation of the JAK (Janus kinases) family of tyrosine kinases, which
results in activation of STAT (signal transducers and activators of
transcription) proteins that subsequently migrate to the nucleus where
they initiate specific gene transcription (see (1) and (2) for review). The overall signaling pathway is utilized by
the interferons (IFNs) ()and a variety of other cytokines,
including the interleukins, some colony stimulating factors
(granulocyte colony-stimulating factor and granulocyte-macrophage
colony-stimulating factor), leukemia inhibitory factor (LIF),
oncostatin M, and erythropoietin, as well as the peptide hormones
prolactin and growth hormone. The JAK family of kinases includes JAK1,
JAK2, JAK3, and Tyk2, and, to date, six STAT proteins have been
identified.
One of the most studied cytokine signaling systems is
interferon-mediated gene activation. Here it is known that interaction
of IFN- or IFN-
with the IFN-
/
receptor activates
JAK1 and Tyk2 with resulting tyrosine phosphorylation of the 91-kDa
STAT1 and the 113-kDa STAT2 proteins, which are then thought to form a
STAT heterodimer that associates with a 48-kDa DNA-binding protein.
This multiprotein unit is called the interferon-stimulated gene factor
3, and it appears to be the primary positive regulator of
interferon-stimulated response element-controlled
genes(3, 4, 5) . Thus, the STAT proteins are
essentially latent transcription factors residing outside of the
nucleus(6) , and in vivo activation is associated with
interferon-stimulated gene factor 3 translocation to the
nucleus(5, 7) .
We started the work we report
herein asking whether we could use the gentle techniques of cell
rupture and immunoadsorption in hypotonic buffer that we have used to
study steroid receptor and protein kinase heterocomplexes (8) to detect possible complexes between STAT proteins and
hsp90. We were not able to detect any association of STAT1, -2, or -3
with hsp90, but we serendipitously made some fundamental observations
regarding the formation of STAT protein complexes themselves. We found
that rupture of HeLa cells in a hypotonic buffer followed by
immunoadsorption of STAT1 yields coimmunoadsorption of STAT2 and vice versa. This suggests that in untreated cells the two
proteins exist together in a complex but that the association is weak,
as the complex is not observed after cell rupture in buffer containing
1% Triton X-100 and 150 mM NaCl. After stimulation with
IFN- or IFN-
, however, the STAT1-STAT2 complex survives the
``harsh'' rupture conditions with detergent and salt. After
stimulation by IFN-
, which is thought to induce gene expression
through an interaction of tyrosine-phosphorylated STAT1 homodimers with
activation sequences(9) , no STAT1-STAT2 complex is seen
under the harsh rupture conditions.
Our data point to a revision of
the standard model where it has been assumed that signaling by
IFN-/
receptors leads to STAT protein phosphorylation with
subsequent heterocomplex formation. Rather, tyrosine phosphorylation
seems to increase the stability of a preexisting, latent signaling
complex. In the absence of cytokine stimulation, we have found that
STAT1 and STAT2 associate with each other in an equilibrium fashion, as
do STAT1 and STAT3, but STAT2 and STAT3 exist only in separate
heterocomplexes with STAT1. This may imply that STAT1 contains a common
binding site for other STAT proteins.
Figure 1:
STAT1 and STAT2 are associated with
each other prior to stimulation with IFN- or IFN-
. Untreated
HeLa cells or HeLa cells treated with IFN-
, IFN-
, or
IFN-
were assayed for STAT protein complexes. Aliquots (200
µl) of cytosol prepared from hypotonic lysates (lanes
1-3) or from detergent/salt lysates (lanes 4 and 5) were immunoadsorbed with nonimmune rabbit serum,
anti-STAT1, or anti-STAT2. After washing the immunopellets, STAT
proteins were resolved by SDS-polyacrylamide gel electrophoresis and
Western blotted with anti-STAT1 or anti-STAT2, as designated to the left. Under each condition of cell treatment, cytosols were
immunoadsorbed with nonimmune serum (lane 1), anti-STAT1 (lane 2); anti-STAT2 (lane 3); anti-STAT1 (lane
4), and anti-STAT2 (lane 5).
Because STAT1 and STAT2 were coimmunoadsorbed from hypotonic cytosols of untreated cells, we asked if the two proteins would spontaneously associate with each other when mixed together. STAT1 and STAT2 were transcribed and translated in rabbit reticulocyte lysate and then immunoadsorbed with anti-STAT antisera. As shown in Fig. 2(lanes 7-9), when the two proteins were translated in the same mix, immunoadsorption of either STAT1 or STAT2 yielded coimmunoadsorption of the other. It is unlikely that protein folding reactions are required to form the STAT protein heterocomplex because simply mixing two aliquots of reticulocyte lysate containing newly translated STAT1 or STAT2 at 0 °C yields a heterocomplex that can be immunoadsorbed (Fig. 2, lanes 10 and 11).
Figure 2:
In vitro translated STAT1 and
STAT2 form a complex in rabbit reticulocyte lysate. STAT1 and STAT2
were transcribed and translated in rabbit reticulocyte lysate. Shown is
an autoradiogram of [S]methionine-labeled
proteins in aliquots of reticulocyte lysate translation mixture or in
immunoprecipitates prepared from the translation mixture. Lane
1, 5 µl of STAT1 translation mixture; lane 2, 5
µl of STAT2 translation mix; lanes 3 and 4,
preimmune and anti-STAT1 immunoadsorption of STAT1 translation mix; lanes 5 and 6, nonimmune and anti-STAT2
immunoadsorption of STAT2 translation mix; lanes 7-9,
nonimmune, anti-STAT1, or anti-STAT2 immunoadsorption from a
translation mixture containing both STAT1 and STAT2 cDNAs; lanes 10 and 11, separate STAT1 and STAT2 translation mixtures
combined on ice and then immunoadsorbed with nonimmune serum (lane
10) or anti-STAT1 antiserum (lane
11).
Figure 3: STAT1 and STAT3 exist in a preformed complex in 3T3-F442A cell cytosol. Untreated or LIF-treated 3T3 cells were ruptured in hypotonic buffer (lanes 1-3) or buffer with detergent and salt (lanes 4-6), and the cytosols were immunoadsorbed with nonimmune serum (lanes 1 and 4), anti-STAT1 serum (lanes 2 and 5), or anti-STAT3 serum (lanes 3 and 6).
As shown in Fig. 4when STAT1 and STAT3 were cotranslated in the same translation mixture, immunoadsorption of STAT3 yielded coimmunoadsorption of STAT1 (lanes 4 and 5). Despite the fact that heterocomplexes could be demonstrated in a STAT1/STAT3 cotranslation mix as well as in a STAT1/STAT2 cotranslation mix (lanes 6 and 7), no heterocomplex could be demonstrated in STAT2/STAT3 cotranslation (lanes 8 and 9).
Figure 4:
STAT1 and STAT3, but not STAT2 and STAT3,
are coimmunoadsorbed after cotranslation in reticulocyte lysate. Shown
is an autoradiogram of [S]methionine-labeled
proteins in aliquots of reticulocyte lysate translation mixture or
immunoprecipitates prepared from the translation mixture. Lanes
1-3, 5 µl of STAT1, STAT2, and STAT3 translation
mixtures, respectively; lanes 4 and 5, STAT1 and
STAT3 cotranslated in the same aliquot of reticulocyte lysate, which
was then immunoadsorbed with nonimmune serum or anti-STAT3 serum; lanes 6 and 7, STAT1 and STAT2 cotranslation
immunoadsorbed with nonimmune or anti-STAT2 serum; lanes 8 and 9, STAT2 and STAT3 cotranslation immunoadsorbed with nonimmune
or anti-STAT2 serum.
Figure 5: STAT1 is present in complexes with STAT2 or STAT3 but not both proteins simultaneously, and STAT2 and STAT3 compete for association with STAT1. A, STAT2 and STAT3 are in separate complexes with STAT1. 3T3 cells were ruptured in hypotonic buffer and 200-µl aliquots of cytosol were immunoadsorbed with nonimmune serum (lane 1), anti-STAT3 (lane 2), anti-STAT1 (lane 3), or anti-STAT2 (lane 4), and immunoadsorbed proteins were detected by immunoblotting. B, STAT3 competes for STAT2 binding to STAT1. The three STATs were translated separately in reticulocyte lysate. STAT2 and STAT3 were then mixed in a ratio of 1:1 (lane 1), 1:2 (lane 2), or 1:10 (lane 3) prior to being mixed with a fixed amount of STAT1. The volumes of each reaction were equalized using untranslated reticulocyte extract. After 30 min on ice, the mixtures were immunoadsorbed with anti-STAT1 serum, and immunoadsorbed proteins were detected by immunoblotting.
To determine if STAT2 and STAT3 compete for binding to STAT1, each protein was translated in vitro, and STAT2 and STAT3 were mixed with a constant amount of STAT1. As shown in Fig. 5B, mixing of increasing amounts of STAT3 with STAT2 resulted in more STAT3 and less STAT2 being coimmunoadsorbed with STAT1 (c.f. lanes 1 and 3). As expected, the same amount of STAT1 was immunoadsorbed from each reaction.
Figure 6:
Mutation of Tyr of STAT1 to
phenylalanine does not affect formation of the heterocomplex with
STAT2. Wild-type STAT1, the Y701F STAT1 mutant, and STAT2 were
translated singly or in combination in reticulocyte lysate with
[
S]methionine, and the STAT proteins were
immunoadsorbed. Lanes 1 and 2, wild-type STAT1
translation mix immunoadsorbed with nonimmune serum or anti-STAT1; lanes 3 and 4, Y701F STAT1 translation immunoadsorbed
with nonimmune serum or anti-STAT1; lanes 5 and 6,
STAT2 translation immunoadsorbed with nonimmune serum or anti-STAT2, lanes 7 and 8, wild-type STAT1 and STAT2
cotranslation immunoadsorbed with nonimmune serum or anti-STAT1; lanes 9 and 10, Y701F STAT1 and STAT2 cotranslation
immunoadsorbed with nonimmune or
anti-STAT1.
The notion that interferon-/
treatment induces the
formation in the cytoplasm of STAT1-STAT2 heterocomplexes is part of
the developing basic model of signal transduction via STAT
proteins(1, 2) . The data presented in Fig. 1,
however, are consistent with a variation of the model in which STAT1
and STAT2 may be preassociated as a heterodimer, with IFN-
or
IFN-
treatment increasing the stability of the complex, as
indicated by its resistance to dissociation by the detergent/salt
conditions of a radioimmune precipitation assay buffer. Phosphorylation
of Tyr
on STAT1 is required for its nuclear
translocation(19) , but phosphorylation does not seem to be
required for STAT1 to form at least the low affinity complex with
STAT2, as indicated by the observation that the Y701F mutant binds to
STAT2 in reticulocyte lysate (Fig. 6). It seems likely that
Tyr
phosphorylation is required for the high affinity,
detergent/salt-resistant STAT1-STAT2 complex demonstrated in Fig. 1.
STAT1 and STAT2 appear to bind to each other when mixed in solution (Fig. 2), as do STAT1 and STAT3 (Fig. 4). Although it is reduced, the STAT1-STAT3 complex is not eliminated under the harsh conditions of the detergent/salt buffer (Fig. 3), indicating that it is of higher affinity than the STAT1-STAT2 complex shown in Fig. 1. At this time, it is unclear whether STAT protein phosphorylation as a result of LIF treatment affects the affinity of the STAT1-STAT3 interaction. At least, under our experimental conditions, we have not observed a LIF effect on the salt sensitivity of the heterocomplex. It is interesting that immunoadsorption of STAT2 or STAT3 yields coimmunoadsorption of STAT1 but not each other (Fig. 5A). Although this could be explained by the location of epitopes for the immunoprecipitating antibodies in a STAT2/STAT3 interaction site, it is more likely that STAT2 and STAT3 form separate heterocomplexes with STAT1. As the binding of one of these STATs to STAT1 seems to preclude binding of the other, the binding sites may overlap, or there may be a common binding site on STAT1 for the other STAT proteins as suggested by the competition experiment of Fig. 5B.
The work we report here raises the notion that STAT protein heterocomplexes preexist in the cytoplasm. It also has been shown by coimmunoadsorption that JAK kinases are constitutively associated with several members of the cytokine receptor superfamily(20, 21) , and it is possible that the STAT protein heterodimers are associated, albeit weakly, with this receptor-attached multiprotein structure. Such a notion is consistent with the fact that STATs can be activated in vitro using only plasma membrane-enriched fractions of a variety of cultured cells (22) and with recent evidence of Stahl et al.(23) that direct interactions exist between STATs and modular tyrosine-based motifs in a number of cytokine receptors. One could then conceive of signals being passed via the phosphorylation of entirely preassociated proteins. Whether the STAT proteins then move through the cytoplasm by diffusion or in association with a general protein movement system, as suggested for the steroid receptors(24) , is unknown.