Sequential Activation of Rac-1, SEK-1/MKK-4, and Protein Kinase Cδ Is Required for Interleukin-6-induced STAT3 Ser-727 Phosphorylation and Transactivation

Abstract

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).

Protein kinase C-δ is involved in the inflammatory effect of IL-6 in mouse adipose cells

“…An extensive search for known potential kinases inducing Ser-727 phosphorylation of STAT3 in response to IL-6 was performed by using inhibitors to PKCδ, ERK, JNK, p38 MAPK and mTOR (data not shown), as they have previously been described to induce this phosphorylation [18][19][20][21][22]. Our study revealed PKCδ as the main target molecule.…”

“…In this study, we further characterised Ser-727 phosphorylation of STAT3 by IL-6 and its potential role in mouse 3T3-L1 adipocytes by using specific inhibitors for the kinase pathways presented above [18][19][20][21][22]. We concluded that PKCδ was not only important for Ser-727 phosphorylation, but also for the Tyr-705 phosphorylation, nuclear translocation and transcriptional properties of STAT3.…”

“…In HepG2 cells, Schuringa et al [18] found no inhibition of IL-6-induced STAT3 Tyr-705 phosphorylation while Xu et al [33] found that pretreatment with rottlerin totally blocked IL-13-induced STAT3-DNA binding in monocytes. Another report showed that prolactin-or rat placental lactogen 1-induced Tyr-705 phosphorylation of STAT3 is reduced in response to rottlerin in granulosa cells and, thus, also STAT3-DNA binding [34].…”

“…There are very few reports describing an IL-6-induced phosphorylation of PKCδ. Schuringa et al [18] showed in HepG2 cells that PKCδ Thr-505 is already phosphorylated in the nucleus 2 min after IL-6 addition. However, we were not able to confirm these results in differentiated 3T3-L1 cells.…”

“…The additional Ser-727 phosphorylation has been reported to increase the transcriptional activation of STAT3 [17][18][19][20][21]. Depending on cell origin, many different kinases have been shown to increase Ser-727 phosphorylation of STAT3, such as protein kinase C-δ (PKCδ), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and the mammalian target of rapamycin (mTOR) [18][19][20][21][22].…”