The Association and Nuclear Translocation of the PIAS3-STAT3 Complex Is Ligand and Time Dependent

Abstract

The epidermal growth factor (EGF) receptor activation of downstream signal transducers and activators of transcription 3 (STAT3) plays a crucial role in the pathogenesis of lung cancer. STAT3 transcriptional activity can be negatively regulated by protein inhibitor of activated STAT3 (PIAS3). We investigated the time-dependent PIAS3 shuffling and binding to STAT3 in an EGF-dependent model in lung cancer by using confocal microscopy, immunoprecipitation, luciferase reporter assay, and protein analysis of segregated cellular components. We also explored the role of phosphorylation at Tyr705 of STAT3 in the formation and intracellular shuffling of the PIAS3-STAT3 complex. In a growth factor-free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. On exposure to EGF, we observed STAT3 phosphorylation and rapid formation of the PIAS3-STAT3 complex. Within 5 minutes, there was a progressive translocation of the complex to the nucleus, and by 10 minutes, PIAS3 was uniquely localized to the nuclear compartment. After 30 minutes, PIAS3 returned to the cytoplasm. Using site-directed mutagenesis, we substituted Tyr705 of STAT3 with a phenylalanine. Despite EGF stimulation, we observed a significant decrease in PIAS3-STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in the capacity of PIAS3 to reduce STAT3-mediated gene transcription. In wild-type STAT3 cells, increasing concentrations of PIAS3 resulted in a proportional decrease in STAT3 phosphorylation. These data suggest an important role for the negative regulatory effect of PIAS3

Identification of Novel miR-21 Target Proteins in Multiple Myeloma Cells by Quantitative Proteomics

“…55 Recently, Dabir et al demonstrated that increasing concentrations of PIAS3 result in a proportional decrease in STAT3 phosphorylation and transcription activity. 44 Their results suggest that PIAS3 may inhibit STAT3 by accelerating the dephosphorylation process. 44 Consistent with these reports, our results show that PIAS3 overexpression results in decreased STAT3 phosphorylation and transcription activity ( Figure 5), suggesting that PIAS3 may play a role in the dephosphorylation process of STAT3 in myeloma cells.…”

“…44 Their results suggest that PIAS3 may inhibit STAT3 by accelerating the dephosphorylation process. 44 Consistent with these reports, our results show that PIAS3 overexpression results in decreased STAT3 phosphorylation and transcription activity ( Figure 5), suggesting that PIAS3 may play a role in the dephosphorylation process of STAT3 in myeloma cells. However, the precise role of PIAS3 in the regulation of STAT3 phosphorylation and transcription activity in MM cells remains unclear.…”

Low PIAS3 Expression in Malignant Mesothelioma Is Associated with Increased STAT3 Activation and Poor Patient Survival

“…PIAS3 was initially found to interact specifically with phosphorylated STAT3, and not STAT1, in IL6-activated murine myeloblast M1 cells and to decrease STAT3 DNAbinding capacity and transcriptional activity (9). We (10,11) and others (12) have demonstrated that overexpression of PIAS3 can inhibit STAT3 transcriptional activity and promote growth inhibition in vitro. If PIAS3 counteracts transactivation by STAT3, it would be conceivable that PIAS3 expression could be decreased in malignant tissues with high STAT3 activation.…”

“…Cell growth and viability were assessed in manually counted cells by either Trypan blue dye exclusion or the MTS assay, as described previously (10).…”

“…3A), a representative NSCLC cell line (10,11,14,15). Because PIAS3 is thought to be an endogenous inhibitor of STAT3 activity, we examined whether any decreased PIAS3 expression in mesothelioma cells was accompanied by increased p-STAT3 expression.…”