STAT3 regulates Nemo-like kinase by mediating its interaction with IL-6-stimulated TGFbeta-activated kinase 1 for STAT3 Ser-727 phosphorylation

Abstract

Signal transducer and activator of transcription 3 (STAT3) is activated by the IL-6 family of cytokines and growth factors. STAT3 requires phosphorylation on Ser-727, in addition to tyrosine phosphorylation on Tyr-705, to be transcriptionally active. In IL-6 signaling, the two major pathways that derive from the YXXQ and the YSTV motifs of gp130 cause Ser-727 phosphorylation. Here, we show that TGF-beta-activated kinase 1 (TAK1) interacts with STAT3, that the TAK1-Nemo-like kinase (NLK) pathway is efficiently activated by IL-6 through the YXXQ motif, and that this is the YXXQ-mediated H7-sensitive pathway that leads to STAT3 Ser-727 phosphorylation. Because NLK was recently shown to interact with STAT3, we explored the role of STAT3 in activating this pathway. Depletion of STAT3 diminished the IL-6-induced NLK activation by >80% without inhibiting IL-6-induced TAK1 activation or its nuclear entry. We found that STAT3 functioned as a scaffold for TAK1 and NLK in vivo through a region in its carboxyl terminus. Furthermore, the expression of the STAT3(534-770) region in the nuclei of STAT3-knockdown cells enhanced the IL-6-induced NLK activation in a dose-dependent manner but not the TGFbeta-induced NLK activation. TGFbeta did not cause STAT3 Ser-727 phosphorylation, even when the carboxyl region of STAT3 was expressed in the nuclei. Together, these results indicate that STAT3 enhances the efficiency of its own Ser-727 phosphorylation by acting as a scaffold for the TAK1-NLK kinases, specifically in the YXXQ motif-derived pathway.

Fig. 1.

Endogenous TAK1 binds to STAT3 and is activated through the YXXQ motif in gp130. (A) HepG2 cells were untreated (–) or treated with IL-6 for 15 min. WCE was immunoprecipitated (IP) with control IgG (lane 1–2), anti-TAK1 (lanes 3 and 4), or anti-STAT3 (lanes 5 and 6) Abs. Immunoprecipitates were resolved on SDS/PAGE and immunoblotted (IB) with the indicated Abs. (B) HepG2 cells were untreated (–) or treated with 20 ng/ml IL-6, IL-1β, or TGF-β for 10 min. The proteins immunoprecipitated with anti-TAK1 Ab were divided into two aliquots. One aliquot was subjected to an in vitro kinase assay (IVK) with recombinant MKK6 as the substrate (Upper), and the other aliquot was used in an immunoblot to examine the amount of immunoprecipitated TAK1 (Lower). (C) HepG2, HepG2-G108-YRHQ, or HepG2-G108-YSTV cells were transfected with pEF-Flag-TAK1 plasmid, and untreated (–) or treated with the indicated cytokines at 20 ng/ml for 10 min. The anti-Flag immunoprecipitates of WCEs were subjected to the kinase assay (Upper) and immunoblot analysis with an anti-Flag Ab (Lower) as in B.

Fig. 2.

Preferential activation of NLK through the YXXQ-TAK1 pathway. (A) EMSA for NFκB activity. ³²P-labeled oligonucleotides containing an NFκB-binding site and nuclear extracts from HepG2 cells that were untreated (–) or stimulated with IL-1β or IL-6 for 30 min were used. The reaction mixtures were preincubated with no additions (lanes 1, 2, and 5), anti-p65 Ab (lanes 3 and 6), or control rabbit IgG (lanes 4 and 7). (B) HepG2 cells (lanes 1 and 3), HepG2-G108YRHQ (lane 5), HepG2-G108YSTV (lane 6), or HepG2-G108YRHQ with TAK1 KD (lane 4, see also Fig. 3A) cells were infected with LV-Myc-NLK. WCEs from parental (lane 2) and the transfected cells were immunoblotted (IB) with an anti-NLK Ab (Left Upper, lanes 1 and 2), an anti-TAK1 Ab (Right Upper, lanes 3–6) or anti-Myc Ab (Lower). The total amount of endogenous and exogenous NLK in the HepG2-Myc-NLK cells was estimated as ≈2- to 2.2-fold of that of endogenous NLK in the parental cells. (C and D) The various HepG2-Myc-NLK cells shown in B were left unstimulated or stimulated with the indicated cytokines at 20 ng/ml for 10 min. The anti-Myc immunoprecipitates (IP) of WCE were divided into two aliquots. Each aliquot was subjected to the NLK kinase assay in the absence of exogenous substrate (Upper) or immunoblot analysis with the anti-Myc Ab (Lower).

Fig. 3.

TAK1 and NLK are components of the YXXQ-mediated H7-sensitive kinase pathway leading to the phosphorylation of STAT3 Ser-727. (A) HepG2-G108YRHQ cells were infected with LV-U6-small interfering RNA against TAK1 and NLK to deplete TAK1 and NLK (TAK1KD and NLKKD, respectively). The silencing effect was evaluated by immunoblotting with the indicated Ab. (B) HepG2-G108YRHQ cells and the TAK1KD or NLK KD HepG2-G108YRHQ cells were left unstimulated or stimulated with the indicated cytokines at 20 ng/ml (lanes 2, 4, and 5) or at 100 ng/ml (lanes 3, 7, and 9) for 15 min. The WCEs were immunoblotted with the indicated Abs. (C) The Flag-TAK1 immunoprecipitates from unstimulated or IL-6-stimulated HepG2 cells were subjected to the in vitro kinase assay as in Fig. 1B in the absence or presence of 5 μM H7. (D) HepG2-Myc-NLK cells were stimulated with IL-6 at 20 ng/ml for 15 min. The immunoprecipitates of WCEs with anti-Myc Ab were subjected to the in vitro NLK kinase assay as in Fig. 2C (lanes 1 and 2) or the in vitro NLK kinase assay by using GST-STAT3 (534–770) made in E. coli (lanes 3–6) in the presence of the indicated amounts of H7. Phospho-Ser-727 was detected with an antiphospho-Ser-727 Ab (Right Upper). The amount of NLK used for each kinase assay is shown in Lower. (E) HepG2-G108YRHQ cells pretreated with nothing (–) or H7 at 50 μM (+) and HepG2-G108YRHQ-NLK-KD cells were left unstimulated or stimulated with the indicated concentrations of IL-6 (lanes 2–7) or 20 ng/ml G-CSF for 15 min. The WCEs were immunoblotted with the indicated Abs (Left). The intensities of each band were quantified by a densitometer (GS-700, Bio-Rad), and the percentage of STAT3 in the phosphorylated form was calculated (Right). The value in HepG2 cells stimulated with 30 ng/ml IL-6 was defined as 100%, because almost all of the STAT3 was phosphorylated on Ser-727, as judged by its migration pattern on SDS/PAGE (data not shown). Average values of two independent experiments are shown.

Fig. 4.

STAT3 is required for IL-6-induced NLK activation but not for TAK1 activation. (A) HepG2 and HepG2-STAT3-knockdown (KD) cells were left unstimulated or stimulated with IL-6 at 1 (lanes 2 and 5), 100 (lanes 3 and 6), or 20 (lanes 8, 10, 12, and 14) ng/ml. Endogenous TAK1 from WCE extracts (lanes 1–6), cytosolic extracts (Cyt, lanes 7, 8, 11, and 12) or nuclear extracts (Nuc, lanes 9, 10, 13, and 14) was immunoprecipitated with an anti-TAK1 Ab, and the kinase activity was measured by using MKK6 as the substrate (Upper). (Lower) Immunoblotting with the anti-TAK1 Ab. (B) HepG2 (G2) cells, HepG2-STAT3KD (KD), and HepG2-STAT3KD reconstituted with RNAi-resistant STAT3 (3R) were infected with LV-Myc-NLK. The expression levels of STAT3 and Myc-NLK are shown in Left Lower. These HepG2 and derivative cells expressing Myc-NLK were left unstimulated or stimulated with the indicated cytokines at 20 ng/ml for 10 min. Kinase assay (Upper) and Western blotting analysis (Lower) of the anti-Myc-immunoprecipitates of WCE are shown. The radioactivity level of each band in the kinase assay was quantified with the BAS 5000 Bioimaging analyzer (Fujix, Tokyo). The activity of NLK in the IL-6-stimulated HepG2-STAT3 KD was estimated to be 17.6 ± 7.6% of that of IL-6-stimulated HepG2 cells (n = 4).

Fig. 5.

The STAT3 molecule acts as a scaffold for TAK1/NLK in the YXXQ-derived pathway. (A) 293T cells were transfected with pCMV-HA-TAK1, pCMV-Myc-NLK, and pEFGST or pEFGST-STAT3_533–770 plasmid. The GST-labeled proteins were pulled-down with reduced glutathione (GSH)-Sepharose. The eluates were diluted with 50 mM Hepes buffer (pH 7.8) containing 150 mM NaCl, and subjected to immunoprecipitation using an anti-Myc Ab. The WCEs (lanes 1 and 2), the eluates from the GSH-Sepharose (lanes 3 and 4), and the anti-Myc Ab immunoprecipitates (lanes 5 and 6) were subjected to immunoblotting analysis with the indicated Abs. (B) HepG2-MycNLK and HepG2-STAT3KD-MycNLK were treated with 20 ng/ml IL-6 for 10 min. The nuclear extracts (500 μg each) were immunoprecipitated with an anti-TAK1 Ab. Nuclear extracts (20 μg, lanes 1 and 2) and the immunoprecipitates (lanes 3 and 4) were separated and probed with the indicated Abs. (C) HepG2-STAT3KD-MycNLK cells were infected with three doses of lentiviral preparation LV-EFNLS-FlagSTAT3_534–770. HepG2-MycNLK-, HepG2-STAT3KD-MycNLK-, and NLS-FlagSTAT3_534–770-expressing cells were stimulated with IL-6 (lanes 2, 4, 6, 8, and 10), or TGF-β (lanes 12–15) at 20 ng/ml, for 10 min or left unstimulated. The anti-Myc-immunoprecipitates (IP) of WCEs were subjected to the in vitro NLK kinase assay (p-NLK) and immunoblot (IB) with the anti-Myc Ab. (Lower) Immunoblots using an anti-STAT3 Ab indicate that the lysate samples contained increasing amounts of NLS-FlagSTAT3_534–770 protein in the WCE. (D) HepG2 cells expressing NLS-FlagSTAT3_534–770 were left untreated or treated with IL-6 or TGFβ for 15 min. The WCEs were separated on SDS/PAGE and probed with the indicated Abs.