Activation of the signal transducer and activator of transcription 3 (STAT3) in response to interleukin-6 (IL-6) type cytokines involves both phosphorylation of Tyr705, which enables dimerization, nuclear translocation and DNA binding, as well as ser727 phosphorylation. Here, we describe that the 65 C-terminal amino acids of STAT3 can function as an independent transcription activation domain (TAD), particularly when a negative charge is introduced at position 727 by mutation of the serine residue into aspartate. The strong transcriptional activity of the C-terminal STAT3 Ser727Asp TAD is coupled to a constitutive association with the co-activator p300. In HepG2 cells, p300 associates with STAT3 upon IL-6 stimulation, and overexpression of p300 enhances the transcriptional activity of STAT3K K, but not of STAT3L L or STAT3 Ser727Ala. We conclude that Ser727 phosphorylation in the C-terminal region of STAT3 is required for transactivation by association with p300. ß

Signal transducers and activators of transcription (STATs) 1 belong to a family of transcription factors that are activated in response to a variety of cytokines and growth factors (1-4). So far, seven different STATs have been identified in mammals; these STATs contain conserved DNA binding and Src homology 2 domains that include a C-terminal tyrosine phosphorylation site (4). Binding of cytokines to their corresponding receptors induces Jak kinase activity, which results in phosphorylation of STATs on a specific tyrosine residue (5-7). Tyrosine-phosphorylated STATs dimerize, translocate to the nucleus, and bind specific DNA promoter sequences (8, 9).IL-6-induced STAT3 signaling involves the sequential activation of the gp130 receptor complex and the gp130-associated protein-tyrosine kinases Jak1, Jak2, and Tyk2 (4, 10 -12). Tyrosine phosphorylation of STAT3 occurs at a single tyrosine residue (Tyr-705) that is located in conserved Src homology 2 domain, allowing homodimerization as well as heterodimerization with other STAT family members (4, 13). In addition to tyrosine phosphorylation, STAT3 is serine-phosphorylated at a single residue (Ser-727) in response to IL-6 as well as other extracellular factors, including interferon ␥ and epidermal growth factor (13-17). Although the role of serine 727 phosphorylation is not yet unambiguously determined, it has been shown to strongly enhance STAT3 transcriptional activation, possibly by modulating interactions with cofactors (13, 18). The Ser-727 residue of STAT3 is located in a conserved Pro-X-SerPro sequence, which is recognized by the mitogen-activated protein kinase ERK (19). ERKs belong to the family of serine/ threonine kinases positioned at the end point of signal transduction cascades that are initiated at the plasma membrane by ligand-receptor interaction (20). Indeed, it has been demonstrated that epidermal growth factor-induced STAT3 Ser-727 phosphorylation involves the activation of ERKs (14). However, it has also been shown that IL-6-induced STAT3 Ser-727 phosphorylation is an ERK-independent process (14,18,21,22). Lim and Cao (23) have demonstrated that stress treatment could induce STAT3 Ser-727 phosphorylation via JNK-1, whereas we demonstrated that IL-6-induced STAT3 transactivation and Ser-727 phosphorylation involves the activation of the GDP-GTP exchange factor Vav, the GTPase Rac-1, and the kinases mitogen-activated protein kinase/ERK kinase kinase 1 and SEK-1/MKK-4 in human hepatoma cells (18).Recently, it has been described that PKC␦ associates with and phosphorylates STAT3 on Ser-727 in an IL-6-dependent manner (24).

In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 ('reactivating kinase') in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser(727) phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant-negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6-induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress-activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant-negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser(727) phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser(727) phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser(727) phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac-1, MEKK and SEK-1/MKK-4 as signal transduction components.

To explore the activation patterns of signal transducer and activator of transcription 3 (Stat3) in acute myeloid leukemia (AML), we examined whether the phosphorylation of tyrosine705 (Tyr705) and serine727 (Ser727) residues was abnormally regulated in cells from patients with AML. In 5 of 20 (25%) patients with AML, Stat3 was constitutively phosphorylated on Tyr705 and Ser727, which were not further up-regulated by treatment with IL-6. Furthermore, Stat3 was constitutively bound to the IRE response element in these cells as determined by electrophoretic mobility shift assay, and stimulation with IL-6 did not result in increased DNA binding. Interestingly, AML cells with constitutive Stat3 activation also secreted high levels of IL-6 protein. Treating these AML cells with anti-IL-6 resulted in restored IL-6–inducible Stat3 phosphorylation on both Tyr705 and Ser727 with low or undetectable basal phosphorylation levels in unstimulated cells. In contrast, treatment with anti-IL-1 did not result in altered Stat3 phosphorylation patterns. The constitutive IL-6 expression was associated with elevated levels of suppressor of cytokine signaling-1 (SOCS-1) and SOCS-3 mRNA expression, which were not down-regulated by anti-IL-6. These data indicate that the constitutive Stat3 activation in the investigated AML blasts is caused by high IL-6 secretion levels, thus stimulating the Jak/Stat pathway in an autocrine manner, a paracrine manner, or both.

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