Sequential activation of Rac-1, SEK-1/MKK-4, and protein kinase C delta isrequired 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 kinas e pathway, as well as protein kinase C delta (PKC delta), as indicated by P KC delta Thr-505 phosphorylation, However, JNK-1, the end point kinase of t he 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 upo n IL-6 stimulation to become associated with STAT3, Inhibition of PKC delta using rottlerin (6 muM) or by overexpression of dominant negative PKC delt a demonstrates that PKC delta kinase activity is required for STAT3 Ser-727 phosphorylation and transactivation but not for STAT3 Tyr-705 phosphorylat ion or nuclear import. PKC delta signals downstream of Rac-1 and SEK-1/MKK- 4, because enhanced STAT3 transactivation induced by overexpression of cons titutive active RacV12 was strongly abrogated by rottlerin, whereas IL-B-in duced SEK-1/MKK-4 Thr-223 phosphorylation was not affected under these cond itions. Studying the kinetics of STAT3 and PKC delta phosphorylation in cyt oplasmic and nuclear fractions revealed that STAT3 Tyr-705 phosphorylation and nuclear translocation precedes PKC delta Thr-505 and STAT3 Ser-727 phos phorylation. 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 comple x. Our results further indicate that PKC delta -mediated STAT3 Ser-727 phos phorylation is mainly a nuclear event.