Quantitative dynamics of phosphoproteome: the devil is in the details.
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ABSTRACT:
SUBMITTER: Salek M
PROVIDER: S-EPMC3477822 | biostudies-other | 2012 Oct
REPOSITORIES: biostudies-other
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Project description:Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQ(TM)) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.
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Project description:BackgroundAlthough recent progress has been made in treating castration resistant prostate cancer, the interplay of signaling pathways which enable castration resistant growth is incompletely understood. A data driven, multivariate approach, was used in this study to predict prostate cancer cell survival based on the phosphorylation levels of key proteins in PC3, LNCaP, and MDA-PCa-2b cell lines in response to EGF, IGF1, IL6, TNF?, dihydrotestosterone, and docetaxel treatment.MethodsThe prostate cancer cell lines were treated with ligands or inhibitors, cell lyates were collected, and the amount of phosphoprotein quantified using 384 well ELISA assays. In separate experiments, relative cell viability was determined using an MTT assay. Normalized data was imported into Matlab where regression analysis was performed.ResultsBased on a linear model developed using partial least squares regression, p-Erk1/2 was found to correlate with castration resistant survival along with p-RPS6, and this model was determined to have a leave-one-out cross validated R2 value of 0.61. The effect of androgen on the phosphoproteome was examined, and increases in PI3K related phosphoproteins (p-Akt, p-RPS6, and p-GSK3) were observed which accounted for the majority of the significant increase in androgen-mediated cell survival. Simultaneous inhibition of the PI3K pathway and treatment with androgen resulted in a non-significant increase in survival. Given the strong effect of PI3K related signaling in enabling castration resistant survival, the specific effect of mTor versus complete inhibition was examined using targeted inhibitors. It was determine that mTor inhibition accounts for 52% of the effect of complete PI3K inhibition on cell survival. The differences in signaling between the cell lines were explored it was observed that MDA-PCa-2b exhibited far less activation of p-Erk in response to varying treatments, explaining one of the reasons for the lack of castration resistance.ConclusionIn this work, regression analysis to the phosphoproteome was used to illustrate the sources of castration resistance between the cell lines including reduced p-Erk signaling in MDA-PCa-2b and variations in p-JNK across the cell lines, as well as studying the signaling pathways which androgen acts through, and determining the response to treatment with targeted inhibitors.
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Project description:BackgroundBombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive.MethodEmploying tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined.ResultsTotally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D.ConclusionThe phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.
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