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An Evolutionary Proteomics Approach for the Identification of Pka

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AN EVOLUTIONARY PROTEOMICS APPROACH FOR THE IDENTIFICATION OF PKA TARGETS IN SACCHAROMYCES CEREVISIAE IDENTIFIES ATG1 AND ATG13, TWO PROTEINS THAT PLAY A CENTRAL ROLE IN THE REGULATION OF AUTOPHAGY BY THE RAS/PKA PATHWAY AND THE TOR PATHWAY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Joseph Stephan, MS * * * * * The Ohio State University 2008 Dissertation Committee: Approved by Name: Dr. Paul Herman Name: Dr. Michael Ostrowski _______________________________ Adviser Name: Dr. Mark Parthun Molecular Cellular Developmental Biology Graduate Program Name: Dr. Amanda Simcox ABSTRACT A cell in its natural environment spends most of its time in a quiescent resting state known as G0 in mammals and stationary phase in yeast. A constant challenge the cell faces is then to appropriately determine when the conditions, including nutrient availability, are favorable for it to grow. Since growth involves massive energy expenditure and a vast remodeling of the cell’s genetic program, a correct determination very likely means the difference between survival and death. Therefore, this crucial decision is both very tightly regulated and extensively coordinated in time and space. This coordination involves a synthesis between internal factors, and environmental clues such as the availability of essential nutrients and favorable conditions. The cell typically accomplishes this complex task through the combined actions of multiple signaling pathways. These pathways link cells to the extracellular environment by providing nutrient-sensing and noxious stress information and determining the appropriate response to any given situation through the activation of specific cellular programs. In essence, these signaling pathways act as molecular switches that engage and maintain the cell into a growth program when the conditions are favorable. This is done through in part through the downregulation of catabolic processes refractory to growth and the upregulation of anabolic processes conducive to it. ii In the budding yeast Saccharomyces cerevisiae, two of the most important signaling pathways that regulate growth in response to the availability of nutrients are the Tor (Target Of Rapamycin) kinase pathway and the Ras/cAMP dependent protein kinase (PKA) pathway. Inhibition of either of these pathways leads to a growth arrest similar to that induced by nutrient starvation, and the cell typically enters into a resting, stationary phase that allows it to survive for prolonged periods of time in adverse conditions. In order to understand how the Tor and PKA kinases regulate growth, it is essential to identify all the substrates of these enzymes. However, the identification of protein kinase targets has proven to be an extremely difficult task. In Chapter 2, we describe an evolutionary proteomics approach for the identification of PKA targets in the budding yeast, S. Cerevisiae. In this method, we use a previously determined PKA target consensus site and mine the yeast proteome for all occurrences of this consensus. Next, we ask if these sites have been conserved through evolution in related yeast species. We found that evolutionary conservation of a PKA site very strongly correlated with the likelihood of that site being recognized by PKA in an in vitro phosphorylation assay. This approach was successful in identifying 44 novel substrates of PKA in yeast. One particularly interesting subset of targets, the AuTophaGy-related proteins Atg1, Atg13 and Atg18, was involved in the catabolic process of autophagy. Autophagy has been implicated in a number of cellular processes including aging, development and cancer. In yeast, this degradative process is fully induced following nutrient limitation and allows the cell to survive prolonged periods of starvation. Previous work in yeast has shown that both the Tor and the Ras/pathways negatively regulate autophagy during the normal, logarithmic (log) phase of growth. iii However, the precise mechanism of this inhibition is not clear. In the last part of this chapter, we focus on one particular PKA target, Atg1. We show that Atg1 is phosphorylated by PKA, and that this phosphorylation inhibits autophagy in part through the regulation of Atg1 localization. In chapter 3, we extend our analysis to Atg13. We find that this protein is a critical target of the Ras/PKA pathway. Atg13 is also regulated by the Tor pathway, and our data suggest that it might be a nexus of signal integration within the autophagy machinery. Tor and PKA appear to respond to different nutritional cues to provide separate inputs in the regulation of Atg13, thus regulating different aspects of the autophagy process. iv DEDICATION To the memory of my father, And his unquenchable thirst for knowledge To my mother and brother, with love v ACKNOWLEDGMENTS First, I would like to acknowledge my advisor, Dr. Paul Herman. With his dedication and passion for science, he leads by example. His guidance and help throughout the years of my PhD have been of tremendous value to me. Paul was always generous with advice on how to lead a successful career in science. For all these reasons, I am sincerely grateful to him. I would like to thank my committee members, Dr. Michael Ostrowski, Dr. Mark Parthun and Dr. Amanda Simcox, for their time and guidance. I am grateful to my program director, Dr. Dave Bisaro, and the MCDB and MG office staff members Mrs Jan Zinaich, Mrs. Debbie Dotter and Mrs. Jessie Siegman for their help and patience. I would also like to acknowledge Yelena, Yuh Ying, and Vidhya. It’s been a pleasure having them as colleagues, and friends, and they have made my stay in the lab enjoyable. My interaction with Steve was very enriching, and I want to thank him for our interesting discussions, ranging from biology to quantum physics. I would also like to thank Vincent for his friendship. I would like to thank my friends. Mirna, Luc, and Hannah, for making Columbus feel like home, Nadine, for her help and her friendship on the journey to graduation, and also Zak, Danielle, Sleiman, Walid, Eliana, and Mohammed for all the good times. I am especially grateful to Sama for her continued support throughout the years. None of this would have been possible without her, in so many ways. Finally, I would like to acknowledge my late grandmother Martha Stephan and my uncle Joseph Rizk. They always believed, sometimes in the face of all the evidence. vi VITA October 13, 1977............................................Born –Berti, Lebanon 1995 -1999 .....................................................B.S. in Biology American University of Beirut Beirut, Lebanon 2000-2002 ......................................................MSc. in Anatomy and Cell Biology University of Sherbrooke Sherbrooke, Quebec, Canada 2002 -2008 ....................................................Teaching and Graduate Associate The Ohio State University Columbus, Ohio, USA PUBLICATIONS Stephan, J. S. and P. K. Herman (2006) The regulation of autophagy in eukaryotic cells: Do all roads pass through Atg1? Autophagy 2: 146-148. Budovskaya*, Y. V., J. S. Stephan*, S. J. Deminoff and P. K. Herman (2005) An evolutionary proteomics approach identifies novel substrates of the cAMP-dependent protein kinase. Proceedings of the National Academy of Sciences 102: 13933-13938. (* The first two authors contributed equally to this work.) Budovskaya, Y. V., J. S. Stephan, F. Reggiori, D. J. Klionsky and P. K. Herman (2004) The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae. Journal of Biological Chemistry 279: 20663-20671. vii FIELDS OF STUDY Major Field: Molecular Cellular Developmental Biology viii TABLE OF CONTENTS Page Abstract.............................................................................................................................ii Dedication......................................................................................................................... v Acknowledgments............................................................................................................vi Vita..................................................................................................................................vii List of Tables ...................................................................................................................xi List of Figures.................................................................................................................xii Chapters: 1. Litterature review.................................................................................................. 1 2. An evolutionary proteomics approach identifies substrates of the cAMP- dependent protein kinase..................................................................................... 25 2.1 Introduction....................................................................................... 25 2.2 Materials and Methods...................................................................... 27 2.3 Results............................................................................................... 30 2.4 Discussion......................................................................................... 37 3. The Tor and PKA pathways independently target the Atg13 protein to control autophagy activity..............................................................................................
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