An Evolutionary Proteomics Approach for the Identification of Pka

Home , PDE1, Phosphofructokinase 2

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

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.

DEDICATION

To the memory of my father, And his unquenchable thirst for knowledge

To my mother and brother, with love

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.

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...... 76

3.1 Introduction...... 76 3.2 Materials and Methods...... 78 3.3 Results...... 83 3.4 Discussion...... 89

4. Synopsis ...... 101

The evolutionary proteomics approach...... 101 ix Autophagy and its regulation: a paradigm for Tor/PKA crosstalk? ..... 103 Future directions...... 105

Bibliography ...... 107

LIST OF TABLES

Table Page

2.1 The 44 candidate PKA substrates identified by the evolutionary proteomics approach described here...... 47

2.2 A list showing the proteins that contain the 553 consensus PKA sites present in the Saccharomyces cerevisiae proteome ...... 48

2.3 The 85 candidate PKA substrates identified by the evolutionary proteomics approach described here...... 75

LIST OF FIGURES

Figure Page

1.1 Different functions of the TORC complexes in the regulation of growth ...... 22

1.2 Interrelationship between the Ras and Tor pathway in the control of growth ...... 23

1.3 The steps of Autophagy ...... 24

2.1 An evolutionary proteomics approach identified 85 potential substrates of PKA in S. cerevisiae ...... 41

2.2 Proteins with highly conserved consensus PKA sites were more likely to be phosphorylated by PKA than proteins with less conserved sites...... 42

2.3 The autophagy-related protein kinase, Atg1, is a substrate of PKA...... 43

2.4 PKA phosphorylation regulates the association of Atg1 with the preautophagosomal structure, or PAS...... 44

2.5 The full-length Atg1 was phosphorylated by cAMP-dependent protein kinase (PKA) in vitro at the serine residues within the two consensus PKA sites ...... 45

2.6 Alteration of the serine residues within the PKA sites in Atg1 did not influence Atg1 protein kinase activity ...... 46

3.1 The Atg13 protein was a substrate for PKA ...... 93

3.2 The PAS localization of Atg13 was regulated by PKA phosphorylation...... 94

xii 3.3 Inhibition of the Ras/PKA signaling pathway was sufficient to induce autophagy...... 95

3.4 The RAS2val19-mediated inhibition of autophagy was reversed by the presence of the nonphosphorylatable Atg13-AAA variant...... 96

3.5 PKA phosphorylation of Atg13 regulates its association with Atg17 but not Atg1 ...... 97

3.6 PKA activity regulates the PAS localization of Atg13 by modulating its interaction with Atg17 ...... 98

3.7 The PKA-dependent phosphorylation of Atg13 is regulated by carbon source cues...... 99

3.8 Tor and PKA regulate different steps in the induction of autophagy ...... 100

xiii

CHAPTER 1

LITTERATURE REVIEW

The Tor pathway, an important regulator of cell growth in eukaryotic cells

In yeast, inhibition of Tor using the drug rapamycin leads growing yeast cells to arrest rapidly in the G0 phase of the cell cycle, known as stationary phase. This arrest is

characterized by specific hallmarks such as markedly decreased rates of transcription

and translation, changes in cell wall composition, and the accumulation of storage

carbohydrates, and is similar to the growth arrest caused by nitrogen or carbon

starvation [1]. Based on this observation, it is widely believed that Tor links nutrient

information with the cell growth machinery [2]. Tor is therefore a central regulator of

cell growth, and it appears that this function has been conserved through evolution from

yeast to man [3, 4]. In fact, every eukaryotic genome studied to date contains a TOR

gene. In the following section, we review current knowledge in the Tor field in yeast,

starting with the seminal discovery of the Tor inhibitor rapamycin, to the cloning and

functional characterization of TOR. Next, we describe the two Tor complexes and

discuss their roles in the regulation of growth. Finally, we comment on the scarcity of

1 information regarding upstream activators and downstream effectors of the Tor signaling pathway.

The discovery of rapamycin

In the 1970s, a vast search for naturally occurring, biologically active compounds was undertaken, and included the collection of soil from geographically diverse locations for analysis. Research on soil samples gathered by a Canadian team from Rapa Nui (Easter

Island) identified a bacterial strain of Streptomyces hygroscopicus that exhibited potent antifungal properties[5]. Upon purification, the active compound was found to be a macrocyclic lactone and was named rapamycin (for Rapa Nui)[5]. Subsequent studies found that rapamycin possessed strong immunosuppressive as well as growth-inhibitory properties: it demonstrated activity against several tumors and was a potent inhibitor of antigen-induced T and B cell proliferation, as well as antibody production [5]. In the budding yeast S. cerevisiae, rapamycin treatment lead to an irreversible growth arrest that was phenotypically very similar to the arrest brought about by nutrient limitation

[6].

The identification of TOR1 and TOR2 in S. cerevisiae

The mechanism by which rapamycin elicited this broad range of effects was completely unknown, until a genetic screen conducted in S. cerevisiae by the Hall laboratory in

Switzerland identified mutations that conferred resistance to the drug [7]. Three broad classes of mutants were obtained: the first group encompassed recessive mutations in the FPR1 gene. FPR1 encodes FKBP12 (FK506-Binding Protein 12). FKBPs are highly

2 conserved peptidyl-isomerases that function in protein folding and stability [8].

Rapamycin acts as an FK506 analogue and binds to FKBP12. This binding is necessary, but not sufficient to mediate the cytostatic effects of rapamycin. The other two classes of mutants identified in the screen displayed dominant resistance to rapamycin as a result of gain-of-function mutations in either of two genes, TOR1 and TOR2 [7]. TOR1 and 2 encode two large (about 280 kDa), homologous serine/threonine protein kinases

(40-60% identity) that belong to the Phosphatidylinositol Kinase-Related Kinase

(PIKK) family. It was then discovered that the rapamycin-FKBP12 complex normally bound to the Tor proteins and inhibited their functions. The TOR mutations identified in the screen disrupted this binding, thus conferring resistance to the drug [9].

Genetic and molecular characterization of TOR

Genetic analyses of tor mutants gave the first clues to TOR functions. Yeast cells with a deletion of the TOR1 gene (tor1 cells) were slow growing and hypersensitive to stress, but viable, leading to the conclusion that TOR1 is not an essential gene in the budding yeast [8, 10-12]. Cells with a loss of TOR2, on the other hand, were not viable, and died with a random arrest in the cell-cycle [11]. Cells lacking both TOR1 and TOR2 died as well, with a cell cycle arrest in the G0 phase of the cell-cycle [11, 13]. These observations suggested that TOR1 and TOR2 have a partially redundant, rapamycin- sensitive function that is required for progression through the G1 phase of the cell cycle

[11, 13]. Finally, TOR2 appeared to have a separate, essential function that was rapamycin-insensitive: cells with rapamycin-resistant TOR1 alleles were able to grow in the presence of the drug [14].

3 The molecular basis for the genetically-defined rapamycin sensitive and insensitive functions of TOR was understood when the Tor proteins were purified. Two mutually exclusive multiprotein complexes were identified: the rapamycin-sensitive TORC1

(TOr Complex 1) and the rapamycin-insensitive TORC2 (TOr Complex 2). TORC1 consists of either Tor1 or Tor2 and Kog1, Lst8, and TC089 [15-18]. TORC2 contains

Lst8, Avo1, Avo2, Avo3, Bit2, Bit61 and Tor2, but not Tor1 [15, 17-19].

TORC1 in yeast performs essential functions in the temporal regulation of growth, while TORC2 is involved in the spatial regulation of growth (Figure 1.1): yeast cells grow at discrete loci and this results from polarization of the actin cytoskeleton that is regulated by TORC2 activity [4]. In the following sections, the roles of the rapamycin- sensitive TORC1 are analyzed in more detail.

TORC1 inhibits catabolic and growth-refractory processes in S. cerevisiae

TORC1 negatively regulates growth arrest by inhibiting stress-induced transcriptional programs

The availability of rapamycin as a potent TORC1 inhibitor has proved an invaluable tool in understanding the roles of TORC1 in S. cerevisiae. Several genome-wide studies

[20-24] have taken advantage of rapamycin to study TORC1 modulation of the cell’s genetic program. What comes out of these studies is that TORC1 inhibition leads to the induction of transcription factors that normally function during starvation and stress conditions. These transcription factors activate expression of genes that facilitate assimilation of alternative nutrient sources and help the cell cope with stress.

4 The mechanism most widely used by TORC1 to inhibit the expression of stress and

nutrient-responsive genes is the phosphorylation-dependent sequestration of

transcription factors in the cytoplasm. Upon nitrogen starvation, the transcription factor

Gln3 is imported to the nucleus where it induces expression of nitrogen-catabolite

repression (NCR)-sensitive genes, that allow the cell to metabolize less favored nitrogen

sources [25, 26]. In growing cells, TORC1 promotes association of Gln3 with the

cytoplasmic protein Ure2, thus preventing its import to the nucleus and inhibiting NCR-

sensitive gene expression [25]. Similarly, TORC1 regulates the Zinc-finger

transcription factors Msn2 and Msn4 in part through their retention in the cytoplasm

[25, 27]. Msn2 and 4 normally activate Stress-responsive Element (STRE) genes upon nutrient limitation and environmental stress [28, 29]. TORC1 also causes cytoplasmic

sequestration of Rtg1 and Rtg3 by promoting their association with a cytoplasmic protein, Mks1 [30-34]. Rtg1 and Rtg3 are transcription factors that activate the glutamine biosynthesis pathway [22, 35]. Upon starvation or TORC1 inactivation,

Mks1 is dephosphorylated and associates with Rtg2, thus allowing Rtg1 and 3 to

translocate to the nucleus and activate their target genes [22, 34, 36, 37]. Finally,

TORC1 sequesters Ime1 in the cytoplasm: this transcription factor normally activates

genes involved in meiosis and sporulation, processes that diploid yeast cells undergo

when no good carbon or nitrogen sources are available [38].

5 TORC1 negatively regulates autophagy

Macroautophagy (herafter referred to as autophagy) is a process where bulk

portions of the cytoplasm including whole organelles are delivered to the yeast vacuole,

the equivalent of the mammalian lysosome, for degradation. This process is induced

upon nutrient limitation or TORC1 inhibition with rapamycin. It is thought that autophagy allows the cell to recycle essential amino acids and cellular building blocks

that allow it to survive prolonged periods of starvation. Autophagy and its regulation

will be introduced in more detail in section 1.3.

TORC1 upregulates anabolic and growth-promoting processes in S. cerevisiae

When the conditions are favorable, TORC1 activity maintains and/or upregulates

several processes essential for cell growth and protein synthesis. Notably, TORC1

promotes translation initiation, expression and assembly of the translation machinery

through regulation of transcription factors such as Ifh1, Crf1, and Sfp1, and the activity

of high-affinity amino-acid permeases that pump amino acids inside the cell for use as

cellular building blocks (Reviewed in [6]).

Upstream and downstream of Tor

Despite the vast availability of Tor readouts, a detailed understanding of how

Tor couples nutrient-sensing with particular cellular programs is still lacking. For

example, how Tor senses stress and nutrient status is unknown. Furthermore, as for

most kinases, the identification of direct targets of the TORC1 complex has proven a

particularly difficult task, even though in vitro phosphorylation by Tor 1 and Tor 2 has

6 been reported for a few substrates [39-41]. A recent report has identified the Sch9

kinase as a direct TORC1 target [41]. TORC1 regulation of ribosome biogenesis,

translation initiation and stationary phase entry required Sch9 [41].

Other TORC1 readouts appear to be Sch9-independent, in particular those that are

mediated by the type 2A and 2A-related protein phosphatases, specifically PP2Ac and

the sit4 phosphatase. When nutrients are available, Tor inhibits Sit4 by promoting its

association with the regulatory protein Tap42 by a mechanism that is still unclear. Upon

TORC1 inactivation, Sit4 dissociates from Tap42 and activates several downstream

effectors such as Gcn2 [42, 43], the Rtg1/3 complex [44], Gln3 [25], Npr1 [45] and

Tip41 [25, 45, 46]. In addition, Msn2 nuclear import is regulated by TORC1 via

TAP42-dependent inhibition of PPH21 and PPH22 [27, 44, 47].

In conclusion, and despite extensive research, there is a clear lack of knowledge of

direct targets of the Tor kinase in yeast. Furthermore, the regulation of growth by Tor

appears to be an extremely complex process. Indeed, an emerging paradigm is that Tor

appears to converge with other signaling pathways on the same subsets of downstream

targets, adding further layers of complexity to the problem of understanding Tor function. This convergence and/or crosstalk between signaling pathways will be discussed further in later sections.

7 The Ras/PKA pathway, an important regulator of growth in S. cerevisiae

The first clues that the Ras/PKA pathway played a key role in linking nutrient status to growth control came from the observation that cells with abnormally elevated levels of

Ras/PKA activity were unable to stop growing and enter into stationary phase.

Conversely, mutants with low Ras/PKA activity exhibited stationary phase hallmarks even in the presence of nutrients [48-51]. It is widely accepted that Ras is an important regulator of cell growth in S. cerevisiae. The Ras/PKA signaling pathway, like the Tor pathway, links nutrient status, and especially glucose availability, to particular cellular programs [52, 53].

In this section, we will introduce and describe the Ras/PKA pathway, starting by the cloning and biochemical characterization of Ras and PKA in yeast. Next, we will describe the core molecular machinery involved in the pathway. We will then describe some of the main roles of the Ras/PKA pathway in the regulation of growth and stationary phase biology. Finally, we will review the current knowledge on upstream activators and downstream effectors of the Ras/PKA pathway, and comment on the interrelationship between Ras and Tor.

Early discoveries: genetic and biochemical characterization of the Ras and PKA proteins in S. cerevisiae

The Retrovirus-Associated Sequences (RAS) proto-oncogenes were first identified by their presence in RNA tumor viruses [54], and soon endogenous homologues of these genes were found in vertebrates [55]. Later, and through southern blotting, S. cerevisiae

8 orthologues of the RAS gene family were identified [56]. The yeast RAS1 and RAS2

encode homologous GTP-binding proteins that share 90% homology in their first 180

amino acids, but the proteins diverge towards their C-terminus [57]. Genetic analysis

revealed that RAS1 and 2 performed redundant functions, as deletion of either gene was

not lethal, while the double-mutant ras1 ras 2 strain was not viable [53, 57]. Further research established that the yeast Ras proteins, unlike their mammalian counterparts, regulated the activity of the adenylate cyclase enzyme, Cyr1. This enzyme converts

ATP to cAMP, a ubiquitous second messenger with a wide variety of roles in the cell including cell growth and proliferation. It was found that mutations with elevated Ras

activity, such as RAS2V19, which maintains Ras in its GTP-bound form and leads to high

Ras activity, led to elevated levels of intracellular cAMP; by contrast, ras1 ras2 bcy1

cells had virtually undetectable cAMP levels [49].

The budding yeast cAMP-dependent protein kinase A (PKA) holoenzyme was first

characterized biochemically, and was found to consist of a regulatory moiety and a

catalytic moiety [58]. Binding of cAMP to the regulatory subunit lead to dissociation of

the catalytic subunit, which exhibited serine/threonine kinase activity [58]. Genetic

characterization followed, and the three genes that encode the catalytic subunits of PKA

were identified: TPK1, TPK2 and TPK3 (for Takashi’s Protein Kinase) [59]. Deletion of

any two of those genes was not lethal, while deletion of all three was, suggesting that

they had redundant functions [59]. The observation that high copy plasmids carrying the

TPK1 gene could suppress loss-of-function mutations in RAS2 and CYR1 genetically

linked PKA to the Ras/cAMP pathway [59]. Similarly, the BCY1 gene encoding the

regulatory subunit of PKA was cloned, and bcy1 cells were found to be sensitive to

9 heat shock and starvation [48]. The activity of PKA isolated from those cells was found to be independent of cAMP [48]. It is now known that yeast PKA is a heterotetramer

consisting of two Bcy1 subunits and two catalytic subunits encoded by either of the

TPK genes.

Signal transduction from Ras to PKA

The Ras proteins constantly exist in one of two states: an inactive, GDP-bound state or an active, GTP-bound state. The guanine-nucleotide exchange factor Cdc25 stimulates

Ras activity by catalyzing the exchange of GDP for GTP. Early studies determined that

Ras1 and Ras2 possessed intrinsic GTPase activity, but this activity is markedly stimulated by the yeast GTPase-Activating Proteins (GAPs) Ira1 and 2. Ira1 and 2 convert GTP-bound Ras back to its inactive state. Active, GTP-bound Ras binds to and activates the yeast adenylate cyclase, Cyr1. This leads to an increase in intracellular cAMP levels. cAMP generated by adenylate cyclase binds to cAMP-pockets in the regulatory subunits of the PKA holoenzyme, leading to decreased affinity to the catalytic subunits which are released and go on to phosphorylate their downstream targets. By contrast, the cAMP phosphodiesterases Pde1 and Pde2 reduce cAMP levels, thereby promoting association of the Tpks with Bcy1, thus downregulating PKA activity.

In addition, there is evidence to the existence of several autoregulatory loops within this pathway. For example, Cdc25, Bcy1 and Pde1 appear to be regulated by PKA. PKA

Phosphorylation of the Pde1 phosphodiesterase upregulates its activity, thereby leading to decreased levels of cAMP and less PKA activity [60].

The Ras/PKA pathway inhibits catabolic and growth-refractory processes in S.

cerevisiae

1.2.3.1 The Ras/PKA pathway inhibits transcription of genes involved in nutrient

limitation, stress response, and stationary phase entry

Some of the earliest PKA substrates identified were metabolic enzymes involved in

glucose metabolism. When glucose is plentiful, PKA phosphorylates and inhibits enzymes involved in the process of gluconeogenesis [61-63]. These enzymes include glycogen synthase (Gsy2p) [64], glycogen phosphorylase (Gph1p), trehalose phosphate synthase (Tps1p), and trehalase (Ath1p and Nrh1p) [65-69].

When the conditions are favorable for growth, PKA inhibits transcription of a number

of genes induced upon starvation or stationary phase entry through the zinc-finger

transcription factors Msn2 and Msn4 [70, 71]. These transcription factors recognize

cAMP-responsive elements, the STRE (mentioned earlier), in the promoters of those

genes [72-74]. PKA achieves this inhibition by regulating Msn2 localization through direct phosphorylation [47, 75].

During growth, PKA also inhibits expression of several stationary phase-specific genes through regulation of the transcription factor Gis1. Gis1 recognizes sequences, known as the Post-Diauxic Shift (PDS) elements [76], in the promoters of its target genes.

Gis1 activity is upregulated by the Rim15 kinase [77], a downstream target of PKA that is required for proper entry into stationary phase [78]. When nutrients are available,

PKA phosphorylates Rim15 to keep it inactive. Upon nutrient limitation, PKA activity

11 decreases, and Rim15 phosphorylates and activates Gis1, leading to derepression of

PDS target genes.

Finally, expression of a subset of genes induced upon starvation or stress appears to be

independent of either Msn2/Msn4 or Gis1. Recent research from our laboratory has

identified a novel mechanism by which PKA inhibits induction of stress and stationary

phase-related transcriptional programs, which could explain how these genes are

regulated by the Ras pathway. In addition to targeting particular transcription factors

and other effectors, PKA seems to be able to directly modulate the activity of the core

transcription machinery. The Srb protein complex, a core component of the RNA

polymerase II holoenzyme, is known to negatively regulate transcription of a subset of

stress-induced genes [79]. Data from our laboratory has shown that PKA directly

phosphorylates Srb9, a component of the Srb complex, and that this phosphorylation is essential for the transcriptional regulation of diauxic shift and stationary phase entry

genes [80].

The Ras/PKA pathway inhibits autophagy

The observation that high levels of extracellular cAMP inhibited autophagy in yeast

was the first hint to the existence of a functional link between this process and the

Ras/PKA pathway [81]. Subsequently, work from our lab showed that elevated levels of

Ras signaling such as those in cells harboring the hyperactive RAS2v19 allele or a TPK1

gene on a high copy plasmid resulted in the inhibition of autophagy [82]. This inhibition

appeared to occur in the early steps of the pathway, as normal transport intermediates

that form in the initial, induction phase of autophagy were not observed in RAS2v19

12 Cells [82]. Conversely, inhibition of Ras signaling resulted in the induction of

autophagy even in rich media [82]. Autophagy and its regulation will be discussed

extensively in section 1.3.

The Ras/PKA pathway regulates growth by promoting anabolic processes

When glucose is available, PKA not only inhibits gluconeogenesis, but also actively promotes the activity of enzymes involved in glycolysis, such as fructose-1,6-

bisphosphate (Fbp1p) [83] and phosphofructokinase 2 (Pfk26p) [84].

Upstream and downstream of the Ras/PKA pathway

As for Tor, the mechanism by which the Ras/PKA pathway senses nutrient status is still

unknown. Intriguingly, recent work has identified a G-protein-coupled receptor (GPCR)

system, consisting of a receptor Gpr1 and its Gα protein Gpa2, that appear to be

required for cAMP production in response to extracellular glucose. This GPCR system

signals to Cyr1 in a mechanism similar to that seen in higher eukaryotes. It has been

suggested that Gpr1 might bind glucose directly [85, 86]. This finding, however, has not

helped further our understanding as to the precise nature of the regulation of the Ras

proteins or the exact signals they respond to.

As mentioned above, several in vivo targets of PKA have been identified: these number

about twenty and include, but are not limited to: Rim15, Msn2, Yak1, Cdc25, Cki1,

Bcy1 and Srb9. Considering the number of known PKA substrates, it could be argued

that PKA is one of the most-well characterized kinases in the eukaryotic proteome.

However, many other targets of PKA remain unknown. By comparing the target

13 sequences phosphorylated by PKA, and through a number of other studies, a general

PKA consensus site was derived: this PKA target site has the sequence R-3-R-2-x-1-S/T-

B+1 , where “R” refers to an arginine residue, “x” refers to any amino acid, “S/T” refers

to a serine or a threonine residue that is the site of phosphate addition, and “B” to a

residue with a hydrophobic side chain [87, 88]. This site can in theory be used to look for putative substrates of PKA, but is not an absolute predictor of PKA phosphorylation

since proteins that contain it are not necessarily bona fide PKA targets.

Interplay between the Ras/PKA and Tor pathways

It will have already become apparent to the reader that the Ras/PKA pathway and the

Tor pathway seem to perform similar functions in the cell (Figure 1.2). One the major

challenges in the yeast signaling field today is to understand how different inputs from

both those signaling pathways are integrated by the cell. To this end, the precise

interrelationship between the two pathways must be determined at three levels: first,

what lies upstream of Ras and Tor in the signaling cascade. Second, what is the nature

of the crosstalk, if any, between the two pathways. Third, a more or less complete

picture of all the downstream effectors the two pathways impinge upon. Current

knowledge is very limited regarding those three questions. One emerging paradigm,

however, is that PKA and Tor jointly control growth by independently targeting the same proteins. For example, Tor regulates nuclear export of the Msn2/4 complex, while

PKA regulates its nuclear export [27, 47, 75]. Similarly, Tor regulates Rim15 localization while PKA affects Rim15 kinase activity [89, 90]. Finally, large scale

14 transcriptional profile studies also support the model where PKA and Tor act in parallel

to control several gene clusters [24, 91].

However, crosstalk between the pathways has not been ruled out. In fact, recent work

has shown that TORC1 inhibition leads to Tpk1 accumulation in the nucleus where it is

more likely to form an inactive complex with its regulator Bcy1 [92-94]. Finally, it is

intriguing that both Tor1 and Tor2 contain exact matches to the PKA target consensus

phosphorylation site.

Autophagy and its regulation in S. cerevisiae

Autophagy is an important process of macromolecular degradation that is essential for the survival of starved cells. Autophagy is induced by carbon or nitrogen limitation, and allows the cell to sustain long periods of starvation. This is accomplished through the degradation and recycling of energy-consuming or superfluous components into building blocks, for use in cellular processes critical for survival. Interestingly, inhibition of Tor with rapamycin or inactivation of the Ras/PKA pathway both lead to the induction of autophagy, suggesting that Tor and PKA inhibit this process in conditions conducive to growth. Autophagy is highly conserved from yeast to man, and has been linked to development, cell death and aging. Defects in autophagy have been associated with a number of diseases including cancer, Hungtinton’s disease and

Alzheimer’s disease [95, 96]. These findings have sparked tremendous interest in this degradative process. In this section, we give a brief historical perspective on the beginnings of the autophagy field, from the initial studies in mammals to molecular

15 characterization in yeast. We then describe the molecular machinery involved in this

multistep pathway. Finally, we comment on the regulation of autophagy by the

Ras/PKA pathway and the Tor pathway.

Historical perspective

Autophagy (Self-Eating) was initially characterized by morphological studies in mammalian cells in the 1950s. Electron microscopic analysis showed double-membrane bound vesicles, later called autophagosomes, forming around bulk portions of the cytoplasm and even whole organelles, and then fusing with lysosomes where their contents were “digested” [97-100]. It wasn’t until 1992 that autophagy was described in the budding yeast by Yoshinori Ohsumi’s laboratory in Japan. This initial study showed that yeast autophagy shared all the hallmarks of its mammalian counterpart [101]. This observation opened the door to the study of autophagy in a tractable genetic organism.

The first genetic screen looking for autophagy genes was performed in 1993 by the

same laboratory. This screen was based on the morphological identification of mutants defective in accumulation of autophagic bodies (autophagosomes devoid of their outer membrane) in the vacuole, which is the yeast equivalent of the mammalian lysosome.

This laborious visual identification approach identified mutations in a gene, ATG1

(AuTophaGy-related gene 1), that resulted in defects in autophagic body accumulation

[102]. Characterization of ATG1 showed that it was needed for autophagy in yeast. atg1

cells failed to form spores and exhibited a marked loss of viability upon nutrient

starvation, suggesting that autophagy was needed for stationary phase survival [102].

Using this loss of viability phenotype, Ohsumi’s laboratory identified 14 other ATG

16 genes that were essential for autophagy [102]. This number has since risen to 31 ATG genes known to date [103].

Molecular description of autophagy

Even though autophagy is a dynamic process, it can for the sake of a better description be divided into discrete steps, namely induction of the process, formation and expansion of the autophagosomes, docking and fusion of these vesicles with the vacuole, and finally degradation of the autophagosome contents (Figure 1.3). Because loss of particular Atg proteins blocks the pathway at a specific corresponding step, the various

Atg proteins can be classified into functional groups essential for the completion of particular stages in the process. In this section, we describe each of the steps of autophagy and identify the Atg proteins essential at each of these junctures.

Induction of autophagy

The induction of autophagy occurs upon starvation and is essential for the initiation of autophagosome formation, which occurs at a poorly-defined organelle, the Pre-

Autophagosomal-Structure (PAS) [104, 105]. The Atg1 kinase complex, that includes

Atg1, Atg13 and Atg17, plays a critical role in the induction of autophagy by linking nutrient-status to the autophagy machinery. In nutrient rich conditions, Atg1 and Atg13 are found in the cytoplasm, in contrast to most other Atg proteins that are constitutively localized at the PAS. In these conditions, Atg13 is hyperphosphorylated and has very low affinity towards Atg1 [106-108]. Upon starvation, which signals the induction of autophagy, Atg13 is very rapidly dephosphorylated and interacts with Atg1 [106-108],

17 leading to a marked increase in Atg1 kinase activity [107, 109, 110]. No substrates for the Atg1 kinase, beside itself, have been identified to date, and the precise mechanism by which the Atg1 complex signals the start of the autophagy process thus remains unknown. Under starvation conditions, Atg1 and Atg13 localize to the PAS [104, 105] where they interact with Atg17 [111, 112]. A recent study has shown that several Atg proteins fail to localize properly to the PAS in atg17 cells, suggesting that Atg17 plays a critical role in recruitment of other Atg proteins to that organelle [113]. The Atg1 protein complex also performs additional functions in later phases of autophagy.

Autophagosome formation and expansion

Immediately after induction of autophagy, the autophagosomes begin to form at the PAS and grow around bulk portions of the cytoplasm. The source of the membrane used for autophagosome formation is unknown, although the ER or the mitochondria have been suggested as possible membrane donors. The formation and expansion step is quite complex and involves multiple proteins and protein complexes, including the Atg1 complex, Atg9, Atg23, the Vps34 Phosphatidylinositol 3-Kinase Complex I [114] and two ubiquitin-like conjugation systems, Atg8-phosphatidylethanolamine (Atg8-PE) and

Atg12-Atg5 [115]. Formation of the Atg12-Atg5 conjugate requires the E1-like

(ubiquitin-activating) Atg7 [116] and the E2-like (ubiquitin-conjugating) Atg10 [117].

The Atg12-Atg5 conjugate then binds Atg16 and forms a multimeric Atg12-Atg5/Atg6 complex [118]. Formation of the Atg8-PE conjugate involves Atg7 and the E2-like Atg3

[119].

Both Atg9 and The Vps34 Complex I are important for the recruitment of several

18 proteins to the PAS, including Atg18 [120], the Atg12-Atg5/Atg16 complex [104] and

Atg8-PE [104]. Similarly, the Atg12-Atg5/Atg16 complex is important for the recruitment of Atg8-PE to the PAS [104]. During vesicle expansion, the Atg12-Atg5-

Atg16 may play a role in determining vesicle curvature before dissociating from the autophagosome [121], while it seems the Atg8-PE conjugate remains associated with the complete vesicle [122] and forms a structural component that could ultimately regulates autophagosome size [121].

The Atg1 kinase complex also plays a role in the formation and expansion step. Indeed,

Atg17 plays a role in autophagosome size as atg17 cells form small, aberrant

autophagosomes [111, 112]. Furthermore, the Atg1 complex is essential for recycling of

the membrane proteins Atg9 and Atg23 [123]. These two proteins are found in

punctuate structures throughout the cytoplasm [123-125]. Some of these structures

correspond to the PAS, while the others have been shown to be mitochondria, at least

for Atg9 [126]. Atg23 and Atg9 constantly cycle between the PAS and those spots, and

this cycling has been hypothesized to involve lipid delivery from the mitochondria to

the autophagosome for membrane growth. This cycling appears to be regulated by the

Atg1 complex as Atg9 and Atg23 are found only at the PAS in atg1 or atg13 cells [123].

Docking and fusion

Atg5 is known to dissociate from the autophagosome immediately prior to or right after

its completion [127]. This has been suggested to expose factors on the autophagosome

membrane that lead to docking and fusion with the vacuole, and the subsequent release

of the autophagic body into the vacuolar lumen [121].

Degradation

The membrane of the autophagic body is degraded first, and this requires the putative

lipase Atg15 [128, 129]. The contents of the vesicle are then broken down by the

resident vacuolar hydrolases Prb1 and Pep4 [101].

Regulation of autophagy

Since autophagy is responsible for turnover of large portions of the cell, full induction

of this process during normal growth is probably deleterious to the cell. As a result,

autophagy is very tightly regulated by the concerted action of several signaling pathways. This regulation ensures that autophagy only takes place at times where it is needed by the cell.

The Tor signaling pathway regulates the induction phase of autophagy by regulating the

Atg1 kinase complex. This is accomplished through Tor-dependent phosphorylation of

Atg13 [107, 109], which prevents Atg1 complex formation and inhibits autophagy.

Upon Tor inactivation with rapamycin, Atg13 is very rapidly dephosphorylated, the

Atg1 kinase complex is active and autophagy is induced, even in nutrient-rich media

[107]. Tor also negatively regulates the transcription of several ATG genes through the transcription factor Gln3.

The Ras/PKA pathway also appears to inhibit early steps of autophagy. A study by our lab has shown that high Ras levels blocked autophagy before the autophagosome expansion step [82]. Conversely, inhibition of the Ras/PKA pathway resulted in the induction of autophagy in nutrient-rich conditions [82].

20 Finally, the Snf1 kinase which is required for derepression of genes normally inhibited by glucose, appears to positively regulate autophagy in yeast [129].

Figure 1.1: Different functions of the TORC complexes in the regulation of growth. The arrows indicate positive regulation of the process, while the T-shaped lines indicate inhibition.

Figure 1.2: Interrelationship between the Ras and Tor pathway in the control of growth.

Figure 1.3: The steps of the autophagy process. An autophagosome originates from the Pre-Autophagosomal Structure, grows and engulfes cytoplasm and organelles, and fuses with the vacuole for degradation.

CHAPTER 2

AN EVOLUTIONARY PROTEOMICS APPROACH IDENTIFIES

SUBSTRATES OF THE CAMP-DEPENDENT PROTEIN KINASE

***Note: this chapter was published as a paper in which Yelena Budovskaya and I are co-authors. She is responsible for the sequence comparisons and has contributed equally to this work.

2.1. INTRODUCTION

Protein kinases are key components of signal transduction pathways that regulate many aspects of eukaryotic biology [130, 131]. The protein kinase gene family is one of the largest in eukaryotic organisms and typically constitutes almost 2% of all protein- encoding genes [132, 133]. In general, these enzymes catalyze the transfer of the terminal phosphate from ATP to the hydroxyl group of particular serine, threonine or tyrosine residues in a defined set of protein targets. This phosphorylation ultimately alters cell physiology by modifying the activities associated with these substrate proteins. A complete understanding of the biology of any protein kinase therefore 25 requires the identification of the particular substrates of this enzyme. Unfortunately, this identification process is often a difficult and labor-intensive task and, as a result, we generally know few of the physiologically-relevant substrates of any protein kinase

[134].

The cAMP-dependent protein kinase (PKA) has been extensively studied and is one of the best understood members of the protein kinase family [135, 136]. In S. cerevisiae,

PKA is a key regulator of cell growth and is regulated largely by the small GTP-binding

Ras proteins [52, 71, 137]. The two Ras proteins, Ras1 and Ras2, bind to adenylyl cyclase and stimulate the production of cAMP [138, 139]. This stimulation results in elevated PKA activity and the increased phosphorylation of substrates that are presumably important for cell growth and proliferation [59]. Although several PKA substrates have been described, the biological activities of these proteins are not sufficient to explain the global effect that PKA activity has on S. cerevisiae growth.

In the past decade, there has been a tremendous accumulation of DNA sequence information for a wide variety of organisms. One of the major challenges for modern biology is the development of methods to mine the information inherent in these data so as to further our understanding of basic biology and to provide insights into human disease. Comparative analyses between related species is one approach that appears to hold great promise in this pursuit. By comparing the DNA sequence of organisms separated by a range of evolutionary distances, experimenters have been able to identify important features of both entire genomes and individual genes and their protein products [140-144]. In this report, we describe a comparative approach that uses sequence information to identify the biologically-relevant occurrences of a protein motif

26 of interest. In this approach, the evolutionary conservation of all occurrences of a

particular sequence element in the proteome is systematically assessed within a group of

related organisms. The underlying premise is that a higher degree of sequence

conservation would identify those elements that are functional in vivo [142, 144].

The general utility of this approach was assessed here by examining whether the

evolutionary conservation of a consensus phosphorylation site would identify

physiologically relevant substrates of a particular protein kinase, PKA in S. cerevisiae.

By comparing the sequences of orthologous proteins present in a series of related

budding yeast species, we identified 44 candidate substrates for this protein kinase. A

phosphorylation analysis indicated that all of these candidates can be phosphorylated by

PKA in vitro and suggested that these proteins might be in vivo targets of this enzyme.

A more detailed analysis of one particular target, the autophagy-related protein kinase,

Atg1, showed that this protein was phosphorylated and regulated by PKA in vivo. In

all, these data demonstrate the general potential this type of a comparative approach has

for determining the physiological relevance of any sequence element found in any type

of protein.

2.2. MATERIALS AND METHODS

Protein sequence comparisons

The pattern match program, PatMatch, at the SGD website (www.yeastgenome.org) was

used to identify the consensus PKA sites present in the S. cerevisiae proteome. The

proteins containing these PKA sites were then aligned with their likely orthologs from

27 the other budding yeast species used in this analysis with the Blastp and Dialign

alignment programs. The final sequence alignments were also examined by eye to

ensure that no conserved PKA site had been missed. The protein sequences for the five

Saccharomyces species used in this analysis were obtained from the web site for the

Genome Sequencing Center at Washington University (genome.wustl.edu). The

Candida albicans sequences were obtained from the CandidaDB website

(www.pasteur.fr/ Galar_Fungail/CandidaDB) developed by the Galar Fungail European

Consortium. The PKA consensus site used here, R-3-R-2-x-1-S/T-B+1, was deduced from

a variety of studies including work with combinatorial peptide libraries and an analysis

of known PKA target sites [145-148]. In this site, “x” refers to any amino acid, “B” to a

residue with a hydrophobic side chain and the “S/T” to the serine or threonine residue

that is the site of phosphate addition. A second consensus site of R-6-x-5-x-4-R-3-x-2-x-1-

S/T-B+1 has been identified for mammalian PKA enzymes [149]. However, since it is not yet known if this site is also recognized by the S. cerevisiae enzyme, we have focused on the former consensus site in this study. Also, it should be pointed out that previous studies have indicated that PKA phosphorylation can occur at sequences that differ from both of these potential consensus sites. Such potential targets would also be missed by this analysis.

Alkaline phosphatase-based autophagy assays

Autophagy levels were measured with an alkaline phosphatase-based assay that has been described [82, 150]. Autophagy was induced by transferring cells to a medium

28 that lacks a nitrogen source, SD-N, and alkaline phosphatase levels were assessed after

0 and 15 hrs at 30OC.

Analysis of protein phosphorylation

In general, the in vitro phosphorylation assays were performed with glutathione S-

transferase (GST) fusion proteins that were under the control of the GAL1 promoter in the yeast strain, Y258 (MATa his4-580 ura3-52 leu2-3,112 pep4-3) [151]. The strains were grown to mid log phase in raffinose medium and transferred to galactose for 4 hrs at 300C. The GST fusion proteins were then isolated on glutathione-agarose beads

[152] and incubated with 1 µCi [γ-32P] ATP (Perkin Elmer) and either 0 or 5 U of

bovine PKA catalytic subunit (Sigma) as described [80]. A Western immunoblot was

performed with an α-GST antibody (Cell Signaling) to quantify the relative amount of

GST fusion protein present.

The Protein A (PrA)-Atg1 fusion proteins were constructed by subcloning PCR fragments encoding Atg1 residues 345-559 into a plasmid, pPHY1044, that contains two repeats of the immunoglobulin binding region of PrA from Staphylococcus aureus

[153]. The HA-tagged full-length Atg1 and PrA-Atg1 fusion proteins were expressed in

the protease-deficient strain, TVY614 (MAT∆ his3-∆200 leu2-3,112 ∆lys2-801 suc2-∆9 trp1-101 ura3-52 prc1∆::HIS3 pep4∆::LEU2 prb::hisG) [154]. The site-directed mutageneses were performed as described [153, 155, 156]. For the in vivo phosphorylation experiments, yeast cells were labelled with [32P] inorganic

orthophosphate [157] and the labelled PrA-Atg1 was precipitated as described [80].

29 The PKA-minus strain used for this analysis, NB13-14D (MATa ade8 his3 leu2 trp1

ura3 tpk1::URA3 tpk2::HIS3 tpk3::TRP1 rim15::kanMX2), has been described [78].

Fluorescence microscopy

The CFP-Atg11, RFP-Atg11 and YFP-Atg1 fusions were under the control of the inducible promoter from the yeast CUP1 gene [105]. Expression of these fusion

0 proteins was induced by the addition of 100 µM CuSO4 for 1 hr at 30 C. For the

starvation experiments, the cells were transferred to SD-N medium containing 100 µM

0 CuSO4 for 1 hr at 30 C. The GFP-Atg23 fusion protein has been described previously

[123]. The samples were imaged with an Axioplan 2 Imaging E Mot microscope (Carl

Zeiss, Inc.) equipped with a 100x Plan Neofluar objective (1031-172), filter sets

31044v2 (CFP), 41017 (Endow GFP), 41028 (YFP) (Chroma Technology Corp.) and

model C4742-95-12ERG CCD (Hamamatsu Photonics K.K). Image processing and

contrast enhancement were performed with OpenLab 3 (Improvision Inc.) and Adobe

Photoshop software.

2.3. RESULTS

Assessing the evolutionary conservation of the consensus PKA sites in the S.

cerevisiae proteome

The comparative analysis used here consisted of two major steps. In the first, a pattern

match program was used to identify the S. cerevisiae proteins that contain a consensus

PKA phosphorylation site (see Methods). For this study, we used the consensus site, R-

3-R-2-x-1-S/T-B+1, that had been defined by previous work with PKA enzymes from a

30 variety of sources, including yeast and humans [145-148]. A search of the S. cerevisiae proteome found 553 occurrences of this consensus sequence in 491 proteins (Table 2.2).

Fifty-one proteins were found to have multiple sites, with five sites being the most present in any one protein.

The second stage of this analysis assessed whether these consensus PKA sites were conserved in the likely orthologous proteins present in six different budding yeasts, including five Saccharomyces species that represent the three major subgroups of this genus and the pathogenic yeast, C. albicans (Figure 2.1A) [141, 158]. The

Saccharomyces species used in this analysis were the sensu stricto species, S. mikatae,

S. kudriavzevii and S. bayanus, the sensu lato species, S. castellii and the petite-negative species, S. kluyveri. It is important to point out that recent work has indicated that PKA activity is regulated, at least in part, by the Ras proteins in C. albicans, the yeast that is most distantly related to S. cerevisiae in this analysis [159, 160]. These observations therefore suggest that a Ras/PKA signaling pathway is functional in all of the yeasts being used for this study.

As expected, this analysis found that the number of conserved PKA sites dropped as the evolutionary distance from S. cerevisiae increased (Figure 2.1A). Only 92 of the original 553 sites (~17%) present in S. cerevisiae were conserved in the other

Saccharomyces species and C. albicans. The 85 proteins that contain these conserved sites are involved in a wide variety of processes important for cell growth (Table 2.3).

This observation is consistent with both the highly pleiotropic phenotypes associated with mutations affecting the Ras/PKA pathway and with a model proposing that

Ras/PKA signaling activity might be functioning as part of a general growth checkpoint

31 mechanism in S. cerevisiae [71, 137]. It is important to point out that many of these 85 proteins are highly conserved amongst these budding yeasts, and thus it was unclear whether the observed conservation of their PKA sites was significant. Therefore, we limited the subsequent analysis to those candidates that possessed a conserved PKA site in a region exhibiting less than 50% identity between the S. cerevisiae and C. albicans

proteins. This constraint reduced the number of potential PKA substrates to 44 proteins,

or less than 1% of the total proteome (Table 2.1). Significantly, these candidates

included five of the best-characterized PKA substrates in the S. cerevisiae literature

(Table 2.1).

The candidates identified by the evolutionary proteomics approach were

phosphorylated by PKA in vitro

The underlying premise in this analysis was that the more highly conserved PKA

consensus sites would be correspondingly more likely to represent bona fide PKA

phosphorylation sites. To test this hypothesis, we asked whether proteins with highly

conserved sites were more likely than other S. cerevisiae proteins to be phosphorylated

by PKA in an in vitro assay. Representative proteins from five different groups were

examined: proteins lacking a PKA consensus site; proteins with sites only in S.

cerevisiae; proteins with sites conserved amongst the sensu stricto Saccharomyces

species; proteins with sites conserved to the sensu lato and petite-negative

Saccharomyces species; and proteins with sites conserved to C. albicans.

In general, we found that the likelihood of phosphorylation correlated well with the

degree of conservation of the PKA consensus sites. In particular, all of the candidates

32 tested with sites conserved to C. albicans (23/23) were phosphorylated by PKA (Figure

2.2A,E). In contrast, only 20-33% of the proteins containing less conserved sites were labelled in this assay (Figure 2.2B,C). Finally, none of the proteins that lacked a

consensus site were efficiently phosphorylated by PKA (Figure 2.2D). Thus, the presence of a highly conserved PKA consensus site was a very strong predictor of PKA phosphorylation (Figure 2.2E). The striking correlation between the conservation of the

PKA site and its tendency to be phosphorylated suggested that the candidates with the most highly conserved sites might all be in vivo targets of PKA. Consistent with this

prediction, five of these candidate proteins, Bcy1, Cki1, Msn2, Rim15 and Yak1, have

been previously shown to be substrates of this kinase (Table 2.1) [47, 78, 161-163].

Finally, it is important to point out that several proteins with less conserved sites were

phosphorylated by PKA in this study and could also be relevant targets of this enzyme

in vivo (Figure 2.2B-D). The key point here is that the degree of evolutionary

conservation can be used as a predictive tool to assess the likely physiological relevance

of a given sequence motif in the proteome.

The autophagy-related protein kinase, Atg1, is a substrate of PKA

As a proof of principle, we tested whether the function of one particular candidate was

indeed regulated by PKA phosphorylation in vivo. For this analysis, we chose to

examine the autophagy-related protein kinase, Atg1. Autophagy is a highly conserved,

membrane trafficking pathway responsible for much of the protein and membrane

turnover in eukaryotic cells [164, 165]. Atg1 is a serine/threonine-specific protein

kinase that is a key regulator of the initial induction stage of this degradative pathway

33 [164, 166]. Interestingly, three proteins important for autophagy, Atg1, Atg13 and

Atg18, were all identified in this study as potential substrates for PKA. These

observations suggested that the Ras/PKA pathway was regulating autophagic activity in

yeast cells and recent work from our lab has confirmed this possibility (Figure 2.3A)

[82].

Atg1 has two conserved sequences, R-R-P-S508-L and R-R-L-S515-I, that conform to the

PKA consensus site discussed above. Interestingly, we found that the full-length Atg1 was phosphorylated in vitro by PKA and that this phosphorylation required the presence of the serine residues in these two consensus sites (Figure 2.3B; Figure 2.5). The ability

of PKA to phosphorylate an Atg1 fusion protein in vitro was also dependent upon these

same serine residues (Figure 2.3C). Finally, the in vivo phosphorylation of this Atg1

fusion protein also required the presence of the two PKA consensus sites and PKA

activity (Figure 2.3D). In mutants lacking all three of the PKA catalytic subunits, Atg1 was not phosphorylated appreciably at Ser508 or Ser515. In all, these data indicated that Atg1 was likely a bona fide substrate for PKA in S. cerevisiae.

PKA phosphorylation regulates the association of Atg1 with the pre-

autophagosomal structure (PAS)

Two observations indicated that Atg1 protein kinase activity was not regulated by PKA

phosphorylation. First, the loss of the consensus PKA sites had no significant effect on

Atg1 protein kinase activity in vitro in assays measuring either autophosphorylation or

the phosphorylation of an exogenous substrate (Figure 2.6A). Second, the in vivo level

of Atg1 autophosphorylation was not affected by alterations of the two PKA sites in this

34 protein (Figure 2.6B, which is published as supporting information on the PNAS web

site). Thus, PKA phosphorylation must be affecting some other aspect of Atg1 function

in vivo. Since previous work had indicated that the subcellular localization of Atg1 is

influenced by nutrient availability, we tested whether PKA might be regulating this facet

of Atg1 behaviour.

Atg1 is predominantly cytoplasmic in dividing cells but is recruited to a specialized site,

known as the PAS, upon the induction of autophagy (Figure 2.4A) [104, 105]. The PAS

is thought to be the site of autophagosome formation; the autophagosome is the double-

membrane intermediate responsible for transporting bulk cytoplasm to the

vacuole/lysosome during autophagy [104, 105, 164]. We therefore tested whether PKA

phosphorylation might control the association of Atg1 with the PAS. In support of this possibility, we found that an Atg1 variant lacking both PKA sites, Atg1-AA, was constitutively localized to the PAS (Figure 2.4A). In addition, the introduction of a constitutively-active allele of RAS2, known as RAS2val19, blocked Atg1 localization to

the PAS in a manner that was dependent upon the two PKA sites in Atg1 (Figure 2.4A).

In the RAS2val19 mutant, wild-type Atg1 was not found at the PAS in either growing or

starved cells. PKA phosphorylation therefore appears to control the recruitment of Atg1

to the PAS during nutrient limitation, conditions that normally induce the autophagy

pathway.

The data here suggest that when nutrients are plentiful, Ras/PKA signaling levels are

high and Atg1 is phosphorylated and largely cytoplasmic. Upon nutrient deprivation,

Atg1 would become dephosphorylated and associate with the PAS. This model is

consistent with previous work indicating that PKA activity decreases upon nutrient

35 limitation [167]. We tested the basic tenet of this model by examining the in vivo

phosphorylation of an Atg1 fusion protein in dividing and starved cells. The

phosphorylation of this fusion is completely dependent upon the presence of the PKA

consensus sites and PKA activity (see above). Consistent with the model, we found that

the relative level of Atg1 phosphorylation decreased by more than 10-fold following a

period of nitrogen starvation (Figure 2.4B).

We also tested whether the presence of the RAS2val19 allele had any effect on the

subcellular localization of Atg23. Atg23 is a peripheral membrane protein that has been

shown to cycle between the PAS and unknown structures in the cytoplasm in an Atg1-

dependent manner [123]. In wild-type cells, an Atg23-GFP fusion is found associated

with a number of punctate foci in cells, some that correspond to the PAS and others that

do not (Figure 2.4C) [125]. In contrast, in atg1 mutants, Atg23 is found at the PAS

only and not with the other punctate structures seen in wild-type cells [123]. These

results have been interpreted as evidence that Atg1 is required for the recycling of

Atg23 from the PAS. Consistent with a role for the Ras/PKA pathway in the regulation

of this Atg1 activity, we found that Atg23 was also restricted to the PAS in RAS2val19 mutants (Figure 2.4C). Since Atg1 (and its regulatory partner, Atg13) is the only Atg protein known to be required for the proper recycling of Atg23, these data reinforced the above phosphorylation analysis and suggested that the Ras/PKA pathway was regulating Atg1 activity in S. cerevisiae cells.

2.4. DISCUSSION

This report makes use of a sequence-based, comparative method for finding

functionally relevant sequences within an eukaryotic proteome. In this approach, the evolutionary conservation of all occurrences of a given sequence motif is systematically assessed within a group of related organisms. The underlying premise is that the physiologically-relevant motifs would be distinguished by a higher degree of sequence conservation. The general utility of this approach was assessed here with an attempt to identify candidate substrates for the protein kinase, PKA, in S. cerevisiae. In this study,

the conservation of consensus PKA target sites was assessed within a group of budding

yeast species that are separated by up to 800 million years of evolutionary distance

[168]. This analysis identified 44 proteins as potential substrates of PKA and,

remarkably, we found that all of the candidates tested were phosphorylated by this

enzyme in an in vitro assay. Moreover, our data indicated that the degree of sequence

conservation was a very strong predictor of the likelihood of PKA phosphorylation.

The more highly conserved the PKA site, the more likely it was to be phosphorylated by

PKA. In all, the data suggested that this evolutionary proteomics approach had

successfully identified a number of novel substrates of the S. cerevisiae PKA.

Similar types of comparative approaches have been used extensively to identify

functional motifs in individual proteins. However, this study emphasizes how this

strategy can be systematically applied over an entire proteome. In this case, we were

examining a short sequence motif that was relatively low in information content.

37 Nonetheless, we were able to successfully identify proteins that had been previously

shown to be targets of PKA and many additional candidate substrates. In theory, this

approach should be applicable to any type of sequence element in any protein. In fact,

structure-function studies of proteins often identify similarly short, but biologically

interesting, sequence domains. This study demonstrates how the biological relevance of these elements can be assessed. The only prerequisite is that there must be sequence information available for an appropriate group of evolutionarily-related organisms.

The power of these comparative approaches is illustrated by the fact that more potential

PKA substrates were identified here than had been found in the past two decades by other means. Although further work is obviously needed to show that these proteins are

indeed regulated by PKA phosphorylation, this candidate pool already contains five

previously-identified substrates of PKA. The work presented here adds to this list by showing that the autophagy-related kinase, Atg1, is both phosphorylated and regulated

by PKA in vivo. Interestingly, the activities of several of the other candidates have

already been linked to the Ras/PKA signaling pathway. For example, a recent study has

suggested that Ras/PKA pathway regulates the activity of Ifh1 and Sfp1, two key

regulators of ribosome synthesis and cell size in S. cerevisiae [94, 169]. Finally, it is

important to point out that the proteins identified here are equally likely to be substrates

for PKA in the pathogenic yeast, C. albicans. This possibility is especially interesting

in light of recent work indicating that PKA activity is important for virulence in this

yeast [159, 160].

This study identified three proteins important for autophagy, Atg1, Atg13 and Atg18, as

potential substrates for PKA. These observations led us to investigate the role of the

38 Ras/PKA signaling pathway in the control of this degradative process. Our work

indicates that Ras/PKA activity inhibits an early step in autophagy, a step that precedes

the formation of the autophagosome [82]. Interestingly, all three of these Atg proteins

appear to act at this stage of the autophagy process [102, 120, 164]. In this study, we

found that the PKA phosphorylation of Atg1 regulates the localization of this protein in

accordance with nutrient availability and ensures that Atg1 is associated with the PAS

only under conditions that result in the induction of autophagy. Since most Atg proteins are present constitutively at the PAS, we feel that this PKA phosphorylation is likely

indirectly influencing Atg1 protein kinase activity by regulating its ability to associate

with its most likely substrates in the cell. Finally, it is important to point out that Atg1

does not appear to be the only PKA target important for the control of autophagy. This

assertion follows from our observation that the hyperactive RAS2val19 allele still inhibits

autophagy in mutants carrying the Atg1-AA variant (our unpublished observations). We

are presently testing whether Atg13 and/or Atg18 might be these other relevant targets

of PKA phosphorylation in vivo.

In summary, this report describes a systematic, sequence-based approach that uses a

basic tenet of the theory of natural selection to identify the functionally relevant

occurrences of a given sequence element in an eukaryotic proteome. The success we

had identifying potential substrates of the S. cerevisiae PKA illustrates the potential

inherent in this general strategy. In addition to identifying autophagy as an important

target of the Ras/PKA pathway, the other substrates identified here are likely to provide

fundamental insights into the manner in which this signaling pathway controls the growth of these budding yeasts. Finally, further comparisons between the consensus

39 sites that are phosphorylated by PKA and those that are not, could identify additional sequence elements that are important for PKA substrate recognition.

Figure 2.1: An evolutionary proteomics approach identified 85 potential substrates of PKA in S. cerevisiae. The relative number of S. cerevisiae candidates that have PKA sites conserved in the likely orthologous protein present in each of the indicated budding yeast species is shown. The approximate evolutionary distance to S. cerevisiae is shown for several positions on this phylogenetic tree.

Figure 2.2: Proteins with highly conserved consensus PKA sites were more likely to be phosphorylated by PKA than proteins with less conserved sites. Shown is a phosphorylation analysis of representative proteins from the following five groups identified by the comparative analysis performed here: (i) proteins with consensus PKA sites conserved to C. albicans (A); (ii) proteins with sites conserved to the sensu lato and petite-negative Saccharomyces species; (iii) proteins with sites conserved amongst the sensu stricto Saccharomyces species (B); (iv) proteins with PKA sites in S. cerevisiae only (C); (v) proteins with no consensus PKA sites (D). For this analysis, the amount of label incorporated into a full- length GST fusion protein from [ -32P]ATP was assessed with an in vitro assay performed in the presence or absence of PKA as described in Methods. Note that the labeled protein bands have been appropriately lined up to facilitate comparisons between samples. Western immunoblots were performed with an -GST antibody to quantify the relative amount of fusion protein present. (E) A summary graph indicating the percentage of candidates in each of the above groups that was phosphorylated by PKA.

Figure 2.3: The autophagy-related protein kinase, Atg1, is a substrate of PKA. (A) Elevated levels of Ras/PKA signaling activity inhibited autophagy. Autophagy levels were assessed with an alkaline phosphatase-based assay that has been described. The values shown represent the difference between the alkaline phosphatase levels found in starved and nonstarved cultures of the indicated yeast strains. HC- TPK1, high-copy plasmid encoding a catalytic subunit of PKA. (B) The full-length Atg1 was phosphorylated by PKA in vitro at the serine residues within the two consensus PKA sites. The residues at positions 508 and 515 within the two PKA sites are indicated: S, serine; A, alanine. Note that this experiment was performed with a kinase-inactive variant of Atg1, Atg1-K54A, to avoid the background autophosphorylation signal. (Lower) A Western immunoblot control indicating the relative levels of Atg1 present in each kinase reaction. (C and D) The in vitro (C) and in vivo (D) phosphorylation of an Atg1 fusion protein depended upon both PKA activity and the two PKA sites in Atg1. This fusion protein contained two repeats of the Ig-binding region of protein A fused in frame to residues 345–559 of Atg1. In both cases, the Upper panel is the phosphorylation assay and the Lower is the Western immunoblot control. For the in vivo experiments, yeast cultures were incubated with [32P]orthophosphate, and the amount of label incorporated into the PrA-Atg1 fusion proteins was assessed by autoradiography. The pka strain lacks all three catalytic subunits of the S. cerevisiae PKA enzyme.

Figure 2.4: PKA phosphorylation regulates the association of Atg1 with the preautophagosomal structure, or PAS. (A) A fluorescence microscopy analysis of the subcellular localization of a YFP-Atg1 fusion protein in either log-phase or starved cultures of the indicated strains. The location of the PAS is shown by a CFP-Atg11 reporter construct. (B) The level of Atg1 phosphorylation decreased upon nitrogen starvation. Wild-type cells containing either a control vector (Vector) or a plasmid encoding a Protein A-Atg1 fusion protein (pATG1) were incubated with [32P]orthophosphate either before (Log) or after (Starved) a 2-h incubation in a medium lacking nitrogen. The amount of radiolabel incorporated into Atg1 was assessed by autoradiography. (Lower) The Western immunoblot control. (C) The presence of the RAS2val19 allele resulted in the redistribution of a GFP-Atg23 fusion protein from a number of punctate structures within the cell to the PAS. The identity of these punctate structures is not yet known. The location of the PAS is indicated by an RFP-Atg11 fusion protein

Figure 2.5: The full-length Atg1 was phosphorylated by cAMP-dependent protein kinase (PKA) in vitro at the serine residues within the two consensus PKA sites. The residues at positions 501 and 515 in the two PKA sites are indicated: S, serine; A, alanine. Note that Atg1 is a protein kinase that catalyzes an autophosphorylation reaction. This autophosphorylation retards the mobility of this protein in SDS/PAGE gels and causes the wild-type Atg1 to run as a doublet. The slower migrating species is the autophosphorylated form of the protein. Note that PKA phosphorylation did not affect the mobility of Atg1.

Figure 2.6: Alteration of the serine residues within the PKA sites in Atg1 did not influence Atg1 protein kinase activity. (A) The indicated versions of Atg1 were precipitated, and in vitro phosphorylation reactions were performed as described in Methods. (Top) Autophosphorylation assay assessing label incorporation into Atg1. KD, kinase-dead. (Middle) An Atg1 in vitro phosphorylation assay using the myelin basic protein as substrate. (Bottom) A Western immunoblot control showing the relative levels of Atg1 present in the kinase reactions above. (B) Alteration of the consensus PKA sites did not influence the autophosphorylation of Atg1 in vivo. Shown is a Western immunoblot assessing the relative levels of the autophosphorylated and nonautophosphorylated forms of the indicated Atg1 variants.

Table 2.1: The 44 candidate PKA substrates identified by the evolutionary proteomics approach described here.

Table 2.2 is continued

Table 2.2: A list showing the proteins that contain the 553 consensus PKA sites present in the Saccharomyces cerevisiae proteome. The degree to which each of these sites is conserved among the six budding yeast species analyzed in this study is shown. The sites conserved out to Candida albicans (C. alb.) are highlighted. S. cer., S. cerevisiae; S. bay., S. bayanus; S. kud., S. kudriavzevii; S. mik., S. mikatae; S. cas., S. castelli.

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Continued

Table 2.2 continued

Table 2.3: The 85 candidate PKA substrates identified by the evolutionary proteomics approach described here

CHAPTER 3

THE TOR AND PKA SIGNALING PATHWAYS INDEPENDENTLY TARGET

THE ATG13 PROTEIN TO CONTROL AUTOPHAGY ACTIVITY

3.1. INTRODUCTION

Macroautophagy (hereafter referred to as autophagy) is a highly-conserved membrane trafficking pathway that is responsible for the turnover of bulk cytoplasmic protein and organelles [164, 170]. This pathway was initially identified as a cellular response to nutrient deprivation [171, 172]. However, recent studies indicate that autophagy is involved in a wide variety of physiological processes, including tissue remodeling during development, the removal of protein aggregates and innate immune responses

[103, 173]. Defects in autophagy may contribute to a number of human diseases,

including breast and ovarian cancers, Huntington’s disease and the cardiomyopathic

Danon disease [174, 175]. During autophagy, an isolation membrane emanates from a

nucleation site that is known as the pre-autophagosomal structure (PAS) in S. cerevisiae

76 and the phagophore assembly site in mammals [104, 176]. This double membrane

encapsulates nearby cytoplasm and ultimately targets it to the vacuole/lysosome for

degradation. The breakdown products are then recycled to allow for the synthesis of the

macromolecules needed for survival during the period of starvation [177]. The cellular

components mediating autophagy were initially described in S. cerevisiae, and orthologs of most of these Atg proteins have since been identified in other eukaryotes

[178, 179].

The flux through the autophagy pathway is tightly controlled by multiple signaling

pathways that are responsible for coordinating cell growth with nutrient availability. In

S. cerevisiae, the Tor and cAMP-dependent protein kinase (PKA) pathways play critical

roles in this regulation. The primary target of these pathways appears to be a complex

of proteins that contains the Atg1 protein kinase [81, 82, 107, 180]. Atg1 is specifically recruited to the PAS, and activated, in response to conditions that induce autophagy

[104, 105]. In contrast, most Atg proteins are constitutively localized to this nucleation

structure [181]. Recent work suggests that the mammalian Atg1 proteins, ULK1 and

ULK2, are also recruited to the phagophore assembly site, and activated, upon nutrient

deprivation [182, 183]. A key question that remains is how do these different signaling

pathways work together through Atg1 to ensure the appropriate autophagic response.

A key component of the Atg1 complex is Atg13, a protein that physically interacts with

Atg1 and is required for full kinase activity in vitro [107, 109]. Although the

mechanistic basis of this activation is not yet understood, the Atg1-Atg13 interaction

appears to be regulated by Tor signaling activity. Atg13 is hyperphosphorylated in

growing cells and this phosphorylation is lost upon inactivation of the Tor pathway

77 [107]. Here, we show that Atg13 is also a substrate of PKA, and that this

phosphorylation regulates the association of this protein with the PAS. In all, the data

suggest that the Tor and PKA pathways independently regulate autophagy, and that

Atg13 is a key point of signal integration in this process. Finally, our data indicate that

these signaling pathways are responding to different nutritional cues and that each

regulates distinct aspects of the autophagy process. A model that attempts to explain

how these different inputs might be working together to control the autophagy pathway

is presented.

3.2. MATERIALS AND METHODS

Growth Media

Standard E. coli growth conditions and media were used throughout this study. The

yeast rich growth medium, YPAD, consists of 1% yeast extract, 2% Bacto-peptone, 50

mg/l adenine-HCl, and 2% glucose. The yeast YM glucose and SC glucose minimal

growth media have been described [80, 184]. The nitrogen starvation medium, SD-N,

consists of 0.17% yeast nitrogen base lacking amino acids and ammonium sulfate, and

2% glucose. The carbon starvation medium was SC minimal medium without the

added glucose. Growth media reagents were from DIFCO.

78 Plasmid Constructions

The construction and use of the MET3-RAS2val19 integrating plasmid, pPHY446, was

described previously [185]. The plasmid, pPHY2427, encoded an HA epitope-tagged

version of Atg13 under the control of the promoter from the copper-inducible CUP1

gene. The ATG13 locus was cloned from the plasmid, pRS316-ATG13, that was kindly

provided by Dr. T. Noda. A site-directed mutagenesis was performed to insert an XmaI

site immediately following the start codon of ATG13. The ATG13 coding sequences

and transcriptional terminator were then cloned as a 3.1 kb XmaI - NotI fragment into

pPHY2203 to form pPHY2427. The pPHY2203 plasmid was described previously and

contains the CUP1 promoter and the HA epitope [186]. For the YFP-Atg13 plasmid,

pPHY2395, a 3.4 kb XmaI - NgoMIV fragment of the above mutagenized ATG13

plasmid was subcloned in-frame into pPHY2297. This latter plasmid contains the YFP

locus under the control of the CUP1 promoter. The site-directed mutagenesis of the

ATG13 PKA sites was performed with the Transformer mutagenesis kit (Clontech), and

resulted in the substitution of alanine residues for the relevant serines found in the PKA

phosphorylation sites.

To construct the myc6-tagged Atg17 plasmid, pPHY2806, the ATG17 locus was

amplified by PCR from yeast genomic DNA and a 1.6 kb SalI – NotI fragment was

subcloned into pRS424. An XmaI site was then placed immediately downstream of the

ATG start codon by a site-directed mutagenesis, and a fragment encoding the myc6 epitope tag was inserted into this site to generate pPHY2806. To construct the CUP1 promoter-driven YFP-Atg17 plasmid, pPHY2777, the ATG17 open-reading frame was amplified by PCR from the above pRS424-ATG17 plasmid and subcloned in-frame

79 behind YFP in the pCUYFP(426) plasmid, kindly provided by Dr. D. Klionsky. The

high copy number TPK1 plasmid was created by subcloning the TPK1 gene under the

control of inducible CUP1 promoter in plasmid, pPHY2203.

Yeast strain construction and growth conditions

The yeast strains used in this study were PHY1220 (MAT∆ his3-∆200 leu2-3,112 lys2-

801 trp1-101 ura3-52 suc2-∆9), PHY1942 (PHY1220 prc1::HIS3 pep4∆::LEU2 prb1∆::hisG), TN125 (MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho8∆60), YYK126

(TN125 atg1∆::LEU2), YYK130 (TN125 atg13∆::TRP1) and PHY3687 (TN125 atg1∆::LEU2 atg13∆::kanMX) [107]. Unless otherwise noted, strains were from our lab collection or were derived during the course of this work. Standard yeast genetic methods were used for the construction of all strains [184]. Strains carrying the MET3-

RAS2val19 or MET3-RAS2ala22 alleles were grown in media containing 500 µM

methionine to keep the MET3 promoter in its repressed state. Expression from the

MET3 promoter was induced by transferring these cells to a medium that lacked methionine. Expression from the CUP1 promoter was induced by the addition of 100

µM CuSO4 to the growth medium.

Identification of PKA sites

The evolutionary proteomics approach used to identify the candidate PKA sites in

Atg13 was described previously [187, 188].

80 Western blotting and immunoprecipitations

Protein extracts for Western blotting were prepared by a glass bead lysis protocol described previously [82]. The resulting protein extracts were separated on SDS- polyacrylamide gels and transferred to nitrocellulose membranes (Hybond ECL,

Amersham Biosciences) at 4°C. The membranes were probed with the appropriate primary and secondary antibodies and the Supersignal chemiluminescent substrate

[152] was used to illuminate the reactive bands. The immunoprecipitation experiments were performed as described [80, 186].

Autophagy assays

The alkaline phosphatase (ALP) assay for autophagy activity was performed as described [82, 150]. This assay measures the delivery of an altered form of the Pho8 phosphatase by the autophagy pathway.

PKA phosphorylation assays

In general, the in vitro phosphorylation assays were performed with HA epitope-tagged proteins that were under the control of the yeast CUP1 promoter in the yeast strain,

PHY1942. The strains were grown to mid-log phase in selective SC minimal medium containing 2% glucose, and induced with 100 µM copper sulfate for 90 mins. The HA epitope-tagged Atg13 variants were isolated on an α-HA antibody resin (Roche), and incubated with [α-32P] ATP (PerkinElmer) and 5 U of bovine PKA catalytic subunit

(Sigma), as described previously [186, 187]. A Western immunoblot control was

81 performed with an α-HA antibody (Sigma) to assess the relative amount of Atg13 present in each sample.

The in vivo level of PKA phosphorylation was assessed with an α-PKA substrate antibody (Cell Signaling) as described previously [80, 186]. Briefly, the substrate proteins were isolated from yeast cell extracts with native or denaturing immunoprecipitation protocols and the precipitated proteins were separated on SDS- polyacrylamide gels. The relative level of occupancy at the PKA sites was assessed by

Western blotting with the α-PKA substrate antibody used at a concentration of 1:2000.

Fluorescence Microscopy

The CFP-Atg11, YFP-Atg13 and YFP-Atg17 fusions were under the control of the inducible promoter from the yeast CUP1 gene. Expression of these fusion proteins was induced by the addition of 100 µM CuSO4 for 1 h at 30°C. For the starvation

experiments, the cells were transferred to SD-N medium containing 100 µM CuSO4 for

1 h at 30°C. The samples were imaged with an Axioplan 2 Imaging E Mot microscope

(Zeiss) equipped with a X100 Plan Neofluar objective (1031-172) and filter sets

31044v2 (CFP), 41017 (Endow GFP), and 41028 (YFP) (Chroma Technology,

Rockingham, VT) and model C4742-95-12ERG charge-coupled device (CCD)

(Hamamatsu Photonics, Hamamatsu City, Japan). Image processing and contrast enhancement were performed with OPENLAB3 (Improvision, Lexington, MA) and

Photoshop (Adobe Systems, San Jose, CA) software.

82 3.3. RESULTS

The association of Atg13 with the PAS is regulated by PKA phosphorylation

A recent study identified Atg13 as a candidate substrate for PKA in S. cerevisiae [187].

This identification was based on the presence of evolutionarily conserved matches in

Atg13 to the PKA consensus phosphorylation site (Figure 3.1). Two sites were very

similar to the consensus of R-R-x-S/T-B, where x refers to any amino acid and B to a

hydrophobic residue [145]. A third conserved site that deviates more from the

consensus was also identified (Site 1, Figure 3.1a). Each of these sites was recognized

by PKA in vitro, and alteration of all three sites resulted in a >95% decrease in Atg13

phosphorylation (Atg13-AAA; Figure 3.1a). Similar results were observed in vivo with

an assay that uses an antibody that recognizes phosphorylated PKA sites (Figure 3.1b,c)

[80, 186]. For these experiments, an epitope-tagged Atg13 was immunoprecipitated

from cell extracts and the level of PKA phosphorylation was assessed by Western

blotting with this α-substrate antibody. This latter signal was lost following

phosphatase treatment of the precipitated Atg13 and when the Atg13 was isolated from

cells that lack PKA activity (Figure 3.1c, data not shown). Finally, this in vivo signal

was elevated in cells that contained a hyperactive allele of RAS2, known as RAS2val19

(Figure 3.1d) [49]. The S. cerevisiae Ras proteins, Ras1 and Ras2, regulate cAMP production, and thus PKA activity, by directly binding to, and stimulating adenylyl cyclase [138, 139]. In all, these data indicated that Atg13 was a direct substrate for

PKA.

83 We found that Atg13 was largely cytoplasmic in growing cells and was

associated with the PAS upon nutrient limitation (Figure 3.2). Here, we tested whether this localization to the PAS was regulated by PKA phosphorylation. A clear precedent exists as PKA phosphorylation has been shown to inhibit the PAS association of Atg1

[187]. Consistent with this possibility, the Atg13-AAA variant, unlike the wild-type protein, was localized to the PAS in both growing and nitrogen-starved cells (Figure

3.2). Moreover, the association of the wild-type Atg13 (Atg13-SSS) with the PAS was inhibited by presence of the RAS2val19 allele. This inhibition was dependent upon the

Atg13 PKA sites as the Atg13-AAA protein was still constitutively localized to the PAS

in RAS2val19 cells (Figure 3.2). Altogether, these data demonstrated that PKA

phosphorylation was inhibiting the association of Atg13 with the PAS.

Inactivation of the PKA pathway is sufficient to induce autophagy

Elevated levels of PKA activity have been shown to inhibit the autophagy process [81,

82]. Here, we tested whether inactivation of this pathway was sufficient to induce

autophagy, and compared the relative contributions of the PKA and Tor pathways. To

examine the consequences of inactivating the Ras/PKA pathway, we used an inducible

form of RAS2ala22, a dominant negative allele of RAS2 [189]. This allele was under the

control of the promoter from the MET3 gene, a locus that is repressed when methionine

is present in the growth medium [190]. We found that autophagy was efficiently

induced upon expression of the RAS2ala22 protein (Figure 3.3a,b). The kinetics and

magnitude of the response were similar to that seen upon inactivation of the Tor

pathway with rapamycin (Figure 3.3a). (Note that rapamycin specifically inhibits the

84 TORC1 complex and that we will be referring to this complex when we discuss Tor

signaling in this report [4]). Slightly different times of incubation were used for the

rapamycin and RAS2ala22 treatments to correct for the degree of growth arrest observed

in the respective cultures. To examine this further, we used a lower concentration of

rapamycin that resulted in a growth arrest profile that was very similar to that seen in

the MET3-RAS2ala22 cells (data not shown). Under these arrest conditions, the kinetics

and the magnitude of the autophagy responses were essentially identical (Figure 3.3b).

As with other autophagy-related processes, we found that the autophagy induced in

RAS2ala22 cells required the presence of Atg1 (Figure 3.3c). Finally, we observed higher levels of autophagy in cells that were treated with both rapamycin and the RAS2ala22 protein (Figure 3.3b). This latter result is consistent with these pathways working independently of each other, even though both appear to be targeting Atg13 (see below). In all, the data indicated that the PKA and Tor pathways make very similar contributions to the regulation of overall autophagy activity.

Atg13 is the primary target for PKA in the autophagy machinery

Our work to date has identified two proteins required for autophagy, Atg1 and Atg13, as substrates for PKA. To examine the relative importance of these substrates, we assessed autophagy activity in RAS2val19 cells that contained the variants, Atg1-AA

and/or Atg13-AAA, that were not phosphorylated by PKA. The RAS2val19 allele has

been shown to effectively inhibit starvation-induced autophagy [82]. We found that the presence of the Atg13-AAA variant was sufficient to fully reverse the inhibitory effects of RAS2val19 on autophagy (Figure 3.4). In contrast, the presence of Atg1-AA restored

85 very little, if any, autophagy activity to these cells (Figure 3.4). These results therefore

suggested that Atg13 was the primary target for PKA signaling in the autophagy pathway.

PKA phosphorylation influences the association with the PAS by interfering with the Atg13 interaction with Atg17

Previous studies have shown that Atg13 interacts with a number of proteins, most notably Atg1 and Atg17 [111, 112]. These studies have also suggested that Atg17 might play a key role in organizing the PAS [113, 191, 192]. Therefore, we tested whether PKA phosphorylation might be influencing the interaction between Atg13 and either Atg1 or Atg17. As reported, we found that the amount of Atg17 associated with

Atg13 increased upon nitrogen starvation (Figure 3.5a) [111]. However, this difference was lost in cells expressing the Atg13-AAA variant. Equivalent levels of Atg17 were detected in Atg13-AAA precipitates from both growing and starved cell extracts (Figure

3.5a). Moreover, the relative amount of Atg17 detected in either condition was greater than that associated with the wild-type Atg13. Furthermore, the presence of high PKA activity did not affect the Atg1-Atg13 interaction (Figure 3.5b) while high PKA activity affected the interaction of Atg13 with Atg17 (Figure 3.5c). Therefore, PKA activity was

specifically influencing the Atg13-Atg17 interaction. Finally, The Atg13-AAA variant

interacted with Atg17 to a similar extent whether PKA levels were high or not (Figure

3.5c). The above data suggested that the Atg13-AAA protein might be constitutively at

the PAS as a result of its increased interaction with Atg17. Consistent with this

possibility, we found that the Atg13-AAA variant was less efficiently targeted to the

86 PAS in cells that lacked Atg17 (Figure 3.6a). Atg17 was found to be associated with

the PAS in both growing and nitrogen-starved cells, and this localization was not

inhibited by the presence of the RAS2val19 allele (Figure 3.6b) [112, 191]. Therefore,

PKA phosphorylation was apparently regulating the PAS association of Atg13, at least

in part, by interfering with its interaction with Atg17.

We also tested whether Atg13 was required in vivo for the activation of Atg1 kinase activity. To assess Atg1 activity, we took advantage of a previous observation concerning the mobility of Atg1 in SDS-polyacrylamide gels [187]. In particular, autophosphorylation was found to retard the mobility of Atg1 in these gels, and the presence of this slower-migrating band serves as an indicator of Atg1 kinase activity in vivo. Consistent with previous in vitro studies, we found that Atg1 activity in vivo

increased upon rapamycin treatment, as a evidenced by the appearance of a slower

migrating band (Figure 3.6c). This band was lost upon phosphatase treatment or when a

kinase-dead variant of Atg1 (Atg1KD) was used, suggesting that its presence was

dependent on autophosphorylation. This activation did not occur in cells that lacked

either Atg13 or Atg17; these mutants were the only atg strains tested that exhibited a

significant defect in this Atg1 autophosphorylation reaction. Therefore, Atg13 was

required in vivo for full Atg1 protein kinase activity.

The PKA- dependent phosphorylation of Atg13 is responding to the levels of the

carbon source present

Several observations indicated that the PKA and Tor pathways were targeting different

phosphorylation sites on Atg13. First, a recent study (82) suggested that the Tor-

87 dependent phosphorylation of Atg13 was independent of PKA. Atg13 normally is

hyperphosphorylated in growing cells, and this latter phosphorylation causes Atg13 to

migrate anomalously as a broad “smear” in SDS-polyacrylamide gels run under the

right conditions [107]. Upon inactivation of the Tor pathway, this smear collapses into a tight, faster-migrating band on these gels (82). Interestingly, this change in gel

mobility still occurred upon expression of the RAS2v19 protein in the rapamycin-treated

cells, even though autophagy was inhibited [82]. Moreover, the in vitro

phosphorylation of Atg13 by PKA did not alter the mobility of this protein in SDS-

polyacrylamide gels, despite the incorporation of high amounts of phosphate (Figure

3.1a, Figure 3.7a). Finally, the degree of phosphorylation at the Atg13 PKA sites in vivo

did not decrease upon inactivation of the Tor pathway (Figure 3.7). Therefore, although

the precise locations of the Tor-dependent phosphorylation sites on Atg13 have not yet

been identified, these positions appear to be distinct from the PKA sites described here.

Previous studies found that the starvation for either a nitrogen or carbon source resulted

in the induction of autophagy activity [101]. Since Atg13 appears to be targeted by both

the PKA and Tor pathways, we assessed how these starvation regimens influence Atg13

phosphorylation. The ultimate goal was to obtain insight into the manner in which

nutritional signals are conveyed to the autophagy machinery. Although the PKA and

Tor pathways are generally thought to play a role in coordinating cell growth with

nutrient availability, it is not yet clear what the precise inputs are into each of these

pathways [137, 193-195]. There was no decrease in the level of PKA phosphorylation

under nitrogen starvation conditions (Figure 3.7a). The opposite effects were observed

upon carbon source deprivation. These data therefore suggest, that at least for

88 autophagy, the Tor and PKA pathways might be responding to distinct nutritional cues.

In particular, the PKA-dependent phosphorylation of Atg13 was apparently regulated

by carbon source levels and the Tor-dependent phosphorylation by nitrogen availability.

Finally, we found that cells treated with rapamycin and starved for carbon at the same time had higher levels of autophagy than cells treated with rapamycin and starved for nitrogen or cells that were only treated with rapamycin (Figure 3.7b). Similarly, that

additive effect was seen in cells with the Ras2ala22 allele that were also treated with rapamycin (Figures 3.3 and 3.7b). This additive effect was not seen when Ras2ala22 cells were also depleted for carbon (Figure 3.7b). These results confirm the model whereby

Ras and PKA provide independent inputs from distinct nutrient sources to regulate autophagy.

3.4. DISCUSSION

The results here make three major points relevant to the control of cell growth and

autophagy. The first is that Atg13 is a key target of regulation within the autophagy machinery of S. cerevisiae. In fact, the data here suggest that Atg13 is the primary target of the Ras/PKA signaling pathway. Atg13 is directly phosphorylated by PKA, and alteration of the PKA sites in Atg13 renders the autophagy process largely insensitive to Ras/PKA activity. For example, the presence of the Atg13-AAA protein was sufficient to reverse the RAS2val19-mediated inhibition of autophagy. Atg13 is also

the only known target of Tor activity in this pathway [107]. Therefore, the Atg13

protein may function as an important point of signal integration that allows for the

89 appropriate autophagic response to the given environmental conditions. A mammalian

homolog of Atg13 has been identified recently, and it will be interesting to see if this

protein serves a similar function in the autophagy machinery of these cells [179].

The second major point is that the PKA and Tor pathways appear to function independently during the control of the autophagy process. These pathways both target

Atg13, but appear to do so through different sites on this protein. Moreover, the

primary effects of each pathway appear to differ. Whereas PKA regulates the

association with the PAS, the Tor-dependent phosphorylation of Atg13 appears to

primarily modulate the interaction with Atg1 (see below) [107]. It is important to note

that we did not see any evidence indicating that one pathway was completely under the

control of the other [92]. For example, inhibition of the Tor pathway did not have a

significant effect upon the occupancy level of the PKA sites of Atg13. Therefore,

autophagy activity appears to be modulated by at least two independent regulatory

inputs.

The data here also indicate that the PKA and Tor pathways both make important

contributions to the regulation of autophagy. Inhibition of either pathway was sufficient

for a robust induction of autophagy activity (see Figure 3.3). These results differ from

those in a recent study that suggested a more minor role for PKA in this regulation

[196]. This latter study examined autophagy in yeast cells that carried drug-sensitive versions of the PKA enzymes. Although the reason for this discrepancy is not clear, it is important to note that the authors of this latter study did not demonstrate that the drug treatments fully inhibited PKA signaling. Therefore, their results may be due to only a partial inhibition of the Ras/PKA pathway. The work here shows that the efficient

90 inhibition of Ras/PKA signaling can induce autophagy to the same extent as conditions

that similarly inhibit Tor activity.

The third and final point is that the PKA and Tor pathways appear to be responding to different environmental cues. In particular, our data suggest that there is a division of

labor with respect to autophagy with the PKA pathway responding primarily to the level

of the carbon source present and the Tor pathway to nitrogen availability. Therefore,

different environmental conditions may control autophagy by distinct mechanisms. It is

interesting to speculate that the use of multiple regulatory inputs may provide the cell

with greater flexibility in the control of downstream processes, like autophagy. For

example, a stronger and more rapid induction could occur when both pathways are

inactivated (see Figures 3.3 and 3.7b). In addition, the deprivation for either a carbon or

nitrogen source may impose different requirements upon the cell that require distinct

autophagy responses in each case. It will be important to test whether the different

nutritional inputs into the PKA and Tor pathways observed here are specific to

autophagy, or are more generally true for these signaling pathways.

A model for the regulation of autophagy that incorporates the data from this study is

presented in Figure 3.8. In this model, PKA phosphorylation primarily controls the

PAS association of both Atg1 and Atg13. The main role of Tor signaling, on the other

hand, is to regulate the Atg1-Atg13 interaction, and thereby control Atg1 kinase

activity. Therefore, when PKA activity is limiting, Atg1 and Atg13 will localize to the

PAS. However, Tor should still be active under those conditions, and Atg13 should be

less inclined to interact with Atg1. We suggest that this Tor-dependent inhibition might

be overcome by the greatly increased local concentration of Atg1 and Atg13 at the PAS.

91 In contrast, Tor inactivation would allow for the efficient formation of the Atg1-Atg13 complex [107]. The key question in these conditions is how would this complex get to the PAS to induce autophagy? One possibility is that this complex is more efficiently targeted to the PAS than either protein alone. Alternatively, the Tor-dependent phosphorylation of Atg13 could also play a role in the regulation of Atg13 association with the PAS. For example, the aggregate sum of the PKA and Tor-dependent phosphorylation might ultimately control Atg13 localization to the PAS. Consistent with this latter possibility, the amount of Atg13 associated with Atg17 is slightly elevated in either nitrogen-starved, or rapamycin-treated cells, relative to log phase cells

(Figure 3.5a) [111]. Clearly, it will be important to identify the sites of Tor-dependent phosphorylation on Atg13 and to assess the consequences of altering these positions. In all, we feel that this model provides an initial framework to describe the regulation of autophagy, and that future experimentation will continue to resolve the details involved in these processes.

Figure 3.1: The Atg13 protein was a substrate for PKA. (a) The in vitro phosphorylation of Atg13 was dependent upon the presence of the above three PKA sites. The indicated Atg13 variants were precipitated from yeast cells, treated with phosphatase and incubated with bovine PKA and [γ-32P] ATP. S, serine; A, alanine. The relative amount of Atg13 present in each reaction is indicated by a Western blot with an α-HA antibody. (b) The in vivo phosphorylation level at the three PKA sites of Atg13. The indicated Atg13 proteins were immunoprecipitated from yeast cell extracts with an α-HA antibody. The level of PKA phosphorylation was assessed by Western blotting with an α-PKA substrate antibody that recognizes phosphorylated PKA sites. (c) Phosphatase treatment of the precipitated Atg13 protein resulted in a loss of recognition by the α-PKA substrate antibody. (d) The in vivo level of PKA phosphorylation on Atg13 was elevated in a RAS2val19 mutant. Atg13 was precipitated from wild-type and RAS2val19 cells and the amount of PKA phosphorylation was assessed by Western blotting with the α- PKA substrate antibody.

Figure 3.2: The PAS localization of Atg13 was regulated by PKA phosphorylation. Fluorescence microscopy was performed with cells that contained the indicated YFP-Atg13 fusion proteins. The Atg13 proteins had either wild-type (Atg13-SSS) or nonphosphorylatable versions (Atg13-AAA) of the three PKA sites. The CFP-Atg11 fusion protein was present in all cells and served as a marker for the PAS. The RAS2val19 allele was present in the indicated strains. Nitrogen starvation was achieved by transferring the cells from SC glucose minimal medium to the SD-N medium for 1 hr at 300C.

Figure 3.3: Inhibition of the Ras/PKA signaling pathway was sufficient to induce autophagy. (a) Autophagy induction upon either rapamycin-treatment or expression of the dominant negative RAS2ala22 allele. Wild-type cells (TN125) were grown to mid-log phase and treated with 200 ng/ml rapamycin for 4 hr at 300C. Cells carrying the MET3-RAS2ala22 allele were transferred to an SC minimal medium lacking methionine for 6 hr at 300C to induce expression from the MET3 promoter. Autophagy levels were assessed with an alkaline phosphatase-based assay as described in the Methods. (b) Autophagy induction upon inactivation of the Tor and/or PKA signaling pathways. Autophagy levels were assessed following the addition of 10 ng/ml rapamycin and/or the transfer of cells to a minimal medium lacking methionine to induce expression of the MET3-RAS2ala22 allele. (c) The RAS2ala22-mediated induction of autophagy was dependent upon the presence of Atg1. Autophagy activity was assessed in wild-type (TN125) and atg1∆ (YYK126) cells that were either treated with 10 ng/ml rapamycin or subject to the expression of the RAS2ala22 allele for 8 hr at 300C.

Figure 3.4: The RAS2val19-mediated inhibition of autophagy was reversed by the presence of the nonphosphorylatable Atg13-AAA variant. Autophagy levels were assessed in atg1∆ atg13∆ RAS2val19 cells that carried single-copy plasmids expressing the indicated Atg1 and Atg13 proteins. Autophagy was induced by transferring the cells to the SD-N starvation medium for 6 hr at 300C. Autophagy was assessed with the alkaline phosphatase-based assay as described in the Methods.

Figure 3.5: PKA phosphorylation of Atg13 regulates its association with Atg17 but not Atg1. (A) Alteration of the PKA phosphorylation sites in Atg13 influenced the association of Atg13 with Atg17. The myc6-Atg17 was immunoprecipitated from wild-type yeast cells (PHY1942) expressing either the Atg13-SSS or the Atg13-AAA proteins. The cell extracts were prepared from either mid-log phase cultures (L) or from cells that had been transferred to the SD(-N) starvation medium (S) for 6 hours at 300C. The precipitated proteins were separated on SDS-polyacrylamide gels, and the levels of Atg13 present were assessed by Western Blotting with an α-HA antibody. Control Western blots with the α-HA and α-myc antibodies show the relative input level of each protein. (B) The presence of the RAS2val19 allele did not inhibit the interaction of Atg13 with Atg1. The immunoprecipitations were performed as described in (a). The cells were collected in mid-log phase. The RAS2val19 allele was induced for 2.5 hours by transfer to methionine-free medium. (C) High levels of Ras inhibited the interaction between Atg13- SSS (lanes 1 and 2) and Atg17, but did not inhibit the interaction between Atg13-AAA (lanes 3 and 4) and Atg17. The immunoprecipitations were performed as in (a). Cells containing the TPK1 gene on a high copy plasmid or a vector control, and Atg13-SSS or Atg13-AAA, were collected in mid-log phase. as described in the methods section. The TPK1 allele was under the control of the CUP1 promoter and was induced by the addition of 100 µM of copper sulfate to the medium, for 2 hours.

Figure 3.6: PKA activity regulates the PAS localization of Atg13 by modulating its interaction with Atg17. (a) Atg13-AAA and Atg1-AA localized to the PAS in log phase wild-type cells, but not in atg17 cells. The bottom panel shows representative pictures from wild-type or Atg17 cells harboring the CUP1- inducible YFP-Atg13-AAA and YFP-Atg1-AA. (b) The RAS2val19 allele did not affect the presence of YFP-Atg17 at the PAS. (c) Atg1 kinase activity was reduced in atg13 and atg17 cells. Rap is rapamycin treatment. PPase: treatment with phosphatase.

Figure 3.7: The PKA-dependent phosphorylation of Atg13 is regulated by carbon source cues. (A) PKA- dependent phosphorylation of Atg13 was lost in cells deprived of a carbon source (-C), but not in cells treated with rapamycin (Rap) or starved for nitrogen (-N). After collection and extract preparation by native immunoprecipitations with the α-HA antibody, samples were treated with PKA (+) or not (-) before loading on SDS gels. PKA phosphorylation was assessed using the α-substrate antibody, and an α- HA antibody Western blot was performed to determine equal loading. (B) The additive effects of rapamycin and carbon starvation on autophagy induction. Autophagy was assessed in strains carrying either a control vector or the Ras2ala22 allele. Rap: 10 ng/ml rapamycin treatment. –carbon: carbon starvation. –nitrogen: nitrogen starvation. (Off): the allele was repressed by presence of methionine in the medium. (On): the allele was induced by removal of methionine from the growth medium. Autophagy was assessed with the alkaline phosphatase-based assay as described in the Methods.

Figure 3.8: Tor and PKA regulate different steps in the induction of autophagy. Tor regulates the interaction between Atg1 and Atg13, primarily in response to nitrogen levels, while PKA regulates Atg1 and Atg13 localization to the PAS in response to carbon levels.

100

CHAPTER 4

SYNOPSIS

The evolutionary proteomis approach

In a relatively short number of years, several genome-sequencing projects have provided a wealth of information that just a few years back would have been impossible to conceive. Several genomes have been completely sequenced, including the human genome. One of the major challenges facing the scientific community today is the extraction of meaningful biological information from this wealth of data. In this work, we have used a comparative approach to identify biologically relevant targets of PKA in

S. cerevisiae. Our initial query for the presence of a previously identified PKA consensus site in the yeast proteome returned 491 hits, which constitutes a significantly high percentage of all yeast proteins. In order to narrow our search to the most relevant targets of PKA, we then used available sequence information to compare the sequences from this budding yeast to a group of related yeast species, including the pathogenic C. albicans. We reasoned that PKA sites with meaningful biological roles would more likely be conserved across evolutionary distances. We were able to identify 44 proteins

(less than 1% of the budding yeast proteome) that contained PKA sites conserved from budding yeast to C. albicans. Of these 44 proteins, we were able to test 20 for 101 phosphorylation by PKA. Remarkably, 19 out of those 20 proteins were recognized by

that enzyme. Furthermore, the likelihood of phosphorylation by PKA diminished as we

looked at sites that were only conserved to closely related yeast species.

The evolutionary proteomics approach for PKA substrate identification was therefore

extremely successful, and will probably yield insights into the mechanism by which

PKA controls growth and proliferation. One interesting side to our results is that they

could prove useful in understanding the pathogenic biology of C. albicans. The Ras

pathway has been shown to be important for this yeast’s virulence, and as for the

budding yeast, seems to work through PKA. The PKA substrates we have identified

could therefore be potential targets for drug development. It is certainly possible, however, that PKA mediates virulence in C. albicans through a different set of effectors

that are specific to this yeast, and these would be missed by our approach. Finally, this

approach is by no means exhaustive. Indeed, proteins that contain close matches to the

PKA consensus site we used could still be relevant in vivo targets of PKA. Furthermore,

the high stringency criteria we used in the approach probably eliminated several bona

fide PKA substrates from our final list of forty-four candidates. Finally, PKA sites might serve species-specific functions that could be missed by this approach.

In conclusion, the evolutionary approach to substrate identification worked remarkably well. In theory, this strategy could be used for any motif of interest in any species, as long as sequence information exists for that particular species and its relatives.

102 Autophagy and its regulation: a paradigm for Tor/PKA crosstalk?

The Atg13 and Atg1 proteins were identified as substrates of PKA through the evolutionary proteomics approach. These proteins are part of the Atg1 complex, which plays a central role in the induction of autophagy. This process has garnered a lot of interest recently, and is implicated in aging, cell death, development and the pathogenesis of several human diseases. In yeast, autophagy is induced upon nutrient- limitation and is essential for the long-term survival of cells in stationary phase.

We found that PKA is a critical regulator of autophagy. High levels of PKA activity inhibited the starvation-induced initiation of the process, while inhibition of PKA resulted in the induction of autophagy even when nutrients were available. These results suggest that PKA normally inhibits autophagy in conditions favorable to growth.

We have shown that PKA regulates autophagic activity through the direct phosphorylation of Atg1 and Atg13. Upon nutrient limitation, Atg1 and Atg13 localize to the PAS, the site where their activity is needed for the initial stages of autophagy to occur. We found that high levels of PKA activity prevented this localization by inhibiting the interaction of Atg13 (and we suggest, Atg1) with the core PAS component Atg17. Removal of the PKA sites on Atg1 and Atg13 rendered the proteins insensitive to high levels of PKA activity. The Atg1-AA and Atg13-AAA proteins localized at the PAS even in the presence of nutrients, and the interaction of Atg13-

AAA with Atg17 became constitutive. Interestingly, alteration of the Atg13 PKA sites alone was sufficient to reverse PKA inhibition of starvation-induced autophagy, suggesting that Atg13 might be the key point of regulation in the pathway. Atg13

103 phosphorylation is also regulated by Tor in a nutrient-dependent fashion. We provided evidence that the PKA- and Tor-dependent phosphorylation of Atg13 likely occur at distinct sites on the protein. Furthermore, these phosphorylation events appear to be regulated independently and in response to different nutritional cues. Nitrogen starvation or rapamycin treatment did not affect the PKA-dependent phosphorylation status of Atg13. This phosphorylation was mainly responsive to carbon source levels. In support of this idea, rapamycin treatment combined with carbon starvation had an additive effect on the magnitude of the autophagic response observed. This magnitude was similar to that seen in rapamycin-treated cells carrying a dominant-negative RAS allele. In contrast, no additive effects were observed when rapamycin was combined with nitrogen starvation, and the dominant-negative RAS allele with carbon. These data support a model wherein Atg13 integrates independent Tor and PKA inputs, from different nutritional cues, to ultimately drive the autophagy machinery. This data is interesting as very little is known about the exact extracellular signals the Tor and Ras pathways respond to. This mode of regulation could be conserved among other Tor and

PKA effectors. Alternatively, it could be Atg13- and autophagy- specific. Clearly, investigation of other targets of those signaling pathways will be critical for confirmation of these findings.

The Atg13-AAA variant was sufficient to reverse high PKA inhibition of nitrogen- or rapamycin-induced autophagy. However, neither cells with Atg13-AAA nor cells with

Atg13-AAA and Atg1-AAA showed induction of autophagy in nutrient-rich media. By contrast, inactivation of PKA led to such an induction. There are multiple explanations for this observation: first, PKA might regulate autophagy at multiple steps in the

104 process and/or through additional proteins. The Atg18 protein was also identified as a

PKA target in our evolutionary proteomics strategy, and it would be interesting to see if

removal of the sites on all three proteins leads to constitutive induction of autophagy.

This hypothesis does not really explain why Atg13-AAA would be sufficient to reverse

PKA inhibition of starvation-induced autophagy, unless some modulation of Atg18

function occurs in only in starved cells and therefore allows the reversal. This is

certainly possible, considering how tightly the autophagy process must be regulated during normal logarithmic phases of growth. Another possibility, and one that perhaps correlates better with our findings, involves Atg13 as an integrator of signals from two different inputs. In nitrogen-starved or rapamycin-treated cells carrying the hyperactive

RAS allele, Atg13-AAA doesn’t receive any inhibitory input from Tor, which is inactive. Furthermore, Atg13-AAA is insensitive to high PKA, and so the ultimate result is induction of autophagy. In nutrient-rich conditions, Atg13-AAA is insensitive to PKA activity but still receives inhibitory signals from Tor, and therefore autophagy remains inhibited.

Future directions

A recent paper from Michael Snyder’s group has used large-scale, whole proteome

protein chips to map the yeast “kinome”. One surprising finding in that research was

that Tpk1, 2 and 3 had relatively few overlap in the targets they recognized. The Tpks

have redundant functions with regards to growth, but some differences have been

reported. For example, the tpks have antagonistic effects on pseudohyphal growth. We

105 have shown that Tpk1 inhibits autophagy, and it would be interesting to see if Tpk2 and

Tpk3 exhibit any differences in that regard. It is possible that the Ras inhibitory signals

could be mediated by Tpk1 alone, or by any combination of the three subunits.

Beside the elucidation of the intriguing interrelationship between Ras and Tor with

regards to growth in general and autophagy in particular, several interesting questions

remain to be addressed. First, the Atg1 kinase complex is an important regulator of the

induction phase of autophagy, and it is a key point of regulation by many signaling pathways. Furthermore, orthologues of Atg1 and Atg13 have been found in mammals

and possibly perform similar functions in those organisms. However, no substrates of

the Atg1 kinase, beside itself, have been identified to date. Snyder’s group has

identified putative Atg1 substrates by large-scale protein chip analysis, including

proteins involved in autophagy such as Atg18. It would be interesting to see if those

proteins are bona fide targets of Atg1 in vivo. Identification of Atg1 targets will

obviously be crucial for our ultimate understanding of the process of autophagy and its

regulation. It is tempting to speculate that the different inputs from Tor and PKA could be relayed to the Atg1 kinase through Atg13 or other proteins, and that Atg1 activity would be modulated differently depending on those inputs. This could result, for example, in the phosphorylation of different sets of targets based on the input, and could provide a molecular basis for specific autophagic targeting of particular subcellar components based on the particular needs of the cell.

106

BIBLIOGRAPHY

1. Werner-Washburne, M., et al., Stationary phase in Saccharomyces cerevisiae. Mol Microbiol, 1996. 19(6): p. 1159-66.

2. Rohde, J., J. Heitman, and M.E. Cardenas, The TOR kinases link nutrient sensing to cell growth. J Biol Chem, 2001. 276(13): p. 9583-6.

3. Schmelzle, T. and M.N. Hall, TOR, a central controller of cell growth. Cell, 2000. 103(2): p. 253-62.

4. Wullschleger, S., R. Loewith, and M.N. Hall, TOR signaling in growth and metabolism. Cell, 2006. 124(3): p. 471-84.

5. Sehgal, S.N., Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc, 2003. 35(3 Suppl): p. 7S-14S.

6. De Virgilio, C. and R. Loewith, Cell growth control: little eukaryotes make big contributions. Oncogene, 2006. 25(48): p. 6392-415.

7. Heitman, J., N.R. Movva, and M.N. Hall, Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science, 1991. 253(5022): p. 905-9.

8. Helliwell, S.B., et al., TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell, 1994. 5(1): p. 105-18.

9. Dumont, F.J., et al., Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin. J Immunol, 1990. 144(1): p. 251-8.

10. Cafferkey, R., et al., Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol Cell Biol, 1993. 13(10): p. 6012-23.

11. Kunz, J., et al., Target of rapamycin in yeast, TOR2, is an essential 107 phosphatidylinositol kinase homolog required for G1 progression. Cell, 1993. 73(3): p. 585-96.

12. Crespo, J.L., et al., The GATA transcription factors GLN3 and GAT1 link TOR to salt stress in Saccharomyces cerevisiae. J Biol Chem, 2001. 276(37): p. 34441-4.

13. Helliwell, S.B., et al., TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics, 1998. 148(1): p. 99-112.

14. Zheng, X.F., et al., TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell, 1995. 82(1): p. 121-30.

15. Loewith, R., et al., Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell, 2002. 10(3): p. 457-68.

16. Chen, E.J. and C.A. Kaiser, LST8 negatively regulates amino acid biosynthesis as a component of the TOR pathway. J Cell Biol, 2003. 161(2): p. 333-47.

17. Wedaman, K.P., et al., Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae. Mol Biol Cell, 2003. 14(3): p. 1204-20.

18. Reinke, A., et al., TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem, 2004. 279(15): p. 14752-62.

19. Fadri, M., et al., The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol Biol Cell, 2005. 16(4): p. 1883-900.

20. Cardenas, M.E., et al., The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev, 1999. 13(24): p. 3271-9.

21. Hardwick, J.S., et al., Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.

108 Proc Natl Acad Sci U S A, 1999. 96(26): p. 14866-70.

22. Komeili, A., et al., Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors. J Cell Biol, 2000. 151(4): p. 863-78.

23. Shamji, A.F., F.G. Kuruvilla, and S.L. Schreiber, Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins. Curr Biol, 2000. 10(24): p. 1574-81.

24. Chen, J.C. and T. Powers, Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae. Curr Genet, 2006. 49(5): p. 281-93.

25. Beck, T. and M.N. Hall, The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature, 1999. 402(6762): p. 689-92.

26. Carvalho, J. and X.F. Zheng, Domains of Gln3p interacting with karyopherins, Ure2p, and the target of rapamycin protein. J Biol Chem, 2003. 278(19): p. 16878-86.

27. Santhanam, A., et al., PP2A phosphatase activity is required for stress and Tor kinase regulation of yeast stress response factor Msn2p. Eukaryot Cell, 2004. 3(5): p. 1261-71.

28. Mager, W.H. and A.J. De Kruijff, Stress-induced transcriptional activation. Microbiol Rev, 1995. 59(3): p. 506-31.

29. Ruis, H. and C. Schuller, Stress signaling in yeast. Bioessays, 1995. 17(11): p. 959-65.

30. Dilova, I., C.Y. Chen, and T. Powers, Mks1 in concert with TOR signaling negatively regulates RTG target gene expression in S. cerevisiae. Curr Biol, 2002. 12(5): p. 389-95.

31. Dilova, I., et al., Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes. J

109 Biol Chem, 2004. 279(45): p. 46527-35.

32. Tate, J.J., et al., Mks1p is required for negative regulation of retrograde gene expression in Saccharomyces cerevisiae but does not affect nitrogen catabolite repression-sensitive gene expression. J Biol Chem, 2002. 277(23): p. 20477-82.

33. Sekito, T., et al., RTG-dependent mitochondria-to-nucleus signaling is regulated by MKS1 and is linked to formation of yeast prion [URE3]. Mol Biol Cell, 2002. 13(3): p. 795-804.

34. Liu, Z., et al., Retrograde signaling is regulated by the dynamic interaction between Rtg2p and Mks1p. Mol Cell, 2003. 12(2): p. 401-11.

35. Butow, R.A. and N.G. Avadhani, Mitochondrial signaling: the retrograde response. Mol Cell, 2004. 14(1): p. 1-15.

36. Liao, X. and R.A. Butow, RTG1 and RTG2: two yeast genes required for a novel path of communication from mitochondria to the nucleus. Cell, 1993. 72(1): p. 61-71.

37. Sekito, T., J. Thornton, and R.A. Butow, Mitochondria-to-nuclear signaling is regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p. Mol Biol Cell, 2000. 11(6): p. 2103-15.

38. Colomina, N., et al., TOR regulates the subcellular localization of Ime1, a transcriptional activator of meiotic development in budding yeast. Mol Cell Biol, 2003. 23(20): p. 7415-24.

39. Jiang, Y. and J.R. Broach, Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. Embo J, 1999. 18(10): p. 2782-92.

40. Bertram, P.G., et al., Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J Biol Chem, 2000. 275(46): p. 35727-33.

41. Urban, J., et al., Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell, 2007. 26(5): p. 663-74.

110 42. Cherkasova, V.A. and A.G. Hinnebusch, Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev, 2003. 17(7): p. 859-72.

43. Rohde, J.R., et al., TOR controls transcriptional and translational programs via Sap-Sit4 protein phosphatase signaling effectors. Mol Cell Biol, 2004. 24(19): p. 8332-41.

44. Duvel, K., et al., Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. Mol Cell, 2003. 11(6): p. 1467-78.

45. Schmidt, A., et al., The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. Embo J, 1998. 17(23): p. 6924-31.

46. Jacinto, E., et al., TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol Cell, 2001. 8(5): p. 1017-26.

47. Gorner, W., et al., Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor. Embo J, 2002. 21(1-2): p. 135-44.

48. Toda, T., et al., Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol, 1987. 7(4): p. 1371-7.

49. Toda, T., et al., In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell, 1985. 40(1): p. 27-36.

50. Werner-Washburne, M., et al., Stationary phase in the yeast Saccharomyces cerevisiae. Microbiol Rev, 1993. 57(2): p. 383-401.

51. Kataoka, T., et al., Genetic analysis of yeast RAS1 and RAS2 genes. Cell, 1984. 37(2): p. 437-45.

52. Broach, J.R., RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway. Trends Genet, 1991. 7(1): p. 28-33.

111 53. Kataoka, T., et al., Genetic analysis of yeast RAS1 and RAS2 genes. Cell, 1984. 37(2): p. 437-45.

54. Sherwin, S.A., et al., Rescue of endogenous 30S retroviral sequences from mouse cells by baboon type C virus. J Virol, 1978. 26(2): p. 257-64.

55. Langbeheim, H., T.Y. Shih, and E.M. Scolnick, Identification of a normal vertebrate cell protein related to the p21 src of Harvey murine sarcoma virus. Virology, 1980. 106(2): p. 292-300.

56. DeFeo-Jones, D., et al., ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature, 1983. 306(5944): p. 707-9.

57. Powers, S., et al., Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell, 1984. 36(3): p. 607-12.

58. Takai, Y., H. Yamamura, and Y. Nishizuka, Adenosine 3':5'-monophosphate- dependent protein kinase from yeast. J Biol Chem, 1974. 249(2): p. 530-5.

59. Toda, T., et al., Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell, 1987. 50(2): p. 277-87.

60. Ma, P., et al., The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell, 1999. 10(1): p. 91-104.

61. Thevelein, J.M., Signal transduction in yeast. Yeast, 1994. 10(13): p. 1753-90.

62. Thevelein, J.M., The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek, 1992. 62(1-2): p. 109- 30.

63. Broach, J.R. and R.J. Deschenes, The function of ras genes in Saccharomyces cerevisiae. Adv Cancer Res, 1990. 54: p. 79-139.

64. Hardy, T.A. and P.J. Roach, Control of yeast glycogen synthase-2 by COOH- terminal phosphorylation. J Biol Chem, 1993. 268(32): p. 23799-805.

112 65. Zahringer, H., H. Holzer, and S. Nwaka, Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP-dependent protein kinase, Tpk1 and Tpk2. Eur J Biochem, 1998. 255(3): p. 544-51.

66. Wiemken, A. and M. Schellenberg, Does a cyclic AMP-dependent phosphorylation initiate the transfer of trehalase from the cytosol into the vacuoles in Saccharomyces cerevisiae? FEBS Lett, 1982. 150(2): p. 329-31.

67. Francois, J., M.J. Neves, and H.G. Hers, The control of trehalose biosynthesis in Saccharomyces cerevisiae: evidence for a catabolite inactivation and repression of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Yeast, 1991. 7(6): p. 575-87.

68. Francois, J. and H.G. Hers, The control of glycogen metabolism in yeast. 2. A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their interconversion in a cell-free extract. Eur J Biochem, 1988. 174(3): p. 561-7.

69. Francois, J., M.E. Villanueva, and H.G. Hers, The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers. Eur J Biochem, 1988. 174(3): p. 551-9.

70. Smith, A., M.P. Ward, and S. Garrett, Yeast PKA represses Msn2p/Msn4p- dependent gene expression to regulate growth, stress response and glycogen accumulation. Embo J, 1998. 17(13): p. 3556-64.

71. Thevelein, J.M. and J.H. de Winde, Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol, 1999. 33(5): p. 904-18.

72. Belazzi, T., et al., Negative regulation of transcription of the Saccharomyces cerevisiae catalase T (CTT1) gene by cAMP is mediated by a positive control element. Embo J, 1991. 10(3): p. 585-92.

73. Marchler, G., et al., A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions. Embo J, 1993. 12(5): p. 1997-2003.

113 74. Martinez-Pastor, M.T., et al., The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). Embo J, 1996. 15(9): p. 2227-35.

75. Gorner, W., et al., Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev, 1998. 12(4): p. 586-97.

76. Boorstein, W.R. and E.A. Craig, Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae. Mol Cell Biol, 1990. 10(6): p. 3262-7.

77. Pedruzzi, I., et al., Saccharomyces cerevisiae Ras/cAMP pathway controls post- diauxic shift element-dependent transcription through the zinc finger protein Gis1. Embo J, 2000. 19(11): p. 2569-79.

78. Reinders, A., et al., Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase. Genes Dev, 1998. 12(18): p. 2943-55.

79. Holstege, F.C., et al., Dissecting the regulatory circuitry of a eukaryotic genome. Cell, 1998. 95(5): p. 717-28.

80. Chang, Y.W., S.C. Howard, and P.K. Herman, The Ras/PKA signaling pathway directly targets the Srb9 protein, a component of the general RNA polymerase II transcription apparatus. Mol Cell, 2004. 15(1): p. 107-16.

81. Noda, T. and Y. Ohsumi, Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem, 1998. 273(7): p. 3963-6.

82. Budovskaya, Y.V., et al., The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae. J Biol Chem, 2004. 279(20): p. 20663-71.

83. Marcus, F., et al., Yeast (Saccharomyces cerevisiae) fructose-1,6- bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis. J Biol Chem, 1988. 263(13): p. 6058-62.

114 84. Francois, J., E. Van Schaftigen, and H.G. Hers, Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast. Eur J Biochem, 1988. 171(3): p. 599-608.

85. Colombo, S., et al., Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae. Embo J, 1998. 17(12): p. 3326-41.

86. Kraakman, L., et al., A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose. Mol Microbiol, 1999. 32(5): p. 1002- 12.

87. Kemp, B.E., et al., Substrate specificity of the cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A, 1975. 72(9): p. 3448-52.

88. Zetterqvist, O., et al., The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver. Biochem Biophys Res Commun, 1976. 70(3): p. 696-703.

89. Pedruzzi, I., et al., TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0. Mol Cell, 2003. 12(6): p. 1607-13.

90. Wanke, V., et al., Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex. Embo J, 2005. 24(24): p. 4271-8.

91. Zurita-Martinez, S.A. and M.E. Cardenas, Tor and cyclic AMP-protein kinase A: two parallel pathways regulating expression of genes required for cell growth. Eukaryot Cell, 2005. 4(1): p. 63-71.

92. Schmelzle, T., et al., Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast. Mol Cell Biol, 2004. 24(1): p. 338-51.

93. Griffioen, G., et al., Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae. J Biol Chem, 2000. 275(2): p. 1449-56.

115 94. Martin, D.E., A. Soulard, and M.N. Hall, TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell, 2004. 119(7): p. 969-79.

95. Ogier-Denis, E. and P. Codogno, Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta, 2003. 1603(2): p. 113-28.

96. Klionsky, D.J. and S.D. Emr, Autophagy as a regulated pathway of cellular degradation. Science, 2000. 290(5497): p. 1717-21.

97. Ashford, T.P. and K.R. Porter, Cytoplasmic components in hepatic cell lysosomes. J Cell Biol, 1962. 12: p. 198-202.

98. Clark, S.L., Jr., Cellular differentiation in the kidneys of newborn mice studies with the electron microscope. J Biophys Biochem Cytol, 1957. 3(3): p. 349-62.

99. De Duve, C. and R. Wattiaux, Functions of lysosomes. Annu Rev Physiol, 1966. 28: p. 435-92.

100. Novikoff, A.B., The proximal tubule cell in experimental hydronephrosis. J Biophys Biochem Cytol, 1959. 6(1): p. 136-8.

101. Takeshige, K., et al., Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol, 1992. 119(2): p. 301-11.

102. Tsukada, M. and Y. Ohsumi, Isolation and characterization of autophagy- defective mutants of Saccharomyces cerevisiae. FEBS Lett, 1993. 333(1-2): p. 169-74.

103. Klionsky, D.J., Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol, 2007. 8(11): p. 931-7.

104. Suzuki, K., et al., The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. Embo J, 2001. 20(21): p. 5971-81.

105. Kim, J., et al., Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de 116 novo vesicle formation. J Biol Chem, 2002. 277(1): p. 763-73.

106. Scott, S.V., et al., Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting. J Biol Chem, 2000. 275(33): p. 25840-9.

107. Kamada, Y., et al., Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J Cell Biol, 2000. 150(6): p. 1507-13.

108. Abeliovich, H., et al., Dissection of autophagosome biogenesis into distinct nucleation and expansion steps. J Cell Biol, 2000. 151(5): p. 1025-34.

109. Funakoshi, T., et al., Analyses of APG13 gene involved in autophagy in yeast, Saccharomyces cerevisiae. Gene, 1997. 192(2): p. 207-13.

110. Scott, S.V., et al., Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole. Proc Natl Acad Sci U S A, 1996. 93(22): p. 12304-8.

111. Kabeya, Y., et al., Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy. Mol Biol Cell, 2005. 16(5): p. 2544-53.

112. Cheong, H., et al., Atg17 regulates the magnitude of the autophagic response. Mol Biol Cell, 2005. 16(7): p. 3438-53.

113. Suzuki, K., et al., Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells, 2007. 12(2): p. 209-18.

114. Ishihara, N., et al., Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion. Mol Biol Cell, 2001. 12(11): p. 3690-702.

115. Ohsumi, Y., Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol, 2001. 2(3): p. 211-6.

116. Kim, J., et al., Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways. Mol Biol Cell, 1999. 10(5): p. 1337-51. 117 117. Shintani, T., et al., Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. Embo J, 1999. 18(19): p. 5234-41.

118. Mizushima, N., T. Noda, and Y. Ohsumi, Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. Embo J, 1999. 18(14): p. 3888-96.

119. Kirisako, T., et al., The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol, 2000. 151(2): p. 263-76.

120. Guan, J., et al., Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris. Mol Biol Cell, 2001. 12(12): p. 3821-38.

121. Yorimitsu, T. and D.J. Klionsky, Autophagy: molecular machinery for self- eating. Cell Death Differ, 2005. 12 Suppl 2: p. 1542-52.

122. Kirisako, T., et al., Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J Cell Biol, 1999. 147(2): p. 435-46.

123. Reggiori, F., et al., The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure. Dev Cell, 2004. 6(1): p. 79- 90.

124. Noda, T., et al., Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways. J Cell Biol, 2000. 148(3): p. 465-80.

125. Tucker, K.A., et al., Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy. J Biol Chem, 2003. 278(48): p. 48445-52.

126. Reggiori, F., et al., Atg9 cycles between mitochondria and the pre- autophagosomal structure in yeasts. Autophagy, 2005. 1(2): p. 101-9.

127. Mizushima, N., et al., Dissection of autophagosome formation using Apg5- deficient mouse embryonic stem cells. J Cell Biol, 2001. 152(4): p. 657-68.

118 128. Teter, S.A., et al., Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase. J Biol Chem, 2001. 276(3): p. 2083-7.

129. Epple, U.D., E.L. Eskelinen, and M. Thumm, Intravacuolar membrane lysis in Saccharomyces cerevisiae. Does vacuolar targeting of Cvt17/Aut5p affect its function? J Biol Chem, 2003. 278(10): p. 7810-21.

130. Cohen, P., Protein kinases--the major drug targets of the twenty-first century? Nat Rev Drug Discov, 2002. 1(4): p. 309-15.

131. Hunter, T., Signaling--2000 and beyond. Cell, 2000. 100(1): p. 113-27.

132. Hunter, T., A thousand and one protein kinases. Cell, 1987. 50(6): p. 823-9.

133. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34.

134. Manning, B.D. and L.C. Cantley, Hitting the target: emerging technologies in the search for kinase substrates. Sci STKE, 2002. 2002(162): p. PE49.

135. Taylor, S.S., J.A. Buechler, and W. Yonemoto, cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem, 1990. 59: p. 971-1005.

136. Taylor, S.S., et al., PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta, 2004. 1697(1-2): p. 259-69.

137. Herman, P.K., Stationary phase in yeast. Curr Opin Microbiol, 2002. 5(6): p. 602-7.

138. Suzuki, N., et al., Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins. Proc Natl Acad Sci U S A, 1990. 87(22): p. 8711-5.

139. Field, J., et al., Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae. Science, 1990. 247(4941): p. 464-7.

119 140. Cliften, P.F., et al., Surveying Saccharomyces genomes to identify functional elements by comparative DNA sequence analysis. Genome Res, 2001. 11(7): p. 1175-86.

141. Cliften, P., et al., Finding functional features in Saccharomyces genomes by phylogenetic footprinting. Science, 2003. 301(5629): p. 71-6.

142. Hardison, R.C., J. Oeltjen, and W. Miller, Long human-mouse sequence alignments reveal novel regulatory elements: a reason to sequence the mouse genome. Genome Res, 1997. 7(10): p. 959-66.

143. Rubin, G.M., et al., Comparative genomics of the eukaryotes. Science, 2000. 287(5461): p. 2204-15.

144. Tagle, D.A., et al., Embryonic epsilon and gamma globin genes of a prosimian primate (Galago crassicaudatus). Nucleotide and amino acid sequences, developmental regulation and phylogenetic footprints. J Mol Biol, 1988. 203(2): p. 439-55.

145. Denis, C.L., B.E. Kemp, and M.J. Zoller, Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical. J Biol Chem, 1991. 266(27): p. 17932-5.

146. Shabb, J.B., Physiological substrates of cAMP-dependent protein kinase. Chem Rev, 2001. 101(8): p. 2381-411.

147. Songyang, Z., et al., Use of an oriented peptide library to determine the optimal substrates of protein kinases. Curr Biol, 1994. 4(11): p. 973-82.

148. Tegge, W., et al., Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper. Biochemistry, 1995. 34(33): p. 10569-77.

149. Smith, C.M., et al., The catalytic subunit of cAMP-dependent protein kinase: prototype for an extended network of communication. Prog Biophys Mol Biol, 1999. 71(3-4): p. 313-41.

150. Noda, T., et al., Novel system for monitoring autophagy in the yeast

120 Saccharomyces cerevisiae. Biochem Biophys Res Commun, 1995. 210(1): p. 126-32.

151. Zhu, H., et al., Global analysis of protein activities using proteome chips. Science, 2001. 293(5537): p. 2101-5.

152. Wang, Y., et al., Ras and Gpa2 mediate one branch of a redundant glucose signaling pathway in yeast. PLoS Biol, 2004. 2(5): p. E128.

153. Budovskaya, Y.V., et al., The C terminus of the Vps34p phosphoinositide 3- kinase is necessary and sufficient for the interaction with the Vps15p protein kinase. J Biol Chem, 2002. 277(1): p. 287-94.

154. Gerhardt, B., et al., The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner. J Biol Chem, 1998. 273(25): p. 15818-29.

155. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith J.A. & Struhl, K., Current Protocols in Molecular Biology. 1995: John Wiley and Sons, Inc.

156. Kunkel, T.A., Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A, 1985. 82(2): p. 488-92.

157. Herman, P.K., J.H. Stack, and S.D. Emr, A genetic and structural analysis of the yeast Vps15 protein kinase: evidence for a direct role of Vps15p in vacuolar protein delivery. Embo J, 1991. 10(13): p. 4049-60.

158. Barnett, J.A., yeast. 1992. p. 1-23.

159. Rocha, C.R., et al., Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol Biol Cell, 2001. 12(11): p. 3631-43.

160. Leberer, E., et al., Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans. Mol Microbiol, 2001. 42(3): p. 673-87.

121 161. Garrett, S., M.M. Menold, and J.R. Broach, The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle. Mol Cell Biol, 1991. 11(8): p. 4045-52.

162. Kuret, J., et al., Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit. J Biol Chem, 1988. 263(19): p. 9149-54.

163. Kim, K.H. and G.M. Carman, Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A. J Biol Chem, 1999. 274(14): p. 9531-8.

164. Noda, T., K. Suzuki, and Y. Ohsumi, Yeast autophagosomes: de novo formation of a membrane structure. Trends Cell Biol, 2002. 12(5): p. 231-5.

165. Reggiori, F. and D.J. Klionsky, Autophagy in the eukaryotic cell. Eukaryot Cell, 2002. 1(1): p. 11-21.

166. Matsuura, A., et al., Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae. Gene, 1997. 192(2): p. 245-50.

167. Russell, M., et al., Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest. Mol Biol Cell, 1993. 4(7): p. 757-65.

168. Heckman, D.S., et al., Molecular evidence for the early colonization of land by fungi and plants. Science, 2001. 293(5532): p. 1129-33.

169. Jorgensen, P., et al., A dynamic transcriptional network communicates growth potential to ribosome synthesis and critical cell size. Genes Dev, 2004. 18(20): p. 2491-505.

170. Xie, Z. and D.J. Klionsky, Autophagosome formation: core machinery and adaptations. Nat Cell Biol, 2007. 9(10): p. 1102-9.

171. Kopitz, J., et al., Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes. J Cell Biol, 1990. 111(3): p. 941-53.

122 172. Schworer, C.M. and G.E. Mortimore, Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids. Proc Natl Acad Sci U S A, 1979. 76(7): p. 3169-73.

173. Levine, B. and D.J. Klionsky, Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell, 2004. 6(4): p. 463-77.

174. Levine, B. and G. Kroemer, Autophagy in the pathogenesis of disease. Cell, 2008. 132(1): p. 27-42.

175. Mizushima, N., et al., Autophagy fights disease through cellular self-digestion. Nature, 2008. 451(7182): p. 1069-75.

176. Seglen, P.O., P.B. Gordon, and I. Holen, Non-selective autophagy. Semin Cell Biol, 1990. 1(6): p. 441-8.

177. Mizushima, N. and D.J. Klionsky, Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr, 2007. 27: p. 19-40.

178. Klionsky, D.J., et al., A unified nomenclature for yeast autophagy-related genes. Dev Cell, 2003. 5(4): p. 539-45.

179. Meijer, W.H., et al., ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes. Autophagy, 2007. 3(2): p. 106-16.

180. Stephan, J.S. and P.K. Herman, The regulation of autophagy in eukaryotic cells: do all roads pass through Atg1? Autophagy, 2006. 2(2): p. 146-8.

181. Suzuki, K. and Y. Ohsumi, Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS Lett, 2007. 581(11): p. 2156-61.

182. Chan, E.Y., S. Kir, and S.A. Tooze, siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy. J Biol Chem, 2007. 282(35): p. 25464-74.

183. Hara, T., et al., FIP200, a ULK-interacting protein, is required for 123 autophagosome formation in mammalian cells. J Cell Biol, 2008. 181(3): p. 497-510.

184. Kaiser, C., Michaelis, S. & Mitchell, A., Methods in Yeast Genetics. 1994: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;.

185. Howard, S.C., et al., The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme. Genetics, 2001. 159(1): p. 77-89.

186. Deminoff, S.J., et al., Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae. Genetics, 2006. 173(4): p. 1909-17.

187. Budovskaya, Y.V., et al., An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase. Proc Natl Acad Sci U S A, 2005. 102(39): p. 13933-8.

188. Deminoff, S.J. and P.K. Herman, Identifying Atg1 substrates: four means to an end. Autophagy, 2007. 3(6): p. 667-73.

189. Powers, S., K. O'Neill, and M. Wigler, Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol, 1989. 9(2): p. 390-5.

190. Mountain, H.A., et al., Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae. Yeast, 1991. 7(8): p. 781-803.

191. Kawamata, T., et al., Organization of the Pre-autophagosomal Structure Responsible for Autophagosome Formation. Mol Biol Cell, 2008. 19(5): p. 2039-50.

192. Cheong, H., et al., The Atg1 Kinase Complex Is Involved in the Regulation of Protein Recruitment to Initiate Sequestering Vesicle Formation for Nonspecific Autophagy in Saccharomyces cerevisiae. Mol Biol Cell, 2008. 19(2): p. 668-81.

124 193. Santangelo, G.M., Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 2006. 70(1): p. 253-82.

194. Schneper, L., K. Duvel, and J.R. Broach, Sense and sensibility: nutritional response and signal integration in yeast. Curr Opin Microbiol, 2004. 7(6): p. 624-30.

195. De Virgilio, C. and R. Loewith, The TOR signalling network from yeast to man. Int J Biochem Cell Biol, 2006. 38(9): p. 1476-81.

196. Yorimitsu, T., et al., Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol Biol Cell, 2007. 18(10): p. 4180-9.

125