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

Abstract

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.

Interleukin-6: Biology, signaling and strategies of blockade

“…Pre-dimerization of STAT3 does not depend on tyrosine phosphorylation but requires the N-terminal domain of the protein. In clear contrast, IL-6-dependent dimerization requires tyrosine 705 phosphorylation but does not require the N-terminal domain [65]. Also nuclear localization is not simply triggered by tyrosine phosphorylation.…”

“…Instead, also non-phosphorylated STAT3 shuttles continuously between the cytoplasm and the nucleus independently from cytokine stimulation, dimerization and STAT3 tyrosine phosphorylation. However, the N-terminal domain is required for nuclear localization of STAT3 [65]. Nevertheless, the number of tyrosine phosphorylated STAT3 proteins in the nucleus increases in response to IL-6 [66].…”

STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism

“…CBP-acetylated STAT3 translocates into mitochondria where STAT3 associates with PDC E1 and promotes pyruvate oxidation. The N-terminal region of STAT3 is the coiled-coil domain, which is homologous among many structural proteins, enzymatic cofactors, as well as transcription factors535455. The exceptionally tight packing of the coiled-coil domain is favourable for the precise control of enzymatic reactions.…”

The molecular regulation of Janus kinase (JAK) activation

“…Depending on the receptor, and thus the docking sites generated by tyrosine phosphorylation within the cytoplasmic region, any one or more of six STAT family members (STAT1, STAT2, STAT3, STAT4, STAT5 or STAT6) may be recruited via their SH2 domains. STATs exist as preformed dimers [7–9], and by being brought into proximity of the receptor-associated JAK can then be phosphorylated by JAK, leading to a reorientation of subunits within the STAT dimer and translocation into the nucleus where it functions as a transcription factor [10–12]. The resulting transcriptional program dictates whether the cell undergoes proliferation, differentiation, survival or death.…”