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

Activation of signal transducer and activator of transcription 3 (STAT3) by interleukin-6 (IL-6) involves phosphorylation of Tyr-705 and Ser-727, both of which are critical for STAT3 transactivation. Here, we demonstrate that IL-6 activates Rac-1 and SEK-1/MKK-4 of the stress-activated protein kinase pathway, as well as protein kinase Cδ (PKCδ), as indicated by PKCδ Thr-505 phosphorylation. However, JNK-1, the end point kinase of the stress-activated protein kinase pathway signal transduction cascade, is not activated by IL-6. PKCδ was found to be associated with SEK-1/MKK-4 in unstimulated HepG2 cells but rapidly dissociates from SEK-1/MKK-4 upon IL-6 stimulation to become associated with STAT3. Inhibition of PKCδ using rottlerin (6 μM) or by overexpression of dominant negative PKCδ demonstrates that PKCδ kinase activity is required for STAT3 Ser-727 phosphorylation and transactivation but not for STAT3 Tyr-705 phosphorylation or nuclear import. PKCδ signals downstream of Rac-1 and SEK-1/MKK-4, because enhanced STAT3 transactivation induced by overexpression of constitutive active RacV12 was strongly abrogated by rottlerin, whereas IL-6-induced SEK-1/MKK-4 Thr-223 phosphorylation was not affected under these conditions. Studying the kinetics of STAT3 and PKCδ phosphorylation in cytoplasmic and nuclear fractions revealed that STAT3 Tyr-705 phosphorylation and nuclear translocation precedes PKCδ Thr-505 and STAT3 Ser-727 phosphorylation. Furthermore, the IL-6-induced PKCδ Thr-505 and STAT3 Ser-727 phosphorylation were only observed in nuclear fractions of HepG2 cells. These results demonstrate that IL-6-induced STAT3 transactivation involves the sequential activation of Rac-1 and SEK-1/MKK-4, which leads to nuclear translocation of PKCδ by release from a SEK-1/MKK-4-containing complex. Our results further indicate that PKCδ-mediated STAT3 Ser-727 phosphorylation is mainly a nuclear event.