טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMeller Anatoly
SubjectDynamics of the Proteome and Phosphoproteome of the MAPK
Signal Cascades in Budding Yeast
DepartmentDepartment of Biology
Supervisor Professor Arie Admon
Full Thesis textFull thesis text - English Version


Abstract

Responding to input from the environment and responding to stress are fundamental functions of all living cells. While unicellular organisms must react to environmental cues, cells of multicellular organisms need also to transmit signals between them, in order to coordinate their responses. To communicate changes from the environmental conditions into cellular responses, robust signaling systems are required. Therefore, signal transduction pathways play a major role in ensuring the survival and well-being of organisms.

MAPKs are serine/threonine-specific protein kinases that are evolutionarily conserved across all eukaryotic organisms. MAPKs regulate a wide range of cellular programs in response to a plethora of signals, such as during embryogenesis, cell differentiation, cell proliferation, apoptosis, stress response, learning and memory. In humans, MAPKs are a clinically important family, involved in cancer, inflammatory, cardiovascular and neurodegenerative diseases.

In this research, Saccharomyces cerevisiae, the budding yeast, was used as the model organism. There are five MAPK cascades in the budding yeast. This research is focused on the HOG signaling pathway, which plays a major role in the response to hyper-osmotic stress. Hog1, the MAPK of the HOG pathway, is an ortholog of the mammalian p38 family of MAPKs. p38 MAPKs in humans are involved in different processes, including response to osmotic stress.

In this research, mass-spectrometry methods were used to study the changes in the proteomic and phosphoprotemic states of the budding yeast cells that result from activation of the HOG pathway. In order to isolate the effects of HOG pathway, a two-pronged strategy was employed: First, cells expressing wild-type or intrinsically active Hog1 MAPK mutants, developed in the lab of Prof. David Engelberg, were used under stress-free conditions, leading to activation of HOG pathway separately from the other effects of HOG-inducing stress. Second, wild type and knock-out yeast strains (lacking the Hog1 MAPK protein, Δ-hog1) were used. Comparison between the response of the Δ-hog1 and the wild-type strain was aimed to help elucidate the specific Hog1-dependent changes in the cells. For more precise comparison of the relative levels of proteins in both strains, the stable isotope labelling by amino acids in cell culture (SILAC) was used.

In both strategies, the cellular response was followed over-time, quantifying the proteome and phosphoproteome dynamics of the yeast cells.

By combining the strategies, we aimed to improve our understanding of HOG signaling pathway in budding yeast in particular, and of MAPK pathways in general.