ABSTRACT
BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.
Subject(s)
DEAD-box RNA Helicases/genetics , NF-kappa B/metabolism , Receptors, Pattern Recognition/genetics , Signal Transduction , Alkylation , Animals , DEAD-box RNA Helicases/metabolism , DNA Probes , Drug Therapy , Humans , Mice , Receptors, Pattern Recognition/metabolismABSTRACT
The tumor suppressor PTEN plays a critical role in the regulation of multiple cellular processes that include survival, cell cycle, proliferation, and apoptosis. PTEN is frequently mutated or deleted in various human cancer cells to promote tumorigenesis. PTEN is regulated by SUMOylation, but the SUMO E3 ligase involved in the SUMOylation of PTEN remains unclear. Here, we demonstrated that PIASxα is a SUMO E3 ligase for PTEN. PIASxα physically interacted with PTEN both in vitro and in vivo. Their interaction depended on the integrity of phosphatase and C2 domains of PTEN and the region of PIASxα comprising residues 134-347. PIASxα enhanced PTEN protein stability by reducing PTEN ubiquitination, whereas the mutation of PTEN SUMO1 conjugation sites neutralized the effect of PIASxα on PTEN protein half-life. Functionally, PIASxα, as a potential tumor suppressor, negatively regulated the PI3K-Akt pathway through stabilizing PTEN protein. Overexpression of PIASxα led to G0/G1 cell cycle arrest, thus triggering cell proliferation inhibition and tumor suppression, whereas PIASxα knockdown or deficiency in catalytic activity abolished the inhibition. Together our studies suggest that PIASxα is a novel SUMO E3 ligase for PTEN, and it positively regulates PTEN protein level in tumor suppression.
Subject(s)
PTEN Phosphohydrolase/metabolism , Protein Inhibitors of Activated STAT/metabolism , SUMO-1 Protein/metabolism , Sumoylation/physiology , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , G1 Phase Cell Cycle Checkpoints/physiology , HeLa Cells , Humans , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Inhibitors of Activated STAT/genetics , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Resting Phase, Cell Cycle/physiology , SUMO-1 Protein/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Objective To construct the recombinant plasmid pCMV-Myc-PIASX? and express the fusion protein in mammalian cells.Methods PIASx? fragment was digested from the original vector pGADT7 with SalⅠand NotⅠ,and then was inserted into the targeted pCMV-Myc vector by the recombinant DNA technique.After identification,the recombinant plasmid was transfected into CHO cells.The expression of recombinant Myc-PIASx? fusion protein was detected by Western blotting.Results By the restriction enzyme digestion,fragment purification,ligation and transformation,the recombinant plasmid was obtained.The right recombinant plasmid pCMV-Myc-PIAS3 was identified with enzyme digestion and sequencing.By EcoRⅠ digestion analysis,pCMV-Myc-PIASx? showed a 5641 bp band.By XbaⅠdigestion analysis,pCMV-Myc-PIASx? showed two expected band of 3291 bp and 2349 bp.A specific protein expression band at 68 000(PIASx? fusion protein) was showed in Western blotting,which matched recombinant plasmid.Conclusion The recombinant plasmid of pCMV-Myc-PIASx? is sucssesefully constructed,which provids a good tool for further function study on PIAS family.
