ABSTRACT
Sumoylation serves to control key cellular functions, but the regulation of SUMO E3 ligase activity is largely unknown. Here we show that the polycomb group protein Pc2 binds to and colocalizes with homeodomain interacting protein kinase 2 (HIPK2) and serves as a SUMO E3 ligase for this kinase. DNA damage-induced HIPK2 directly phosphorylates Pc2 at multiple sites, which in turn controls Pc2 sumoylation and intranuclear localization. Inducible phosphorylation of Pc2 at threonine 495 is required for its ability to increase HIPK2 sumoylation in response to DNA damage, thereby establishing an autoregulatory feedback loop between a SUMO substrate and its cognate E3 ligase. Sumoylation enhances the ability of HIPK2 to mediate transcriptional repression, thus providing a mechanistic link for DNA damage-induced transcriptional silencing.
Subject(s)
Carrier Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Carrier Proteins/analysis , Carrier Proteins/genetics , Cell Line , DNA Damage , Gene Expression Regulation , Green Fluorescent Proteins/analysis , Humans , Ligases , Phosphorylation , Polycomb-Group Proteins , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/genetics , Recombinant Fusion Proteins/analysis , Repressor Proteins/analysis , Repressor Proteins/genetics , Repressor Proteins/physiology , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Transcription factor NF-kappaB plays a key role for the inducible expression of genes mediating proinflammatory effects and is thus an important target for the development of antiinflammatory drugs. Here, we show that extracts from the plant Plagius flosculosus (L.) Alavi and Heyw. can inhibit the induction of NF-kappaB activity, and we describe the identification of three spiroketal compounds. Of those, only compound 1 could inhibit the phosphorylation and proteasomal degradation of IkappaB, thus preventing the nuclear import and DNA binding of NF-kappaB. Accordingly, compound 1, which is also found in the widely used medicinal herb chamomile, interfered with the LPS-induced production of IL-1, IL-6, TNF, and PGE2 in primary human monocytes.
Subject(s)
Acetylene/analogs & derivatives , Asteraceae/chemistry , Inflammation Mediators/metabolism , NF-kappa B/physiology , Spiro Compounds/pharmacology , Acetylene/pharmacology , HeLa Cells , Humans , I-kappa B Proteins/antagonists & inhibitors , I-kappa B Proteins/metabolism , Jurkat Cells , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Plant Extracts/pharmacology , Polymers/pharmacology , Polyynes , Transcription Factor RelAABSTRACT
HIPK2 shows overlapping localization with p53 in promyelocytic leukemia (PML) nuclear bodies (PML-NBs) and functionally interacts with p53 to increase gene expression. Here we demonstrate that HIPK2 and the PML-NB resident protein Sp100 synergize for the activation of p53-dependent gene expression. Sp100 and HIPK2 interact and partially colocalize in PML-NBs. The cooperation of HIPK2 and Sp100 for the induction of p21(Waf1) is completely dependent on the presence of p53 and the kinase function of HIPK2. Downregulation of Sp100 levels by expression of siRNA does not interfere with p53-mediated transcription, but obviates the enhancing effect of HIPK2. In summary, these experiments reveal a novel function for Sp100 as a coactivator for HIPK2-mediated p53 activation.
