Consequences of the disease-related L78R mutation for dimerization and activity of STAT3.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is centrally involved in diverse processes including haematopoiesis, immunity and cancer progression. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 dimers are stabilized by intermolecular interactions between SH2 domains and phosphotyrosine. These activated dimers accumulate in the nucleus and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Förster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized by homotypic interactions between the N-terminal domains. A somatic mutation (L78R) found in inflammatory hepatocellular adenoma (IHCA), which is located in the N-terminal domain of STAT3 disturbs latent dimer formation. Applying intramolecular FRET, we verify a functional role of the SH2 domain in latent dimer formation suggesting that the protomers in the latent STAT3 dimer are in a parallel orientation, similar to activated STAT3 dimers but different from the antiparallel orientation of the latent dimers of STAT1 and STAT5. Our findings reveal unique structural characteristics of STAT3 within the STAT family and contribute to the understanding of the L78R mutation found in IHCA.

The role of the N-terminal domain in dimerization and nucleocytoplasmic shuttling of latent STAT3.

STAT3 is an important transcription factor involved in immunity and cancer. In response to cytokine stimulation, STAT3 becomes phosphorylated on a single tyrosine residue. Tyrosine-phosphorylated STAT3 accumulates in the nucleus, binds to specific DNA response elements and induces gene expression. Unphosphorylated, latent STAT3 shuttles constitutively between cytoplasm and nucleus. We analysed the importance of previously identified putative nuclear localization sequences (NLS) and nuclear export sequences (NES) for nucleocytoplasmic shuttling of latent STAT3 using STAT3-deficient cells reconstituted with fluorescently labelled STAT3 mutants. Mutation of a putative NLS or NES sequence did not impair nucleocytoplasmic shuttling of latent STAT3. We were also interested in the structural requirements for dimerization of unphosphorylated STAT3 and its relevance for nucleocytoplasmic shuttling. By native gel electrophoresis and dual-focus fluorescence correlation spectroscopy (2f-FCS) we identified the N-terminal domain (amino acids 1-125) to be essential for formation of unphosphorylated STAT3 dimers but not for assembly of tyrosine-phosphorylated STAT3 dimers. In resting cells, the monomeric N-terminal deletion mutant (STAT3-ΔNT) shuttles faster between the cytoplasm and nucleus than the wild-type STAT3, indicating that dimer formation is not required for nucleocytoplasmic shuttling of latent STAT3. STAT3-ΔNT becomes phosphorylated and dimerizes in response to interleukin-6 stimulation but, surprisingly, does not accumulate in the nucleus. These results highlight the importance of the N-terminal domain in the formation of unphosphorylated STAT3 dimers and nuclear accumulation of STAT3 upon phosphorylation.

Dynamics and non-canonical aspects of JAK/STAT signalling.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway directly links ligand-binding to a membrane-bound receptor with the activation of a transcription factor. This signalling module enables the cell to rapidly initiate a transcriptional response to external stimulation. The main components of this evolutionary conserved module are cytokines that specifically bind to cytokine receptors leading to the activation of receptor-associated Janus tyrosine kinases (JAKs). The receptor-bound JAKs activate STAT transcription factors through phosphorylation of a single tyrosine residue. Activated STAT dimers translocate into the nucleus to induce target gene expression. In this article we will review current opinions on the molecular mechanism and on intracellular dynamics of JAK/STAT signalling with a special focus on the cytokine receptor glycoprotein 130 (gp130) and STAT3. In particular we will concentrate on non-canonical aspects of Jak/STAT signalling including preassembled receptor complexes, preformed STAT dimers, STAT trafficking and non-canonical functions of STATs.