Project description:Protein expression profiles of human cancer and NPC cell lines treated with kinase inhibitors, epigenetic and neurodevelopmental compounds.
Project description:Background: Constitutive activation of the alternative NF-?B pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-?B signaling results in the development and progression of cancer. We aimed here to learn about the mechanisms how does the constitutively active alternative NF-?B pathway exert its effects in these malignant processes. Methodology/Principal Findings: To explore the consequences of constitutive alternative NF-?B activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-kB2/p100-deficient (p100-/-) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed 73 differentially regulated genes in p100-/- vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100-/- MEFs direct binding of RelB and p52 to the promoter of the enpp2 gene encoding Enpp2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (enpp2, serpina3g, traf1, rrad), chemotactic/locomotory activity (enpp2, ccl8), and lymphocyte homing activity (enpp2, cd34). Most importantly, biochemical analyses of MEFs and gene expression analyses of mice indicated a crosstalk between classical and alternative NF-?B pathways. Conclusions/Significance: The present results show that uncontrolled alternative NF-?B signaling is further enhanced by classical NF-?B activation, indicating that p100 deficiency alone is insufficient for full induction of a subset of genes by the alternative NF-?B pathway. cell type comparison (wt vs p100-/-) after genetic modification
Project description:A primary step in activating the alternative nuclear factor-kappaB (NF-kappaB) pathway requires NF-kappaB2/p100 processing to generate p52. In most cases, stimuli-induced p100 processing is dependent on NF-kappaB-inducing kinase/IkappaB kinase alpha-mediated phosphorylation and ubiquitination. Here, we report that post-translational modification of p100 at specific sites by the small ubiquitin-like modifier (SUMO) is another determining factor for stimuli-induced p100 processing. The results show that basal SUMO modification is required for stimuli-induced p100 phosphorylation and that blocking SUMOylation of p100, either by site-directed mutation or by short interfering RNA-targeted diminution of E2 SUMO-conjugating enzyme Ubc9, inhibits various physiological stimuli-induced p100 processing and ultimate activation of the alternative NF-kappaB pathway. Together, these findings show the crucial role of SUMO1 modification in p100 processing and provide mechanistic insights into the participation of SUMO1 modification in the regulation of signal transduction.
Project description:Here we describe a bead-based method capable of profiling tyrosine kinase phosphorylations in a multiplexed, high-throughput and low-cost manner. This approach allows for the discovery of tyrosine kinase-activating events, even when the DNA sequence is wild-type. In an effort to pilot the establishment of a tyrosine kinase activation catalog, we profiled tyrosine phosphorylation levels of 62 tyrosine kinases in 130 human cancer lines, and followed-up on the frequent SRC phosphorylation in glioblastoma. Keywords: quantitative measurements of tyrosine phosphorylation levels on tyrosine kinases Total protein lysates were collected from 130 cancer cell lines. Tyrosine phosphorylation levels on 62 tyrosine kinases were measured with the bead assay.
Project description:Protein abundance data from S. cerevisiae cells cultivated in synthetic minimal media with a range of metal concentrations (concentrations of Ca, Cu, Fe, K, Mg, Mn, Na and Zn were changed, one at a time)
