The dysregulation of the ERK1/2 signalling cascade appears to be involved not only in oncogenesis but also in the context of Alzheimer’s disease and amyotrophic lateral sclerosis. The mitogen activated protein kinases ERK1 and ERK2 are fully activated by dual phosphorylation at a conserved threonine-glutamate-tyrosine (TEY) motif. Monophosphorylated isoforms of ERK1/2 are also present in living cells and have appreciable kinase activities in vitro at physiological ATP concentrations. The highly sensitive detection and differentiation of the unphosphorylated, monophosphorylated and diphosphorylated isoforms of ERK1 and ERK2 as well as their semi-quantitative analysis can be achieved by capillary isoelectric focussing followed by immunodetection (CIEF-immunoassay).
In the first part of the present work an optimised working procedure was elaborated for the detailed analysis of phosphorylation of ERK1/2 in cell lysates by CIEF-immunoassay. When different lysing systems were tested, it turned out, that reducing agents can substantially influence the ERK1/2 detection. The commercially available M-PER reagent was most suitable as it possessed higher efficiency in lysing nuclei than other buffers, it contained no reducing agents and offered a high recovery of ERK1/2-phospho-forms in the resulting cell lysates. The reproducibility of the relative quantifications in the CIEF-immunoassay was acceptable (coefficient of variation < 15 %) for signals with a relative abundancy greater than 1 % of the added abundancies of all ERK1 and ERK2 signals, respectively. Generally, the observed variation allowed for reasonable comparison of samples within the same assay as well as in different, independent assays.
An important improvement of previously published analytical possibilities of the CIEF-Immunoassay is the additional differentiation and classification of the threonine- and tyrosine-monophosphorylated isoforms of ERK1 and ERK2, which is presented within this work. The differentiation and unequivocal peak identification were achieved with a combination of synthetic phospho-ERK1/2 peptides and several anti-phospho-ERK1/2 antibodies in competitive blocking experiments, which were performed for the first time in the CIEF-Immunoassay in this form.
As a proof of principle the optimised standard operating procedures were applied to exemplarily analyse the kinetics of ERK1/2 phosphorylation and the time dependent occurrence of phospho-ERK1/2 after stimulation in SH-SY5Y and THP-1 cell lines as well as in peripheral blood mononuclear cells in the second part of the work. The ERK1/2-phospho-form distribution seemed to depend on the particular cell type and the applied stimulus.
The optimised method was furthermore applied to an orienting pilot study on human peripheral blood mononuclear cells from a small clinical cohort of patients with Alzheimer’s disease, mild cognitive impairment, amyotrophic lateral sclerosis and non-demented controls. Despite of the very small sample size, statistically significant differences between the diagnostic groups could be observed, after activation by applying a stimulus, for the inactivation (dephosphorylation) of the diphosphorylated and threonine-monophosphorylated forms of ERK1. These changes could possibly be linked to a disease related dysregulation of phosphatase activity. Due to the small size of the groups within this study, the results have to be considered as preliminary. For additional confirmative studies with a greater sample size an optimisation of several parameters will be necessary. Altogether, the CIEF-immunoassay enables detailed studies concerning the relative ERK1/2-phospho-form distribution in cell lysates.
|Advisor:||Wiltfang , Jens|
|School:||Universitaet Duisburg-Essen (Germany)|
|Source:||DAI-C 81/1(E), Dissertation Abstracts International|
|Keywords:||Phosphorylation state, Mitogen activated protein, Kinases ERK1 and ERK2|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be