Generation and characterization of a constitutively active Stat3 protein.

Stats are latent transcription factors involved in normal cellular signaling in response to cytokine or growth factor stimulation. Constitutive activation of Stats (primarily Stat3 and Stat5) has been implicated in growth dysregulation and oncogenesis. Furthermore, increased activation of Stats has been observed in several human tumors and tumor-derived cell lines. To assess the contribution of aberrant Stat activation in oncogenesis, we have created a chimeric molecule between Stat3beta and a portion of the Herpes simplex virus VP16 activation domain. The resulting protein, Stat3beta-VAD (VP16 activation domain), is tyrosine phosphorylated on Y705 and can bind DNA in the absence of upstream activation by c-Src or epidermal growth factor (EGF). Unlike Stat3alpha and Stat3beta, Stat3beta-VAD robustly activates transcription of several reporter genes without cytokine or growth factor stimulation. In addition, we show marked upregulation of the endogenous c-myc and c-fos genes upon inducible expression of Stat3beta-VAD in COS-7 cells. Our protein displays the constitutive transcriptional activation of Stat3alpha seen in human tumors and will be a valuable tool in screens for Stat3-regulated genes. In response to the established Stat3 involvement in human cancers, Stat3beta-VAD will also facilitate assessing the contribution of other cancer signaling cascades in the context of aberrant Stat3alpha activity in cancer development and progression.