Observation of unphosphorylated STAT3 core protein binding to target <i>ds</i>DNA by PEMSA and X‐ray crystallography

Nkansah, Edwin; Shah, R.; Collie, Gavin W.; Parkinson, Gary N.; Palmer, Jonathan M.; Rahman, Khondaker Miraz; Bui, Tam T.; Drake, Alex F.; Husby, Jarmila; Neidle, Stephen; Zinzalla, Giovanna; Thurston, David E.; Wilderspin, Andrew F.

doi:10.1016/j.febslet.2013.01.065

Cited by 65 publications

(84 citation statements)

References 28 publications

(39 reference statements)

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“…8) phosphorylation (38). Furthermore, recent studies using both atomic force microscopy and X-ray crystallography have directly visualized U-STAT3 binding to GAS elements as an unphosphorylated dimer (37,45 (46). However, the same study also showed that IFN-␥ strongly induces STAT3 Tyr 705 phosphorylation but does not elicit STAT3 binding to the EGR1 promoter, consistent with our results for IL-6 ( Fig.…”

Section: Discussionsupporting

confidence: 91%

Signal Integration and Gene Induction by a Functionally Distinct STAT3 Phosphoform

Waitkus

Chandrasekharan

Willard

et al. 2014

Molecular and Cellular Biology

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Aberrant activation of the ubiquitous transcription factor STAT3 is a major driver of solid tumor progression and pathological angiogenesis. STAT3 activity is regulated by numerous posttranslational modifications (PTMs), including Tyr 705 phosphorylation, which is widely used as an indicator of canonical STAT3 function. Here, we report a noncanonical mechanism of STAT3 activation that occurs independently of Tyr 705 phosphorylation. Using quantitative liquid chromatography-tandem mass spectrometry, we have discovered and characterized a novel STAT3 phosphoform that is simultaneously phosphorylated at Thr

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Section: Discussionsupporting

confidence: 91%

Signal Integration and Gene Induction by a Functionally Distinct STAT3 Phosphoform

Waitkus

Chandrasekharan

Willard

et al. 2014

Molecular and Cellular Biology

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“…This is in contrast to the antiparallel dimers of latent STAT1 and STAT5 where the SH2 domains are localized at the opposite ends of the dimer (Mao et al, 2005;Mertens et al, 2006;Neculai et al, 2005). This parallel association could explain why, under some circumstances, latent STAT3 is capable of DNA binding (Nkansah et al, 2013;Timofeeva et al, 2012) and gene induction (Yang et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

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

Domoszlai

Martincuks

Fahrenkamp

et al. 2014

Journal of Cell Science

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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 Fö rster 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.

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“…However, this form of STAT6 CF neither bound DNA nor did it crystallize after extensive screening. These features of STAT6 are unlike STAT1 and STAT3, which are known to bind DNA regardless of phosphorylation (11,16). Because the unphosphorylated STAT6 CF did not bind DNA, we produced phosphorylated STAT6 CF as described previously (17,18) (Fig.…”

Section: Phosphorylated Stat6mentioning

confidence: 99%

“…S4K). We aligned the DBD of our STAT6 CF -N3 complex structure with the other three reported STAT protein structures in complex with N3 site DNA, i.e., STAT1 (PDB ID code 1BF5) and STAT3 (PDB ID codes 1BG1 and 4E68) (9)(10)(11). Although the proteins exhibit some flexibility, the position of the key residue, H415 in STAT6, N460 in STAT1, and N466 in STAT3, in both the protomers within the dimer of these STATs overlaps (Fig.…”

Section: Phosphorylated Stat6mentioning

confidence: 99%

“…Interestingly, STAT5 has been shown to bind N4 site DNA weakly (8). Thus far, only homodimeric structures of STAT1 and STAT3 in complex with N3 site DNA [phosphorylated STAT1 and STAT3, protein data bank (PDB) ID codes 1BF5 and 1BGl, respectively; unphosphorylated STAT3, PDB ID code 4E68] have been solved (9)(10)(11). In addition, structures of unphosphorylated monomeric STAT1 (PDB ID code 1YVL) and STAT3 (PDB ID code 3CWG), as well as unphosphorylated dimeric STAT5a (PDB ID code 1Y1U) in protein-alone form, have been published (12)(13)(14).…”

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confidence: 99%

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Structural basis for DNA recognition by STAT6

Rodriguez

Niu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6 CF ) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNAbinding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6 CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.STAT6 | N4 site DNA recognition | JAK-STAT pathway | antiviral innate immunity | crystal structure P roteins belonging to the STAT family mediate transmission of signals of numerous cytokines and growth factors from the cell membrane to the nucleus via the classical JAK-STAT pathway (1). Malfunctions in this pathway are known to result in immune system disorder and cancers. Therefore, the JAK-STAT pathway is considered to be of great importance in the development of therapeutic interventions (2). The mammalian STAT family is made up of seven structurally and functionally related proteins named STAT1, 2, 3, 4, 5a, 5b, and 6 (3). All of the STAT proteins share a conserved domain organization (Fig. 1A).STAT6, an important member of the STAT family, plays a crucial role in the differentiation of Th2 cells and has been implicated in the development of asthma (4). This STAT is primarily stimulated by IL-4 and IL-13. A recent study reported that STAT6 plays a pivotal role in antiviral signaling initiated by host cells in response to viral infections (5). STAT6 could be activated by the stimulator of interferon genes/TBK1 cascade via phosphorylation of Y641. Intriguingly, residue S407 located in the DNA-binding domain (DBD) of STAT6 has been shown to be phosphorylated by TBK1. However, its implication for biological function of STAT6 is currently unknown (5). Thus, structural studies on STAT6 and its complex with DNA are essential to address several unanswered q...

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Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X‐ray crystallography

Cited by 65 publications

References 28 publications

Signal Integration and Gene Induction by a Functionally Distinct STAT3 Phosphoform

Signal Integration and Gene Induction by a Functionally Distinct STAT3 Phosphoform

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

Structural basis for DNA recognition by STAT6

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