Dataset Information

V5-tagged LIMD1 in HEK293 cells with or without JNK2 activation

ABSTRACT: The experiment was conducted to identify JNK phosphorylation sites on human LIM domain containing protein-1 (LIMD1). V5-tagged LIMD1 was co-transfected or not with a constitutively active form of JNK2 and purified. The purified proteins were subjected to SDS-PAGE, protease K digestion and MS/MS analysis. The data were searched against Swissprot using Mascot server v2.3. The fixed modification was set to carbamidomethylation of C, and variable modifications were set to oxidation of M, phosphorylation of ST, phosphorylation of Y. Mass values were set as monoisotopic. Peptide mass tolerance was set to +/-10ppm, and MS/MS tolerance was set to +/-0.8Da.

INSTRUMENT(S): instrument model, LTQ

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Gongping Sun

PROVIDER: PXD000384 | Pride | 2014-07-09

REPOSITORIES: Pride

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Publications

Ajuba family proteins link JNK to Hippo signaling.

Sun Gongping G Irvine Kenneth D KD

Science signaling 20130910 292

Wounding, apoptosis, or infection can trigger a proliferative response in neighboring cells to replace damaged tissue. Studies in Drosophila have implicated c-Jun amino-terminal kinase (JNK)-dependent activation of Yorkie (Yki) as essential to regeneration-associated growth, as well as growth associated with neoplastic tumors. Yki is a transcriptional coactivator that is inhibited by Hippo signaling, a conserved pathway that regulates growth. We identified a conserved mechanism by which JNK regu ...[more]

PMID: 24023255

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Project description:Purpose: The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. And quantitatively analyze the cell signaling pathways that regulated by the studied mutants. Methods: total mRNA was extracted from U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E.llumina compatible libraries were prepared by using the KAPA Stranded mRNA-Seq Library Preparation Kit for Illumina® platforms (KAPA Biosystems). In brief, 250 ng of total RNA was enriched for poly-A-tailed mRNA by using Kapa’s mRNA Capture beads. Poly-A-enriched RNA was fragmented to a median size of 150 bp by using chemical fragmentation and converted into double-stranded cDNA with dUTP incorporated into the second cDNA strand. The ends of the double-stranded cDNA were polished, 5’-phosphorylated, and 3’-A-tailed for the ligation of indexed adapters. Adapter-ligated DNA fragments were amplified by 8 cycles of PCR. The strand with incorporated dUTP was not amplified. The resulting libraries were quantified by qPCR and assessed for size distribution by using the 4200 TapeStation (Agilent Technologies), and then multiplexed, 4 per pool, and sequenced on the Illumina’s NextSeq500 by using the mid-output, 75-bp paired-end read format. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the human genome and quantified the expression of protein coding genes in the U251 cells of WT Gcn5 and Gcn5 Y645A mutant/WT DLST and DLST R224A/K226E mutant. Altered expression of 3 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of U251 cells with endogenous Gcn5 depletion and reconstituted expression of WT Flag-rGcn5 or Flag-rGcn5 Y645A / endogenous DLST depletion and reconstituted expression of WT V5-rDLST or V5-rDLST R224A/K226E Transcriptomes. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that Gcn5 Y45A mutant and DLST R224A/K226E mutant, which reduce histone H3 K79 succinylation, can regulate several cell signaling pathways that important for cancer cell proliferation.

Dataset Information

V5-tagged LIMD1 in HEK293 cells with or without JNK2 activation

Publications

Ajuba family proteins link JNK to Hippo signaling.

Similar Datasets

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