Dataset Information

Large scale phosphoproteome analysis of LNCaP human prostate cancer cells.

ABSTRACT: Prostate cancer is the most frequently diagnosed cancer among men in the western world. The androgen receptor, a phosphoprotein, is suspected to be involved in all stages of the prostate cancer. Androgen receptor activity can be modulated by various kinases such as PKA, MAPK, AKT, and Src. Phosphorylation is an important post-translational modification and serves as a molecular on-off switch to regulate signaling. Disruptions of cellular phosphorylation are associated with various diseases such as cancer and kinases provide important drug targets. Here we present an analysis of the phosphoproteome in LNCaP human prostate cancer cells. The analytical strategy employed here used proteomics based methodologies with a combination of detergents and chaotropic reagents during trypsin digestion followed by titanium dioxide enrichment of phosphopeptides. Over the course of multiple analyses by mass spectrometry we identified a total of 746 phosphorylation sites in 540 phosphopeptides corresponding to 116 phosphoproteins, of which 56 had not been previously reported. Phosphoproteins identified included transcription factors, co-regulators of the androgen receptor, and cancer-related proteins that include ?-catenin, USP10, and histone deacetylase-2. The information of signaling pathways, motifs of phosphorylated peptides, biological processes, molecular functions, cellular components, and protein interactions from the identified phosphoproteins established a map of phosphoproteome and signaling pathways in LNCaP cells.

SUBMITTER: Myung JK

PROVIDER: S-EPMC3433406 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Large scale phosphoproteome analysis of LNCaP human prostate cancer cells.

Myung Jae-Kyung JK Sadar Marianne D MD

Molecular bioSystems 20120614 8

Prostate cancer is the most frequently diagnosed cancer among men in the western world. The androgen receptor, a phosphoprotein, is suspected to be involved in all stages of the prostate cancer. Androgen receptor activity can be modulated by various kinases such as PKA, MAPK, AKT, and Src. Phosphorylation is an important post-translational modification and serves as a molecular on-off switch to regulate signaling. Disruptions of cellular phosphorylation are associated with various diseases such ...[more]

PMID: 22699815

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Project description:BackgroundProtein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during vegetative growth, especially in biofuel plants, is far from comprehensive.ResultsIn this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total of 1470 phosphorylation sites in 950 phosphoproteins were identified, and these phosphoproteins were implicated in various molecular functions and basic cellular processes by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Among the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. Conservation analysis showed that 93.4% of the 950 phosphoproteins had phosphorylation orthologs in other plant species. Motif-X analysis of the phosphorylation sites identified 13 significantly enriched phosphorylation motifs, of which 3 were novel phosphorylation motifs. Meanwhile, there were 91 phosphoproteins with both multiple phosphorylation sites and multiple phosphorylation motifs. In addition, we identified 58 phosphorylated transcription factors across 21 families and found out 6 significantly over-represented transcription factor families (C3H, Trihelix, CAMTA, TALE, MYB_related and CPP). Eighty-four protein kinases (PKs), 8 protein phosphatases (PPs) and 6 CESAs were recognized as phosphoproteins.ConclusionsThrough a large-scale bioinformatics analysis of the phosphorylation data in seedling leaves, a complicated PKs- and PPs- centered network related to rapid vegetative growth was deciphered in B. distachyon. We revealed a MAPK cascade network that might play the crucial roles during the phosphorylation signal transduction in leaf growth and development. The phosphoproteins and phosphosites identified from our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocots.

Project description:Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G0/G1 cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.

Dataset Information

Large scale phosphoproteome analysis of LNCaP human prostate cancer cells.

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Large scale phosphoproteome analysis of LNCaP human prostate cancer cells.

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