Supervisors info:
Παναγιώτου Γεώργιος, Ερευνητής Α', Τομέας Μοριακής Ογκολογίας, Ερευνητικό Κέντρο Βιοϊατρικών Επιστημών "Αλέξανδρος Φλέμινγκ"
Κωστούρου Βασιλική, Ερευνήτρια Β', Τομέας Ανοσολογίας, Ερευνητικό Κέντρο Βιοϊατρικών Επιστημών "Αλέξανδρος Φλέμινγκ"
Κουτσιλιέρης Μιχαήλ, Καθηγητής, Ιατρικής, ΕΚΠΑ
Summary:
The Mitogen Activated Protein Kinase (MAPK) pathway is involved in many different biological processes, including cell proliferation, differentiation, survival, apoptosis and inflammation and is associated with a variety of pathological diseases, like cancer and neurodegeneration. It consists of a three-level cascade of phosphorylation events which either lead to the activation of a nuclear transcription factor for gene expression regulation or the activation of a cytoplasmic protein. The MAPK pathway includes three major subfamilies: the extracellular-signal regulated kinase (ERK1/2), the p38 kinase and the c-Jun N-terminal kinase (JNK) subfamilies. Even though each pathway is involved in different biological processes, events of co-activation and co-regulation are quite frequent, especially in the case of JNK/p38 kinases, which are also called stress-activated protein kinases (SAPKs). MAP kinase activity is regulated by a specific group of phosphatases called mitogen activated protein kinase phosphatases (MKPs). MKPs belong to a larger group called dual specificity phosphatases (DUSPs). This three-part project focuses on DUSP8, a JNK/p38 specific phosphatase, and its inhibition by arsenite, a JNK pathway-enhancing and oxidative stress inducing agent. The first part concerned the optimization of a phosphoproteomics workflow from cell culture to data analysis. Immunoblotting with specific antibodies of lysates from differentially starved and stimulated HEK293T cells provided information about the arsenite stimulation conditions, where a clear effect of phosphorylation was observed. Arsenite was found to be effective in the overall stimulation of phosphorylation. The following steps were to determine the mass spectrometry (MS) parameters and the sample preparation method with the highest protein and peptide yield after phosphopeptide enrichment and nanoLC-MS/MS analysis. Three methods were examined: Filter-aided sample preparation (FASP), solid-phase-enhanced sample preparation (SP3) and Trifluoroethanol-based digestion (TFE). Determined stimulation periods, MS parameters and sample preparation methods were then implemented in the second part of the project, which involved the phosphoproteome LC-MS/MS analysis of primary mouse embryonic fibroblasts derived from DUSP8-KO mice generated by CRISPR technology, and their WT littermates. Increased phosphorylation on a large number of proteins was detected in DUSP8-KO MEFs. Almost no phosphotyrosine residues were detected, possibly due to their transient nature. Also, increased phosphorylation of ERK2 substrates in DUSP8-KO MEFs suggested a possible regulation of ERK2 activity by DUSP8. In the last part, a comparative whole proteome analysis was conducted in tissues of DUSP8-KO mice and their WT littermates. The results suggested that overactivation of the JNK pathway due to DUSP8 deficiency in the brain leads to mitochondrial apoptosis and neurodegeneration. Additionally, the ERK1/2-driven response to oxidative stress in the brain of DUSP8-KO mice is possibly mediated by increased expression of B-Raf, one of the identified proteins in the comparative whole proteome analysis.
Keywords:
DUSP8, MKP, MAPK signaling, Phosphoproteomics, Arsenite
