Abstract 279: Use of GFP-STAT3βtc for EMSA analysis of protein-protein and protein-DNA interactions in tumorigenic signalling pathways

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL STAT3 (Signal transducer and activator of transcription 3) is a homodimeric transcription factor that binds to the response element of a number of genes that promote tumorigenesis when over-expressed. Tumorigenesis, in many cases, correlates with an aberrant signalling cascade that leads to uncontrolled activation of STAT3. Activation is widely understood to be a result of phosphorylation at tyrosine 705 which leads to dimerisation and DNA binding. We have investigated the dimerisation and DNA binding of STAT3βtc in an isolated cell-free system in order to understand the conditions which modulate these interactions, and to facilitate the discovery of small molecules capable of interfering with these interactions. We designed a GFP-STAT3βtc construct in which the N-terminal 126 amino acids of STAT3 were replaced with spectral variants of the GFP (green fluorescent protein) to act as a reporter. The constructs also lacked the unstructured C-terminal activation domain of STAT3 to avoid aggregation of the purified protein. Phosphorylated and non-phosphorylated GFP-STAT3βtc proteins were purified from BL21(DE3)TKB1 and Rosetta strains of E. Coli, respectively. Soluble expression was observed when tagged with GFP at the N-terminus but not the C-terminus. Using both conventional ELISA and fluorescence-based microtiter plate assays, we demonstrated that both active (phosphorylated) and inactive (non-phosphorylated) fluorescent STAT3βtc can bind to immobilised dsDNA (M67; a modified c-fos sis inducible enhancer) or an immobilised surrogate receptor (a phosphotyrosyl peptide derived from the interleukin-6 receptor subunit gp130), respectively. Furthermore, we were able to demonstrate disruption of a pre-formed non-phosphorylated GFP-STAT3βtc/DNA complex with a phosphotyrosyl peptide at an IC50 of 30 µM. In contrast, 500 µM peptide appeared to have no effect on phosphorylated GFP-STAT3βtc bound to M67. In summary, we have developed an EMSA assay for the STAT3/STAT3 transcription factor based on a protein mobility shift rather than a DNA mobility shift, and which utilizes fluorescent tagged STATs. Both non-phosphorylated and phosphorylated fluorescent STAT3 proteins form a stable complex with M67 DNA, but only the non-phosphorylated STAT3/DNA complex can be disrupted with a phosphotyrosyl peptide in the EMSA assay. This assay is now being utilized for the discovery of novel small molecule inhibitors of the STAT3/STAT3 interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 279. doi:10.1158/1538-7445.AM2011-279