The anti-parallel dimer binding interface in STAT3 transcription factor is required for the inactivation of cytokine-mediated signal transduction.

Signal transducer and activator of transcription 3 (STAT3) gain-of-function mutations have been widely reported in patients with tumors and haematological malignancies. However, the molecular mechanisms of these pathogenic mutations remain largely uninvestigated. In this study, we have extensively characterized two STAT3 missense mutations, namely a valine-to-alanine exchange in the amino-terminal region (V77A) and a phenylalanine-to-alanine substitution (F174A) in the coiled-coil domain. The two mutants displayed elevated levels of tyrosine phosphorylation, premature nuclear accumulation, and differential transcriptional responses following stimulation of cells with interleukin-6 and interferon-ɣ. In line with their hyper-phosphorylated status, a greater fraction of V77A and F174A proteins was bound to DNA on high-affinity binding sites termed sis-inducible elements (SIE) as compared to the wild-type (WT) protein. Unexpectedly, these STAT3 variants displayed similar kinetics using in vitro kinase and dephosphorylation assays performed with recombinant Janus kinase 2 (JAK2) and Tc45 phosphatase, respectively. This indicates that the two mutations neither affected the susceptibility of STAT3 to the enzymatic activity of the inactivating tyrosine phosphatase nor to the activating kinase. However, experiments triggering intracellular dephosphorylation by the addition of the tyrosine-kinase inhibitor staurosporine to cytokine-pretreated cells showed that the two mutants partially resisted dephosphorylation. From these data, we propose that the F174A missense mutation hinders the exchange from a parallel to an anti-parallel dimer conformation, thereby increasing the ratio of tyrosine-phosphorylated molecules bound to DNA and enhancing gene-dependent transcription. Our data point to the physiological importance of the anti-parallel dimer conformation in the inactivation of the cytokine-induced STAT3 signalling pathway. • The STAT3 F174A mutation hinders the exchange to an anti-parallel dimer conformation. • The ratio of phosphorylated STAT3 bound to DNA is increased in the F174A mutant. • The anti-parallel dimer is involved in the inactivation of STAT3 signalling. • There is a physiological relevance of the anti-parallel STAT3 dimer. [ABSTRACT FROM AUTHOR]

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