Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540223
Title: In vitro studies of STAT3 dimerisation and DNA binding
Author: Nkansah, Edwin
ISNI:       0000 0004 2708 8735
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2011
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Abstract:
STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor that homodimerises and binds to the response element of a number of genes that promote tumourigenesis when over-expressed. Tumourigenesis, in many cases, correlates with an aberrant signalling cascade that leads to unregulated activation of STAT3. Activation is widely understood to be a result of phosphorylation at tyrosine 705 which leads to dimerisation and DNA binding. The dimerisation and DNA binding of STAT3βtc in an isolated cell-free system was investigated in order to understand the conditions which modulate these interactions, and to facilitate the discovery of small molecules capable of interfering with these interactions. A GFP-STAT3βtc construct was designed 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. Phosphorylated and unphosphorylated GFP STAT3βtc was purified from BL21 (DE3) TKB1 and Rosetta strains of E. coli respectively. The isoforms were structurally characterized by size exclusion chromatography as dimers when phosphorylated (~ 250 kDa) and as monomers when unphosphorylated (~ 100 kDa). Using both conventional ELISA and fluorescence-based microtiter plate assays, it was demonstrated that both phosphorylated and unphosphorylated GFP-STAT3βtc can bind to immobilised dsDNA (M67; a modified c-fos sis inducible enhancer) or to an immobilised surrogate receptor (a phosphotyrosyl peptide derived from the interleukin- 6 receptor subunit gp130), respectively. A DNA binding assay (PEMSA) was developed for the STAT3 transcription factor based on detection of a protein mobility shift rather than a dsDNA mobility shift, and which utilizes fluorescently tagged STATs. Using PEMSA, DNA binding was demonstrated for both phosphorylated and unphosphorylated GFP-STAT3βtc isoforms. Disruption of a pre-formed unphosphorylated GFP-uSTAT3βtc/dsM67 DNA complex was achieved with a phosphotyrosyl peptide at a DB50 of 30 μM. In contrast, 1000 μM peptide appeared to have no effect on phosphorylated GFP-pSTAT3βtc bound to the dsM67 DNA. FRET was used to directly study dimerisation using the eCFP-uSTAT3βtc/eYFP- uSTATSβtc pair in vitro. Low pH/ low salt buffer as well as co-factors including dsM67 DNA promoted and stabilised STAT3/STAT3 assembly. FRET was also observed between eCFP-uSTAT3βtc and a 5'-FAM labelled phosphotyrosyl peptide which could also form the basis of a high throughput screening assay.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.540223  DOI: Not available
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