Cytokines play several roles in developing and/or reinforcing premature cellular senescence of young cells. One such cytokine, interleukin-6 (IL-6), regulates senescence in some systems in addition to its known functions of immune regulation and promotion of tumorigenesis. In this review, we describe recent advances in studies on the roles of IL-6 and its downstream signal transducer and activator of transcription 3 (STAT3) in regulating premature cellular senescence. IL-6/sIL-6Rα stimulation forms a senescence-inducing circuit involving the STAT3-insulin-like growth factor-binding protein 5 (IGFBP5) as a key axis triggering and reinforcing component in human fibroblasts. We describe how cytokines regulate the process of senescence by activating STAT3 in one system and anti-senescence or tumorigenesis in other systems. The roles of other STAT members in premature senescence also will be discussed to show the multiple mechanisms leading to cytokine-induced senescence.

c-Fos is regulated by phosphorylation and multiple turnover mechanisms. We found that c-Fos was ubiquitylated in the cytoplasm during IL-6/gp130 stimulation under MEK inhibition and sought the mechanisms involved in the regulation. We show that sustained ERK5 activity and the E3 ligase UBR1 regulate the stability and subcellular localization of c-Fos. UBR1, rapidly induced by STAT3, interacts with and ubiquitylates c-Fos in the cytoplasm for its accelerated degradation. ERK5 inhibits the nuclear export of c-Fos by phosphorylating Thr232 in the c-Fos NES(221-233) and disrupts the interaction of c-Fos with UBR1 by phosphorylating Ser32. Moreover, UBR1 depletion in HeLa cells, which constitutively express UBR1 at a high level, enhances both c-Fos expression and cell growth, whereas ERK5 depletion reduces both of them. Interestingly, an NES mutant of c-Fos, but not wild-type, substitutes ERK5 activity for HeLa cell proliferation. Thus, this spatiotemporal regulation of c-Fos by ERK5 and UBR1 is critical for the regulation of c-Fos/AP-1.

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-␤-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 TGF␤-induced NLK activation. TGF␤ 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.gp130 ͉ YXXQ motif ͉ signaling specificity T he signal transducer and activator of transcription (STAT) family plays pivotal roles in a variety of systems and in development, in response to cytokines and growth factors. STAT family members are activated in the cytoplasm by tyrosine phosphorylation, form dimers, and enter the nucleus, where they act as DNA-binding transcription factors (1). Of the seven known STATs, STAT1, 3, 5A, 5B, and 6 are phosphorylated at one or two serine residues in their carboxyl-terminal transactivation domain (1, 2) and at a critical tyrosine. The serine phosphorylation enhances the transcriptional activity in the case of STAT1 (3), STAT3 (3, 4), and STAT6 (5). STAT3 can be activated by a variety of cytokines, including the IL-6 family, by using gp130 as a common receptor subunit (6), Granulocyte colony-stimulating factor (G-CSF) and erythropoietin, and growth factors, EGF, platelet-derived growth factor, and hepatocyte growth factor, and cytoplasmic tyrosine kinases, including Src and v-Eyk (reviewed in ref. 7). IL-6 uses STAT3 in its major signaling pathway and concomitantly activates the Ras͞Raf͞ ERK and PI3-kinase pathways (7). IL-6, therefore, activates multiple genes, including acute-phase reactants, and the junB, tis11, stat3, c-myc, and c-fos genes, mostly through STAT3 (8-14). In the IL-6 receptor system, the tyrosine-phosphorylated YXXQ motif of gp130 is critical for recruiting STAT3 for subsequent phosphorylation at Tyr-705 by the associated Jak kinases (15). I...

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Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor. It is activated by cytokines, including interleukin-6 (IL-6) through phosphorylation at Tyr705 (pY705), which is required for its dimerization and nuclear translocation. However, the role of Ser727 phosphorylation, occurring during activation, remains poorly understood. Using a combination of HepG2-stat3-knockdown cells reconstituted with various STAT3 mutants and protein kinase inhibitors, we showed that phospho-S727 has an intrinsic mechanism for shortening the duration of STAT3 activity, in turn shortening the duration of socs3 mRNA expression. Both STAT3WT and STAT3Ser727Asp (S727D) but not STAT3Ser727Ala (S727A) showed rapid dephosphorylation of pY705 after the inhibition of tyrosine kinases. We found that the nuclear TC45 phosphatase is most likely responsible for the phospho-S727-dependent pY705 dephosphorylation because TC45 knockdown caused prolonged pY705 with sustained socs3 mRNA expression in STAT3WT but not in STAT3S727A, and overexpressed TC45 caused rapid dephosphorylation of pY705 in STAT3WT but not in STAT3S727A. We further showed that phospho-S727 did not affect the interaction of TC45 with STAT3, and that a reported methylation at K140 of STAT3 occurring after phospho-S727 was not involved in the pY705 regulation. These findings indicate that phospho-Ser727 determines the duration of STAT3 activity largely through TC45.

The present results indicate that S1-1 contains multiple NLSs that act cooperatively. Among them, the OCRE is a hitherto unreported NLS. The nuclear localisation of S1-1 appears to be regulated under certain circumstances. We discuss these NLSs in relation to the biochemical processes they are involved in.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705–SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT–CTT intermolecular and the CTT–SH2 intramolecular interactions that support pY705–SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705–SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705–SH2 interaction. Thus, pS727 triggers pY705–SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation–inactivation for proper function of STAT3.

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