Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

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Structural basis of the p53 DNA binding domain and PUMA complex

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2021.02.049 ◽

2021 ◽

Vol 548 ◽

pp. 39-46

Author(s):

Chang Woo Han ◽

Han Na Lee ◽

Mi Suk Jeong ◽

So Young Park ◽

Se Bok Jang

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain

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Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056-6067.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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Role of GCR2 in transcriptional activation of yeast glycolytic genes.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3834 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3834-3842 ◽

Cited By ~ 45

Author(s):

H Uemura ◽

Y Jigami

Keyword(s):

Dna Binding ◽

Fusion Protein ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Gene Products ◽

Lacz Expression ◽

Genetic Method ◽

Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1209-1217.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1209-1217

Author(s):

C F Hardy ◽

D Balderes ◽

D Shore

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transcriptional Activation ◽

Binding Sites ◽

Binding Protein ◽

Binding Domain ◽

Dominant Negative Effect ◽

Dna Binding Domain ◽

Wild Type ◽

Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

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An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation

Molecular Endocrinology ◽

10.1210/mend.12.1.0046 ◽

1998 ◽

Vol 12 (1) ◽

pp. 34-44 ◽

Cited By ~ 3

Author(s):

Ying Liu ◽

Akira Takeshita ◽

Takashi Nagaya ◽

Aria Baniahmad ◽

William W. Chin ◽

...

Keyword(s):

Thyroid Hormone ◽

Dna Binding ◽

Ligand Binding ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Binding Domain ◽

Dna Binding Domain ◽

Chimeric Receptor ◽

Receptor System ◽

Inhibitory Region

Abstract We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor β ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-β (TRβ), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRβ derepressed transcription to basal level. Using this system and a battery of TRβ mutants, we found that TRβ/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRβ ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRβ DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRβ from achieving transcriptional activation above basal level in this chimeric receptor system.

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A position-dependent transcription-activating domain in TFIIIA

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496-7506.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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Functional domains of interferon regulatory factor I (IRF-1)

Biochemical Journal ◽

10.1042/bj3350147 ◽

1998 ◽

Vol 335 (1) ◽

pp. 147-157 ◽

Cited By ~ 67

Author(s):

Fred SCHAPER ◽

Sabine KIRCHHOFF ◽

Guido POSERN ◽

Mario KÖSTER ◽

André OUMARD ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Consensus Sequence ◽

Binding Domain ◽

Functional Domains ◽

Dna Binding Domain ◽

Transcriptional Activation Domain

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitroand was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

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The lack of transcriptional activation of the v-erbA oncogene is in part due to a mutation present in the DNA binding domain of the protein

Nucleic Acids Research ◽

10.1093/nar/18.15.4489 ◽

1990 ◽

Vol 18 (15) ◽

pp. 4489-4497 ◽

Cited By ~ 16

Author(s):

H. d. Verneuil ◽

D. Metzger

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain

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A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3354 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3354-3362 ◽

Cited By ~ 118

Author(s):

M Green ◽

T J Schuetz ◽

E K Sullivan ◽

R E Kingston

Keyword(s):

Heat Shock ◽

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Chimeric Proteins ◽

Dna Binding Domain ◽

Regulatory Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domains

Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.

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