טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMelamed Kadosh Dganit
SubjectPhosphoproteomic Analysis of the MAPK Signaling
Cascades
DepartmentDepartment of Biology
Supervisor Professor Arie Admon
Full Thesis textFull thesis text - English Version


Abstract

Cells use protein phosphorylation as a major regulatory mechanism for diverse biological processes, such as metabolism, proliferation, differentiation, cell growth and morphogenesis. Definition of the phosphoproteome includes the identities of the phosphorylated proteins, their phosphorylation sites and dynamics in responses to stimuli. Phosphoproteomics is a very challenging technology, which includes proteolysis of the proteins, followed by enrichments of the phosphopeptides, and capillary chromatography coupled to tandem mass spectrometry (LC-MS-MS) analyses. The general goal of this research was focused on deciphering the unique signaling cascades of the Mitogen Activated Protein Kinases (MAPK) using large-scale proteomics and phosphoproteomics.

The MAPK super-family consists of four sub-families of serine/threonine protein kinases: ERK1/2, p38s, JNKs and ERK5. We focused on the stress-activated p38 and its adaptation to continuous signaling. Since the same MAPK-Kinases activate different p38 MAPKs, two experimental proteomics approaches were employed, both based on using Stable Isotope Labeling of Amino acids in tissue Culture (SILAC) with mouse embryonic fibroblasts (MEFs): The phosphoproteins analysis was first performed on cultured MEFs expressing HA-tagged wild-type p38α or p38β, or their intrinsically active variants. This way, the signaling cascades induced by each p38 could be elucidated, in spite of interference by other extracellular stimulations or upstream activations. A total of 14,387 phosphorylation sites were identified, and the changes in the active-variant/wild-type ratio of the proteins and phosphopeptides pointed to newly discovered changes in the proteome and phosphoproteome that took place only by the intrinsically active mutants. The effects of the intrinsically active variants of p38 provided indications useful for elucidating the signaling events taking place in cells that are exposed to continuous activation of these MAPK and to the responses involving adaptation to this stress-inducing situation. The adaptation process includes modulation by the intrinsically active variants of the other members of this kinases family, increasing the expression and activities of different phosphatases, and by other mechanisms involving alterations in the molecular interactions of the p38s. These observations help to broaden our understanding of the molecular mechanisms involved in adaptation to continuous signaling, which are possibly significant for coping with chronic inflammation, continuous stress conditions and cancer.

The phosphoproteins analyses were also performed with MEFs that were p38α and p38β knocked-out and with wild-type MEFs. The cells were uniformly and rapidly activated by treatment with Anisomycin at five time points while following the response dynamics to the stress drug treatment which strongly activates these MAPK. The total of 21,507 phosphorylation sites identified in this experiment enabled us to better define the signaling through the different p38 isoforms. A. Clustering of the expression profiles of the phosphorylation sites detected ten patterns of posttranslational modifications, pointing to specificities of the different p38 MAPKs. The main observation of this experiment indicated that the dynamics of the phosphorylation were similar in the three cell lines. This similarity is a result of redundancy between the different p38s. Yet, unique signaling events activated by each of the kinase, could be observed. A number of new and important phosphorylations sites were discovered in these studies.