Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate.

To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3ca) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3ca and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing.

Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors.

Background: Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma.

Results: Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates.

Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells.

Kinases determine the phenotypes of many cancer cells, but the frequency with which individual kinases are activated in primary tumors remains largely unknown. We used a computational approach, termed kinase-substrate enrichment analysis (KSEA), to systematically infer the activation of given kinase pathways from mass spectrometry-based phosphoproteomic analysis of acute myeloid leukemia (AML) cells. Experiments conducted in cell lines validated the approach and, furthermore, revealed that DNA-dependent protein kinase (DNA-PK) was activated as a result of inhibiting the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling pathway.

Global profiling of protein kinase activities in cancer cells by mass spectrometry.

Protein kinases have important functions in the control of cell biology and are implicated in several diseases including cancer. Here we describe a technique to quantify protein kinase activity in a global fashion and without preconception of the kinases that may be active in the cell or tissue under investigation. In Global Kinase Activity Profiling (GKAP), protein kinases present in experimental cell lysates phosphorylate endogenous substrates, also present in the lysate, under defined conditions.

Phosphoproteomic analysis of leukemia cells under basal and drug-treated conditions identifies markers of kinase pathway activation and mechanisms of resistance.

Protein kinase signaling is fundamental to cell homeostasis and is deregulated in all cancers but varies between patients. Understanding the mechanisms underlying this heterogeneity is critical for personalized targeted therapies. Here, we used a recently established LC-MS/MS platform to profile protein phosphorylation in acute myeloid leukemia cell lines with different sensitivities to kinase inhibitors.

Relevance of the MEK/ERK signaling pathway in the metabolism of activated macrophages: a metabolomic approach.

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition.

Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics.

Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates.

Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation.

Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differential activations result in the expression of specific sets of genes involved in the release of pro- or anti-inflammatory stimuli.

In silico strategy to rationally engineer metabolite production: A case study for threonine in Escherichia coli.

Genetic engineering of metabolic pathways is a standard strategy to increase the production of metabolites of economic interest. However, such flux increases could very likely lead to undesirable changes in metabolite concentrations, producing deleterious perturbations on other cellular processes. These negative effects could be avoided by implementing a balanced increase of enzyme concentrations according to the Universal Method [Kacser and Acerenza (1993) Eur J Biochem 216:361-367].

Quantification of intracellular phosphorylated carbohydrates in HT29 human colon adenocarcinoma cell line using liquid chromatography-electrospray ionization tandem mass spectrometry.

The quantitative understanding of the role of sugar phosphates in regulating tumor energetic metabolism at the proteomic and genomic level is a prerequisite for an efficient rational design in combined drug chemotherapy. Therefore, it is necessary to determine accurately the concentration of the main sugar phosphate pools at the lower concentrations present in the often-limited volume of tumor cell samples. Taking as an example the human adenocarcinoma cell line HT29, we here report a fast and reliable quantitative method based on the use of liquid nitrogen, a weak acid extraction, and liquid chromatography-electrospray ionization tandem mass spectrometry to quantify simultaneously the intracellular concentration of sugar phosphate pools.