GC Box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase

Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Cell (Impact Factor: 32.24). 11/1990; 63(1):155-65. DOI: 10.1016/0092-8674(90)90296-Q
Source: PubMed


Efficient transcription of SV40 early genes requires transcription factor Sp1. Here, we report that SV40 infection induces Sp1 phosphorylation. While characterizing this modification, we discovered that Sp1 becomes quantitatively phosphorylated in an in vitro transcription extract. Multiple processive phosphorylation of Sp1 depends on binding of Sp1 to GC box-containing DNA. Cell fractionation and column chromatography reveal that the Sp1 kinase is a nuclear DNA binding protein that corresponds to a previously identified DNA-dependent protein kinase. Because only some trans-activators are phosphorylated by this kinase, Sp1 belongs to a specific subgroup of factors that are phosphorylated upon binding to promoter sequences. Finally, efficient phosphorylation of Sp1 requires both a functional DNA binding domain and a region containing the transcriptional activation domains. Coupling of phosphorylation to DNA binding may represent a novel mechanism for regulating transcriptional initiation.

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    • "Studies further identified DNA-PKcs as a modulator of cancer-associated pathways distinct from DNA repair, including hypoxia, metabolism, inflammatory response, and transcriptional regulation (Goodwin and Knudsen, 2014). Notably, DNA-PKcs was originally discovered and characterized as part of SP1 transcriptional complexes (Jackson et al., 1990) and as a regulatory component of transcriptionally poised RNA polymerase II (RNAPII) (Dvir et al., 1992); accordingly, recent studies revealed that DNA-PKcs is recruited to active sites of transcription (Ju et al., 2006). DNA-PKcs can interact with the basal transcriptional machinery (Maldonado et al., 1996) and both binds and modulates the function of multiple sequence-specific transcription factors (e.g., AIRE, p53, and ERG) as well as select nuclear receptors (including the glucocorticoid, progesterone, estrogen [ER], and androgen receptors [AR]) (Goodwin and Knudsen, 2014). "
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    ABSTRACT: Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Jul 2015 · Cancer cell
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    • "There is growing evidence that the Spfamily plays an important role in proliferation and differentiation and participates in the regulation of genes that are both ubiquitously expressed, as well as those expressed in a tissue specific manner [11] [12]. Sp1 can be phosphorylated, a modification that affects its binding to the DNA [13] [14] [15] and Oglycosilated [16], which confers resistance to proteosome dependent degradation [17]. The GC-rich boxes bound by Sp1 are also recognized by the Sp3 transcription factor, competing for DNA binding. "

    Full-text · Dataset · Sep 2014
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    • "It remains possible that other histone modifications or proteins localized to breaks before fixation are responsible for the observed in situ localized DNA-PK activity. Alternatively, it is possible that in situ reconstitution of γH2AX foci by DNA-PK result from local allosteric activation of DNA-PK via binding to DNA termini (34–36). We reasoned that if local kinase activation by DNA DSBs were responsible for γH2AX foci reconstitution, then introduction of new DSBs into the genome should alter γH2AX in situ phosphorylation patterns. "
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    ABSTRACT: Rapid phosphorylation of histone variant H2AX proximal to DNA breaks is an initiating event and a hallmark of eukaryotic DNA damage responses. Three mammalian kinases are known to phosphorylate H2AX in response to DNA damage. However, the mechanism(s) for damage-localized phosphorylation remains incompletely understood. The DNA-dependent protein kinase (DNA-PK) is the most abundant H2AX-modifying kinases and uniquely activated by binding DNA termini. Here, we have developed a novel approach to examine enzyme activity and substrate properties by executing biochemical assays on intact cellular structures. We apply this approach to examine the mechanisms of localized protein modification in chromatin within fixed cells. DNA-PK retains substrate specificity and independently generates break-localized γH2AX foci in chromatin. In situ DNA-PK activity recapitulates localization and intensity of in vivo H2AX phosphorylation and requires no active cellular processes. Nuclease treatments or addition of exogenous DNA resulted in genome-wide H2AX phosphorylation, showing that DNA termini dictated the locality of H2AX phosphorylation in situ. DNA-PK also reconstituted focal phosphorylation of structural maintenance of chromatin protein 1, but not activating transcription factor 2. Allosteric regulation of DNA-PK by DNA termini protruding from chromatin constitutes an autonomous mechanism for break-localized protein phosphorylation that generates sub-nuclear foci. We discuss generalized implications of this mechanism in localizing mammalian DNA damage responses.
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