Phosphorylation of the Gal4 DNA-binding domain is essential for activator mono-ubiquitylation and efficient promoter occupancy

Ferdous, Anwarul; O'Neal, Melissa; Nalley, Kip; Sikder, Devanjan; Kodadek, Thomas; Johnston, Stephen Albert

doi:10.1039/b809291e

Cited by 8 publications

(9 citation statements)

References 55 publications

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“…3b). Our data corroborate a previous report demonstrating that serine 22 is phosphorylated in vitro and that this phosphorylation event is important for subsequent monoubiquitination of Gal4 and formation of a stable Gal4-DNA complex (Ferdous et al, 2008). 1.…”

Section: Resultssupporting

confidence: 92%

“…Phosphorylation of Ser22 in the Gal4 DNA-binding domain has been demonstrated in vitro, and this modification has been shown to be essential for Gal4 monoubiquitylation. Ubiquitylation at lysine residue K23 in the DNA-binding domain is required to inhibit proteasomal destabilization of the activator-DNA complex (Ferdous et al, 2008). Interestingly, this study showed that phosphorylation of Ser22 does not require the Gal4 activation domain, while ubiquitylation requires both a DNA template and the activation domain.…”

Section: Introductionmentioning

confidence: 74%

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Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin vivo

et al. 2013

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The yeast transcriptional activator Gal4 has long been the prototype for studies of eukaryotic transcription. Gal4 is phosphorylated in the DNA-binding domain (DBD); however, the molecular details and functional significance of this remain unknown. We mutagenized seven potential phosphoserines that lie on the solvent-exposed face of the DBD structure and assessed them for transcriptional activity and DNA binding in vivo. Serine to alanine mutants at positions 22, 47, and 85 show the greatest reduction in promoter occupancy and transcriptional activity at the MEL1 promoter containing a single UASGAL . Substitutions with the phosphomimetic aspartate restored DNA-binding and transcriptional activity at serines 22 and 85, suggesting that they are potential sites of Gal4 phosphorylation in vivo. In contrast, the serine to alanine mutants, except serine 22, were fully proficient for binding to the GAL1-10 promoter, containing multiple UASGAL sites, although they had a reduced ability to activate transcription. Collectively, these data show that at the GAL1-10 promoter, functions of the DBD in transcriptional activation can be uncoupled from roles in promoter binding. We suggest that the serines in the DBD mediate protein-protein contacts with the transcription machinery, leading to stabilization of Gal4 at promoters.

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Section: Resultssupporting

confidence: 92%

Section: Introductionmentioning

confidence: 74%

Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin vivo

et al. 2013

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“…When the Gal4-VP16⅐DNA complex was incubated with HeLa nuclear extract, an essentially quantitative monoubiquitylation of the activator was observed (32). Moreover, this event was found to be dependent on a preceding phosphorylation event at Ser 22 , located in the same domain (34). Remarkably, a DNA complex containing phosphorylated and monoubiquitylated Gal4-VP16 was completely resistant to disruption by the proteasomal ATPases (32).…”

Section: How Does Monoubiquitylation Stimulate Activated Gene Expressmentioning

confidence: 87%

No Splicing, No Dicing: Non-proteolytic Roles of the Ubiquitin-Proteasome System in Transcription

Kodadek

2010

Journal of Biological Chemistry

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The ubiquitin-proteasome pathway (UPP) is responsible for most programmed turnover of proteins in eukaryotic cells, and this activity has been known for some time to be involved in transcriptional regulation. More recently, intersections of the UPP and transcription have been discovered that are not proteolytic in nature and appear to revolve around the chaperoninlike activities of the ATPases in the 19 S regulatory subunit of the proteasome. Moreover, monoubiquitylation, which does not signal degradation, has been found to be a key modification of many transcription factors and histones. These various nonproteolytic roles of the UPP in transcription are reviewed here, and plausible mechanistic models are discussed.Most non-lysosomal protein degradation occurs via the UPP 2 in eukaryotic cells (1). In most cases, the protein targeted for degradation is modified with a Lys 48 -linked poly-Ub chain, a modification catalyzed by ULs, of which there are three types. E1 and E2 ULs are involved in activating the ubiquitin molecule for conjugation, whereas E3 ULs act as matchmakers between the activated Ub-E2 intermediate and substrate proteins. Polyubiquitylated proteins interact with the proteasome, a large multiprotein complex with three proteolytic active sites sequestered inside a barrel-like core 20 S complex (2). The opening to the interior of the barrel is so small that the substrate protein must be unfolded and "fed" through by the action of six AAA class ATPases, now called Rpt1-6. These are part of the 19 S RP that sits immediately atop the 20 S CP. The 19 S RP itself can be dissociated by high salt buffers into a base, which includes the six ATPases, Rpn1, Rpn2, and a "lid" subcomplex (3). All six of the genes that encode these proteins are essential in yeast, indicating that they have non-identical functions. The classical proteolytic activity of the UPP has long been known to regulate transcription indirectly, e.g. by keeping the levels of gene-specific transactivators low under basal conditions. More recently, a more intimate relationship between UPP-mediated turnover of activators, coactivators and other transcription actors has been found to be highly stimulatory for the expression of some genes (4 -6). However, this minireview focuses on a rather different intersection of the UPP and eukaryotic transcription, which involves non-proteolytic activities of UPP proteins.The first hint of such a radical idea was the finding by Johnston and co-workers in 1992 (7) that specific alleles of the Saccharomyces cerevisiae SUG1 and SUG2 genes (sug1-1 and sug2-1), which encode Rpt6 and Rpt4, respectively, suppress the "no growth on galactose" phenotype of gal4D. gal4D encodes a truncated version of the yeast Gal4 activator (8) that lacks about two-thirds of the C-terminal AD (Fig. 1) (7). At the time of this study, it was thought that Sug1 and Sug2 were coactivators whose interaction with Gal4 was weakened by the truncation in Gal4D and reconstituted by a point mutation. When it was discovered soon thereafter that S...

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“…For example, phosphorylation within the basic region can be either a positive (e.g. Gal4) (43) or negative (PRH/Hex) (31) influence. It has also been proposed that dimerization of STAT1 is regulated by phosphorylation (32,33), although others suggest that STATs exist as inactive dimers in the absence of phosphorylation (44,45).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation within the MafA N Terminus Regulates C-terminal Dimerization and DNA Binding

Guo

Vanderford

Stein

2010

Journal of Biological Chemistry

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Phosphorylation influences the activity of many transcription factors. However, the precise manner by which this posttranslational modification impacts regulation has been defined mechanistically in only a few cases (1, 2). Here, we have focused on understanding how phosphorylation affects the DNA-binding potential of MafA and MafB, whose basic-leucine zipper (b-Zip) 2 region defines their dimerization and DNA-binding properties. There are two subfamilies of mammalian musculoaponeurotic fibrosarcoma (i.e. Maf) proteins, termed large and small Mafs (3). The small Maf proteins (MafF, MafG, MafK, and MafT) lack a transactivation domain and affect transcription through dimerization with related and distinct proteins (4 -7). The large Maf proteins (MafA, MafB, c-Maf, and NRL) contain an N-terminal transactivation domain (8 -11), which has considerable identity among MafA, MafB, and c-Maf (12, 13).Large Mafs are required in promoting many distinct physiological processes by binding as dimers to Maf-responsive elements and activating transcription (14, 15). Among other properties, chicken L-Maf (termed MafA in mammals) is involved in lens development (16), mammalian MafB is required for segmentation of the hindbrain (17), mammalian c-Maf contributes to chondrocyte differentiation (9, 18), and mammalian NRL functions in eye rod formation (10). Moreover, MafA and MafB have recently been shown to be essential within the mammalian pancreas, with islet ␣ and ␤ cell production requiring the actions of MafB during development and adult ␤ islet activity uniquely MafA (19 -21). In addition, large Maf proteins mediate cellular transformation in vitro and are overexpressed in human angioimmunoblastic T cell lymphomas and multiple myeloma and contribute directly to cancer progression (22-24).The activity of MafA is regulated by a variety of post-translational modification mechanisms, including phosphorylation, ubiquitination, and sumoylation (25-29). The best studied MafA modification is phosphorylation, which impacts protein stability (26 -28), transactivation (26, 29), and DNA binding (30). For example, a priming phosphorylation at serine 65 in MafA (or Ser 70 in MafB) is necessary for both ubiquitin-mediated degradation (26) and glycogen synthase kinase 3-mediated phosphorylation (27,28), the latter enhancing transactivation and transformation potential (29). In addition, the in vitro DNA-binding capabilities of MafA are reduced by endogenous and exogenous phosphatases (30). Inhibition could entail phosphorylation directly within the basic region of MafA, as found for a variety of different transcription factors, including c-Myb (31), PRH/Hex (31), and HNF4 (31). Alternatively, this modification might influence MafA dimer formation and, as a result, DNA-binding potential. Such a mechanism has been described for STAT1, wherein tyrosine phosphorylation of cytoplasmic STAT1 potentiates dimerization and transcriptional activation (32,33).Large Maf proteins appear to be heavily phosphorylated (27,28). Here, we first used mass spectr...

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Phosphorylation of the Gal4 DNA-binding domain is essential for activator mono-ubiquitylation and efficient promoter occupancy

Cited by 8 publications

References 55 publications

Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin vivo

Solvent-exposed serines in the Gal4 DNA-binding domain are required for promoter occupancy and transcriptional activationin vivo

No Splicing, No Dicing: Non-proteolytic Roles of the Ubiquitin-Proteasome System in Transcription

Phosphorylation within the MafA N Terminus Regulates C-terminal Dimerization and DNA Binding

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