Sequential activation of Rac-1, SEK-1/MKK-4, and protein kinase Cdelta 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 Cdelta (PKCdelta), as indicated by PKCdelta 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. PKCdelta 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 PKCdelta using rottlerin (6 microm) or by overexpression of dominant negative PKCdelta demonstrates that PKCdelta kinase activity is required for STAT3 Ser-727 phosphorylation and transactivation but not for STAT3 Tyr-705 phosphorylation or nuclear import. PKCdelta 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 PKCdelta phosphorylation in cytoplasmic and nuclear fractions revealed that STAT3 Tyr-705 phosphorylation and nuclear translocation precedes PKCdelta Thr-505 and STAT3 Ser-727 phosphorylation. Furthermore, the IL-6-induced PKCdelta 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 PKCdelta by release from a SEK-1/MKK-4-containing complex. Our results further indicate that PKCdelta-mediated STAT3 Ser-727 phosphorylation is mainly a nuclear event.
Schuringa
J J
JJ
Department of Genetics, Biological Center, Kerklaan 30, 9751 NN Haren, The Netherlands.
Dekker
L V
LV
Vellenga
E
E
Kruijer
W
W
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism*; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D050796:STAT3 Transcription Factor; D012694:Serine / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D007527:Isoenzymes; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D012694:Serine; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D020830:rac1 GTP-Binding Protein