Real Time Analysis of STAT3 Nucleocytoplasmic Shuttling

Abstract

The Janus kinase/STAT 1 pathway plays a major role in cytokine and growth factor signaling. In particular, the family members of the ␣-helix bundle cytokines comprising the hematopoietins and interferons exert their biological effects by the activation of STAT transcription factors (for review, see Ref. 1). According to the canonical model (2), signaling through the Janus kinase/STAT pathway is triggered by the binding of a cytokine to class I or class II cytokine receptors which leads to the activation of receptor-associated tyrosine kinases of the Janus kinase family. The activated Janus kinases phosphorylate the receptor on tyrosine residues followed by the recruitment of STAT monomers to these phosphorylated tyrosine motifs. While bound to the receptor, STATs are phosphorylated at a single tyrosine residue and subsequently form dimers by intermolecular phosphotyrosine-SH2 domain interactions. The STAT dimers freely diffuse through the cytosol (3), associate with importin-␣ (4), and translocate via the nuclear pore complexes to the nuclear compartment to transactivate target genes.This canonical model has been challenged by accumulating data suggesting that STATs dimerize prior to activation. The preformation of STAT dimers seems to be independent of tyrosine phosphorylation (5-10). Furthermore, recent observations from several laboratories suggest that a subpopulation of STAT proteins is located in the nucleus of unstimulated cells (8, 10 -15). The latter finding is either the result of a static subcellular distribution or a continuous dynamic shuttling of STAT molecules between the cytosol and the nucleus.STAT3 is one of the seven mammalian STAT proteins. It acts as a signal transducer for many cytokines and growth factors (16) and is of particular importance for the family of IL-6-type cytokines (17). STAT3 participates in a variety of biological processes such as the induction of acute phase protein synthesis in hepatocytes (18), the regulation of hematopoiesis and the immune response (16,19), embryo implantation (20, 21), and development (22, 23). As a consequence of these diverse functions, STAT3 plays a crucial role in inflammatory, autoimmune, and certain neoplastic diseases (24). Mice having a targeted deletion of STAT3 die early during embryonic development (22).STAT proteins consist of six domains: an N-terminal domain involved in cooperative DNA binding, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a C-terminal transactivation domain. The structure of a truncated, tyrosine-phosphorylated, dimeric STAT3 bound to DNA has been solved by x-ray crystallography (25). The dimerization of STAT3 molecules is enforced by reciprocal binding of the phosphotyrosine-containing regions to the SH2 domains, which is a prerequisite for nuclear accumulation and DNA binding of STAT3. The activity of the C-terminal transactivation domain is modulated by phosphorylation at serine 727 (26).The major part of the data concerning the nucleocytoplasmic transport mechanisms of STAT protein...

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“…10A) (33)(34)(35). STAT3 then activates the transcription of neuroprotective genes (e.g., bcl-2, bcl-xl, IAP2, Pim-1, Reg), neurodegenerative genes (e.g., GAP-43), and neurodevelopmental genes (e.g., GFPAF) (36)(37)(38)(39)(40)(41). In this report the inhibitor of STAT3 blocks its posttranslational modification.…”

Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma

“…Activated STATs shuttle more rapidly than non-activated ones [143,144]. Direct interaction of unphosphorylated STATs with the nuclear pore proteins (nucleoporins) Nup153 and Nup214 allows carrier-independent nuclear translocation.…”

“…STAT activation with nuclear accumulation terminates within minutes [145,146]. Recently, nucleocytoplasmic shuttling of STAT3 was shown by fluorescence localization, after photobleaching (FLAP), to be a dynamic process that involves constitutive shuttling of unstimulated STAT3 in the absence of cytokine stimulation [144]. Both import and export signals contribute to the balanced shuttling and IL-6 induction reduces the nuclear export signal, resulting in nuclear accumulation of STAT3.…”

General Nature of the STAT3-Activated Anti-Inflammatory Response

“…1b) and serine phosphorylation (data not shown) of STAT3 followed by expedient transit to the nucleus (our unpublished observation and Ref. 41). However, measurement of STAT3 activation by immunoblotting, EMSA, or microscopy does not necessarily give a strong correlation with the downstream process of specific gene activation (our unpublished data).…”