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

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 C delta (PKC delta), as indicated by PKC delta 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, PKC delta 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 PKC delta using rottlerin (6 muM) or by overexpression of dominant negative PKC delta demonstrates that PKC delta kinase activity is required for STAT3 Ser-727 phosphorylation and transactivation but not for STAT3 Tyr-705 phosphorylation or nuclear import. PKC delta 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-B-induced SEK-1/MKK-4 Thr-223 phosphorylation was not affected under these conditions. Studying the kinetics of STAT3 and PKC delta phosphorylation in cytoplasmic and nuclear fractions revealed that STAT3 Tyr-705 phosphorylation and nuclear translocation precedes PKC delta Thr-505 and STAT3 Ser-727 phosphorylation. Furthermore, the IL-6-induced PKC delta Thr-505 and STATE 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 PKC delta by release from a SEK-1/MKK-4-containing complex. Our results further indicate that PKC delta -mediated STAT3 Ser-727 phosphorylation is mainly a nuclear event.