Abstract
BackgroundThe mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS). P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely.MethodsWe have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro.Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3).In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR.This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.ResultsThe combination of these techniques allowed for:(1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer simulation using MD techniques allowed us to get functional models of both structure and interactions of the previously mentioned phosphorylated kinases and the differences between their phosphorylated and un-phosphorylated forms.ConclusionMany research studies are necessary to unfold the whole signalling network (human proteome), which is so important to advance in clinical research, especially in the cases of malignant diseases.
Highlights
As with other mitogen activated protein kinase (MAPK) pathways, the p38 signalling cascade involves sequential activation of MAPK kinase kinases (MAP3Ks) and MAPK kinases (MKKs) including MKK3, MKK4, and MKK6, which directly activate p38 through phosphorylation in a cell-type- and stimulus-dependent manner [1,2]
Purification and Kinase assay Recombinant glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified using standard protocols. p38beta was activated with MalE-MKK6DD (5:1 ratio) in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT pH 7.5 and 200 uM ATP for 1 hour at 30°C
Identified phosphopeptides (a) The aim of this study was to establish, as a routine path, a method for identification and characterization of individual phosphorylated kinases p38 and HuR in vitro using: TiO2, Immobilized Metal Affinity Capture (IMAC), Sequential Elution from IMAC (SIMAC) coupled to Multistage activation (MSA) and MS3NL on the LTQ ion Trap mass spectrometer (Thermo)
Summary
As with other MAPK pathways, the p38 signalling cascade involves sequential activation of MAPK kinase kinases (MAP3Ks) and MAPK kinases (MKKs) including MKK3, MKK4, and MKK6, which directly activate p38 through phosphorylation in a cell-type- and stimulus-dependent manner [1,2]. Bioinformatics offers a range of methods to analyze and to simulate structural properties of the studied phosphoproteins Both unphosphorylated and phosphorylated states of a residue can be generated “in silico” and included in the appropriate 3D protein context. P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. It has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely
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