A Novel Mechanism of STAT3 Activation by Oncogenic Signaling

CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling. We previously found that UV cross-linking of proteins from HeLa cervical cancer cells resulted in STAT3 interacting with the CARP-1 (614–638) peptide. Mutagenesis and co-IP-WB experiments revealed that CARP-1 interacts with a 40-amino-acid epitope from positions 441–480 (CE Epitope) located in the STAT3 DNA-binding domain. Overexpression of mutant STAT3 with in-frame deletion of the CE epitope (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to the nucleus in IL-6-treated cells. The small GTPase p21Rac1 interacts with and regulates STAT3 activation and nuclear translocation. Here we report the interaction of p21Rac1 with the CE epitope of STAT3 and the CARP-1 (600–650) region, suggesting that CARP-1 is part of a dynamic STAT3-p21Rac1 complex that functions in STAT3 activation and nuclear translocation. Expression of a STAT3 (ΔCE) mutant abolished STAT3 Y705 phosphorylation in cells that were treated with EGF or IL-6. Fine mapping revealed that scrambling the CE epitope peptide or a small peptide from positions 456–465 within the CE epitope resulted in abrogation of STAT3 Y705 phosphorylation by IL-6. Moreover, STAT3 phosphorylation by EGF or IL-6 was diminished in multiple CARP-1 null cancer cells. Importantly, incubation of a TAT-tagged STAT3 (454–467) peptide but not its scrambled version resulted in a reduction in STAT3 Y705 phosphorylation by IL-6/EGF. Taken together, our data demonstrates that the STAT3 CE epitope interacts with CARP-1 and p21Rac1, harbors novel sequences that activate STAT3 and promotes its nuclear translocation by IL-6/EGF.