(Received for publication, October 4, 1995; and in revised form, January 2, 1996)
From the
Interleukin-6 (IL-6) induces tyrosine phosphorylation and activation of the latent transcription factor Stat3 in HepG2 cells. Mutation of Stat3 tyrosine 705 to phenylalanine (Y705F) inhibits IL-6-induced tyrosine phosphorylation of this Stat3 mutant in transfected HepG2 cells. In cotransfections of HepG2 cells, the Stat3 mutant Y705F causes a reduction of the tyrosine phosphorylation of wild type Stat3-FLAG. Moreover, Y705F inhibits the action of endogenous Stat3 in cotransfected cells, reducing IL-6 induction of a Stat3-responsive reporter construct. Y705F therefore acts as a dominant negative mutation of Stat3.
Cytokines and growth factors affect target cells through
attachment to specific cell surface receptors with subsequent formation
of receptor complexes and transduction of signals into the interior of
the cell(1) . One signal transduction pathway that leads to
induction of target gene expression after cytokine stimulation is the
recently described Janus kinase (Jak)-STAT pathway (for reviews, see (2, 3, 4) ). Although the cytoplasmic domains
of cytokine receptors lack apparent enzymatic activity, ligand binding
activates associated Jak tyrosine kinases, which phosphorylate each
other as well as target tyrosines in the receptor
chains(5, 6, 7) . Inactive cytoplasmic
transcription factors or STATs are then selectively recruited to the
receptor chains via interaction between the SH2 ()domain of
the STATs and the receptor tyrosine
phosphopeptides(2, 3, 4) . Recruited STATs
are phosphorylated on a specific tyrosine residue and then dissociate
from the receptor, form homo- and heterodimers that translocate to the
nucleus, bind to cognate DNA response elements, and activate target
gene transcription (2, 3, 4) .
Stat3 can be activated by a number of different cytokines and growth factors, including the IL-6 family of cytokines (IL-6, IL-11, ciliary neurotrophic factor, oncostatin M, and leukemia inhibitory factor) that share the common gp130 receptor subunit, and epidermal growth factor (1, 8, 9) . Stat3 is activated through specific receptor phosphopeptide sequences with the consensus sequence Y*-X-X-Q, which is present four times in the gp130 receptor chain as well as twice in the leukemia inhibitory factor receptor chain and twice in the epidermal growth factor receptor(10, 11, 12) .
We report here the construction of a dominant negative form of Stat3 capable of competitively interfering with wild type Stat3 activation by IL-6. This dominant negative mutation will prove to be a useful tool to investigate the role of Stat3 in cytokine-dependent induction of target genes.
Figure 1: Schematic representation of Stat3, Y705F, and the FLAG-tagged derivatives. The SH2 and SH3 domains are indicated, and the tyrosine 705 is marked. The FLAG present at the COOH terminus is also indicated (hatched box at COOH terminus).
Figure 2: Phosphorylation of tyrosine 705 of Stat3 upon activation by IL-6. A, Hep G2 cells, transiently transfected with 8 µg of pTL1 or expression vectors containing FLAG-tagged Stat3 or Y705F cDNA, were incubated 10 min in the presence or absence of IL-6 (25 ng/ml). Cell extracts were immunoprecipitated with a monoclonal antibody directed against FLAG (M2) and after SDS-PAGE and blotting, probed with the antibody to FLAG or an anti- phosphotyrosine (P-Tyr) monoclonal antibody (4G10). B, Hep G2 cells transiently transfected with 1 µg of the expression vector containing FLAG-tagged Stat3 cDNA, together with 10 µg of pTL1 or the Y705F expression vector. Conditions were as described in A.
Co-transfection of the IIP6-tk-cSPAP reporter construct and the
Stat3 expression vector in HepG2 cells resulted in a more elevated
promoter activation by IL-6, measured in a SPAP assay, as compared to
HepG2 cells co-transfected with the empty expression vector pTL1 and
the SPAP reporter construct (Fig. 3A, IL-6 dose
response, 9.4-fold versus 5.6-fold maximal induction; and
3B, time course after IL-6 addition). When Y705F was
transfected in HepG2 cells a strong inhibition is observed in the
IL-6-induced promoter activation (Fig. 3A, activation
is reduced to 2-fold). These results indicate that phosphorylation
of tyrosine at position 705 of STAT3 is essential in IL-6 signaling and
that the mutant Y705F reduces the signal transduction via endogenous
Stat3. Transfection of HepG2 cells with increasing amounts of Y705F
expression vector inhibits IL-6 induction of the SPAP reporter in a
dose-dependent manner, indicating competition of Y705F with endogenous
Stat3 upon IL-6 activation (Fig. 4). Stat3 and Y705F have no
effect on expression of a cotransfected control reporter construct
RSV-lactamase whose expression remains unaltered by IL-6 treatment
(data not shown).
Figure 3: Y705F inhibits IL-6 induction of IIP6-tk-SPAP reporter. HepG2 cells were transiently transfected with 8 µg of IIP6-tk-cSPAP, 4 µg of RSV-lactamase, 8 µg of pTL1 or expression vectors containing Stat3 or Y705F cDNA. Subsequently cells were retrypsinized and replated in wells of 96-well microtiter dishes. A, IL-6 dose response. The results are expressed as relative SPAP activity with respect to the untreated wells (100%). SPAP activity in each well is normalized with respect to control lactamase production. Each point is the average of quadruplicate wells, and error bars are indicated. B, time course of IL-6 induction. Cells were incubated in the presence of IL-6 (10 ng/ml) for the indicated time in hours (h). Results are expressed as normalized SPAP activity (arbitrary units) induced by IL-6 (normalized SPAP activity in the absence of IL-6 is subtracted from that in the presence of IL-6). Each time point represents the average of quadruplicate wells, and error bars are indicated.
Figure 4: Y705F competitively inhibits IL-6 induction of IIP6-tk-SPAP reporter. Hep G2 cells were transiently transfected with 8 µg of IIP6-tk-cSPAP, 4 µg of RSV-lactamase, and with increasing amounts of Y705F expression vector as indicated. Total amounts of expression vector was standardized throughout by adding various quantities of pTL1. SPAP activity is calculated as in Fig. 3A, normalized for lactamase secretion.
Mutation of tyrosine 705 to phenylalanine in Stat3 leads to reduced levels of tyrosine-phosphorylated mutant protein following IL-6 treatment, indicating that this residue is a site of phosphorylation. This is in accordance with the alignment with other members of the Stat family, in particular Stat1, where tyrosine 701, and Stat2 where tyrosine 690 are phosphorylated, respectively, following cytokine treatment(16, 17, 27) . Moreover, immunoprecipitation studies show that Y705F inhibits phosphorylation of Stat3-FLAG following IL-6 treatment in cotransfected HepG2 cells (Fig. 2B). In addition, the Y705F mutation of Stat3 acts in a dominant negative manner to inhibit IL-6 activation of the IIP6-tk-cSPAP reporter construct in HepG2 cells (Fig. 3). Y705F competitively inhibits IL-6 signal transduction as measured in our transcriptional assay (Fig. 4) and in immunoprecipitation experiments measuring Stat3-FLAG tyrosine phosphorylation in cotransfections with increasing quantities of Y705F (data not shown). This demonstrates that tyrosine 705 in Stat3 is critical for activation by IL-6.
Recent studies indicate that Stat1 and Stat6 bind directly
to tyrosine phosphopeptides derived from the IFN- receptor and the
IL-4 receptor, respectively(19, 20) . Current models
suggest that inactive cytoplasmic STATs are recruited to the activated
receptor by docking of the STAT SH2 domain to selected receptor
tyrosine phosphopeptides, where they are in turn phosphorylated on a
single tyrosine by Jak kinases(10, 19, 20) .
Stat1 is phosphorylated on Tyr-701, and mutation of this tyrosine to
phenylalanine inactivates this mutant Stat1(16) . Domain
swapping experiments between Stat1 and Stat2 demonstrate that the SH2
domain is critical for selective recruitment to activated receptor
chains(21) .
The amino acid sequences surrounding the
phosphorylated tyrosine dictate substrate recruitment via specificity
of SH2 domain/tyrosine phosphopeptide interactions ((22) ; see (23) for review). Recent work indicates that Stat3 can be
activated via receptor tyrosine phosphopeptides with glutamine in the
+3 position (Y*XXQ) (10) . The glutamine at
+3 is believed to play a critical role in Stat3 recruitment since
its alteration to alanine prevents Stat3 tyrosine phosphorylation in
chimeric receptor constructs(10) . Interestingly, this target
sequence differs from the internal Stat3 tyrosine phosphopeptide
sequence that may be used in back-to-back Stat3 homodimer formation
(Y*LKT). This finding also applies to Stat1, which is recruited by
a Y*DKPH motif on the IFN-
receptor(19, 24) , but
when forming a homodimer binds to Y*IKT(E) and in heterodimers with
Stat3 may bind Y*LKT(K). The tyrosine 705 residue that is
phosphorylated during Stat3 activation is thought to be immediately
distal to the carboxy boundary of the SH2 domain, a distance too short
to allow an intramolecular interaction between the SH2 domain and the
phosphotyrosine residue(25) . This close proximity suggests
that phosphorylation of tyrosine 705 may lead to a conformational
change of the Stat3 SH2 domain that alters its selectivity for tyrosine
phosphopeptides. After tyrosine phosphorylation, such an induced
conformational change might encourage dissociation of Stat3 from the
receptor and enhance formation of Stat dimers by altering selectivity
from Y*XXQ, the receptor sites that are likely to be bound by
non-activated Stat3, to Y*LKT the tyrosine phosphopeptide present in
Stat3 that allows STAT dimerization by interacting with the SH2 domain
of the dimeric partner. Such an alteration in the selectivity of an SH2
domain for target tyrosine phosphopeptides has been reported for the
tyrosine kinase p56
(26) .
Our current model for the dominant negative action of Y705F (Fig. 5) implies that the mutated Stat3 can still bind to IL-6-activated gp130 tyrosine phosphopeptides, since the SH2 domain is unaltered, but the critical residue at position 705 can no longer be tyrosine-phosphorylated by Jak kinases. Consequently, Y705F may block recruitment of Stat3 to receptor phosphopeptide docking sites and hence prevent activation of wild type Stat3. If phosphorylation of tyrosine 705 of Stat3 leads to a conformational change of its SH2 domain leading to reduced affinity for the receptor docking sites, then the Stat3 mutant Y705F/receptor complex may have a longer half-life than its wild type homolog.
Figure 5: Model for the mode of action of the dominant negative Stat3 mutation Y705F. A, activation of wild type Stat3. IL-6 treatment causes Stat3 recruitment to receptor tyrosine phosphopeptides (gp130) where it is phosphorylated on tyrosine 705 (Y) by Jak kinase. Stat3 dissociates from the receptor, forms dimers, and migrates to the nucleus where it binds response elements in target genes. Stat3 is shown schematically. B, two possible levels of dominant negative activity of Y705F. Y705F may compete with Stat3 for binding to the tyrosine phosphopeptides on gp130, and when in excess exclude Stat3 recruitment. Y705F may also be able to form a non-functional weak heterodimer with activated Stat3.
It cannot be excluded that Y705F may also form heterodimers with activated Stat3 through a single SH2/phosphotyrosine interaction (Fig. 5B, bottom). Such putative heterodimers with weakened molecular interactions may be susceptible to phosphatase deactivation or may simply be unable to bind DNA target sequences.
By altering the tyrosine residue that is phosphorylated in the different STATs, it should be possible to create similar dominant negative mutations, that will allow investigation of the role of individual STATs in the cellular responses to various cytokines and growth factors.