The Pennsylvania State University. The Graduate School. Graduate Program in Integrative Biosciences

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1 The Pennsylvania State University The Graduate School Graduate Program in Integrative Biosciences THE EIF2α KINASES GCN2 AND PERK REGULATE PHOSPHORYLATION OF TOR TARGET PROTEINS AND DIFFERENTIAL TRANSLATION OF MRNAS A Thesis in Integrative Biosciences by An Ngoc Dang Do 2008 An Ngoc Dang Do Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2008

2 COMMITTEE PAGE The thesis of An Ngoc Dang Do was reviewed and approved* by the following: Leonard S. Jefferson Evan Pugh Professor and Chair of Cellular and Molecular Physiology Thesis Adviser Chair of Committee Douglas R. Cavener Professor and Head of Biology Robert A. Gabbay Professor of Endocrinology Ralph L. Keil Professor of Biochemistry and Molecular Biology Scot R. Kimball Professor of Cellular and Molecular Physiology Peter J. Hudson Willaman Professor of Biology Head of the Integrative Biosciences Program *Signatures are on file in the Graduate School. ii

3 Abstract In eukaryotes, regulation of mrna translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eifs) and their associated proteins. These 11 eifs catalyze the joining of the trna, mrna and rrna into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mrnas, and specialized, in targeting translation of mrnas with regulatory features such as a 5 terminal oligopyrimidine tract (TOP), upstream open reading frames (uorfs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mrna translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mrnas, whereas a family of eif2 kinases does so with mrnas containing uorfs or an IRES. TOR targets translation of mrnas that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eif2 kinase family increase translation of mrnas coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eif2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eif2 kinases regulate the translation of both a global and a specific set of mrnas. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. Also unexplored is the interaction between stress-induced inhibition of translation initiation by the eif2 kinases and its effect on TOR signaling. This is particularly relevant given that both pathways are regulated by some of the same environmental signals, such as nutrient availability. The work presented in this thesis addresses these two topics. The resulting data demonstrate a dependency on translation initiation status for the regulation of downstream targets of TOR, and identify sets of mrnas whose translation is commonly or uniquely regulated by two of the eif2 kinases. iii

4 Table of Contents List of Figures List of Tables Abbreviations Acknowledgements vii ix x xi Chapter 1. Introduction Overview of translation Translation initiation Messenger RNA Transfer RNA Ribosomes Heteromultimeric eif Mechanisms of translation initiation Upstream regulations of translation initiation TOR kinase Regulators of TOR Growth signals Cellular energy status Cellular nutrient status Effectors of TOR eif2α kinases Heme-regulated inhibitor or eif2α kinase 1 (HRI or EIF2K1) Double-stranded RNA-activated protein kinase or eif2α kinase 2 (PKR or EIF2K2) PKR-like ER-resident kinase or eif2α kinase 3 (PERK or EIF2K3) General control nonderepressible 2 or eif2α kinase 4 (GCN2 or EIF2K4) Downstream effects of regulation of translation initiation TOR and translation of mrnas containing 5 terminal oligopyrimidine tract (TOP) eif2α kinases and translation of mrnas containing an IRES and/or uorfs Clinical correlates Diseases associated with mrna Diseases associated with factors involved in regulation of ternary complex formation Diseases associated with factors involved in regulation of TOR Thesis objectives 23 iv

5 Chapter 2. Materials and Methods Animals Liver perfusion Guanine nucleotide exchange activity assay SDS-PA gel electrophoresis and Western blotting Sucrose density gradients 34 Chapter 3. Amino acid regulation of TOR targets is dependent on eif2α kinase activity Rationale Materials and Methods Cell culture Sucrose density gradient Measurement of intracellular amino acid concentration Statistical analysis Results Deprivation of different essential amino acids disparately affects translation initiation and regulation of GCN2 and TOR targets in perfused mouse liver Regulation of TOR targets following amino acid deprivation depends on the presence of GCN Amino acid deprivation-induced regulation of 4E-BP1 and p70s6k1 in Gcn2 -/- MEFs is TOR-dependent Amino acid deprivation inhibits translation initiation in Gcn2 +/+ and elongation in -/- MEFs Intracellular branched-chain amino acid (BCAA) concentrations do not explain the effects on 4E-BP1 and p70s6k1 in Gcn2 -/- MEFs following amino acid deprivation Inhibition of translation initiation restores the regulation of TOR targets in Gcn2 -/- to the same as that in +/+ MEFs Inhibition of translation elongation promotes hyperphosphorylation of TOR targets Amino acid alcohols do not regulate GCN2 and TOR targets similarly as does amino acid deprivation Discussion 49 Chapter 4. GCN2 and PERK differentially regulate mrna translation Rationale Materials and Methods Liver perfusion Protein synthesis measurement RNA extraction PCR evaluation of Xbp-1 mrna splicing 78 v

6 Hybridization arrays Quantitative RT-PCR Statistical analysis Results The optimal perfusion condition for Gcn2 and Perk mice is methionine deprivation and 40 µm of tbuhq, respectively GCN2 or PERK is the only kinase responsible for eif2α phosphorylation following amino acid deprivation or ER stress, respectively, in mouse livers Regulation of eif2b activity in mouse liver following amino acid deprivation or ER stress depends on the presence of GCN2 or PERK, respectively Polysome profiles of perfused Gcn2 and Perk mouse liver samples correlate with changes in eif2α phosphorylation and eif2b activity The absence of GCN2 affects mrna abundance more profoundly than that of PERK GCN2 and PERK recruit different functional groups of mrnas for translation following stress GCN2 and PERK affect the translation of both common and distinct sets of mrnas Discussion 95 Chapter 5. Conclusions Remaining puzzlers Regarding the model for the effects of translation initiation effects on TOR Regarding the differential regulation of mrna translation by GCN2 and PERK Take-home message 144 References 146 Appendix A. Components of liver perfusion buffers. 162 Appendix B. Antibody conditions for Western blotting 163 Appendix C. Regulation of GCN2 and TOR targets following combined treatment with MG-132 and amino acid deprivation in MEFs. 164 Appendix D. Regulation of GCN2 and TOR targets following combined treatment with puromycin and amino acid deprivation in MEFs. 165 Appendix E. Primer sequences and gene-expression assay code for QRT-PCR and PCR reactions. 166 Appendix F. Surveys of the 5 noncoding region (NCR) of mrnas selected for validation by QRT-PCR. 168 vi

7 List of Figures Figure 1.1. Interactions between the mrna being translated and the eukaryotic initiation factors (eifs) that form the translation initiation complex 24 Figure 1.2. Formation of the translation initiation ternary complex 25 Figure 1.3. Upstream regulators and downstream effectors of mtor 26 Figure 1.4. A model of heme-regulated inhibitor (HRI) kinase activation 27 Figure 1.5. A model of double-stranded RNA-activated kinase (PKR) activation 28 Figure 1.6. A model of PKR-like ER-resident kinase (PERK) activation 29 Figure 1.7. A model of general control nonderepressible 2 (GCN2) kinase activation 30 Figure 3.1. Polysome profile of C57Bl/6 mouse liver perfused with different amino acid conditions 53 Figure 3.2. Regulation of eif2α and eif2b in C57Bl/6 mouse livers perfused with different amino acid conditions 54 Figure 3.3. Regulation of 4E-BP1 and p70s6k1 in C57Bl/6 mouse livers perfused with different amino acid conditions 55 Figure 3.4. Regulation of GCN2 and TOR targets following histidine or leucine deprivation in MEFs 56 Figure 3.5. Regulation of GCN2 and TOR targets following combined treatment with rapamycin and amino acid deprivation in MEFs 58 Figure 3.6. Polysome profiles of amino acid-deprived Gcn2 +/+ and -/- MEFs 59 Figure 3.7. Intracellular branched-chain amino acids concentration following histidine or leucine deprivation in MEFs 60 Figure 3.8. Regulation of GCN2 and TOR targets following combined treatment with clasto-lactacystin β-lactone and amino acid deprivation in MEFs 61 Figure 3.9. Regulation of GCN2 and TOR targets following combined treatment with thapsigargin and amino acid deprivation in MEFs 62 Figure Polysome profiles of combined thapsigargin-treated, amino acid-deprived Gcn2 +/+ and -/- MEFs 64 Figure Regulation of GCN2 and TOR targets following combined treatment with arsenite and amino acid deprivation in MEFs 65 Figure Regulation of GCN2 and TOR targets following combined treatment with cycloheximide and amino acid deprivation in MEFs 67 Figure Regulation of GCN2 and TOR targets following combined treatment with histidinol and amino acid deprivation in MEFs 69 Figure Regulation of GCN2 and TOR targets following combined treatment with leucinol and amino acid deprivation in MEFs 70 Figure A model for the regulation of TOR targets by translation initiation state. 71 vii

8 Figure 4.1. Figure 4.2. Figure 4.3. Figure 4.4. Figure 4.5. Figure 4.6. Figure 4.7. Regulation of protein synthesis, eif2α and eif2b in C57Bl/6 mouse livers perfused with different doses of 2,5-di-tert-butylhydroquinone. 102 Regulation of Xbp-1 mrna splicing in mouse livers perfused with 2,5-di-tert-butylhydroquinone. 103 Regulation of eif2α and eif2b in perfused Gcn2 and Perk mouse livers following methionine deprivation or 2,5-di-tert-butylhydroquinone treatment, respectively. 104 Polysome profiles of Gcn2 and Perk mouse livers following methionine deprivation or 2,5-di-tert-butylhydroquinone treatment, respectively. 107 Functional categories of mrnas whose abundance changed in eif2α kinase knockout versus wild-type perfused livers. 112 Functional categories of mrnas whose shift into polysomes following stress is dependent on GCN Functional categories of mrnas whose shift into polysomes following stress is dependent on PERK. 124 viii

9 List of Tables Table 4.1. Table 4.2. Table 4.3. Table 4.4. Table 4.5. Table 4.6. Table 4.7. Table 4.8. Table 4.9. Table Number of mrnas whose abundance changed in eif2α kinase knockout versus wild-type perfused livers. 111 Top ten percent of gene probes whose expression changed in Gcn2 -/- versus +/+ perfused livers. 113 Top ten percent of gene probes whose expression changed in Perk -/- versus +/+ perfused livers. 115 Quantitative RT-PCR validation of abundance of selective mrnas in eif2 kinase wild-type versus knockout perfused livers. 116 Number of mrnas that shift into polysomes following stress in the presence of GCN2 or PERK. 117 Top ten percent of gene probes whose expression changed in the polysome fraction following methionine deprivation and in the presence of GCN Top ten percent of gene probes whose expression changed in the polysome fraction following 40 µm 2,5-di-tert-butylhydroquinone treatment and in the presence of PERK. 126 Common functional categories whose mrnas translation is regulated by either GCN2 or PERK activation a comparative summary of Figures 4.5 and mrnas whose translation is commonly regulated by either GCN2 or PERK activation. 132 Quantitative RT-PCR validation of polysome expression of selective mrnas in eif2 kinase wild-type versus knockout perfused livers. 134 ix

10 Abbreviations 5 NCR 5 non-coding region 4E-BP eif4e binding protein AA amino acid/s AMPK AMP-activated kinase BCAA branched-chain amino acid cdna complementary deoxyribonucleic acid CLBL clasto-lactacystin β-lactone crna complementary ribonucleic acid DMEM Dulbeco s modified Eagle's medium eef2 eukaryotic elongation factor 2 eif eukaryotic initiation factor ER endoplasmic reticulum GCN2 general control nonderepressible 2 GDP guanine nucleoside diphosphate GTP guanine nucleoside triphosphate HRI heme-regulated inhibitor IRE iron regulatory element IRES internal ribosomal entry site IRP IRE regulatory protein MEFs mouse embryonic fibroblasts Met-tRNA i methionyl initiating trna mrna messenger RNA NCR non-coding region p70s6k rps6 kinase of 70 kda PERK PKR-like ER resident kinase PIC pre-initiation complex PKB protein kinase B PKR double-stranded RNA-activated protein kinase QRT-PCR quantitative real time-polymerase chain reaction RNA ribonucleic acid rps6 ribosomal protein S6 rrna ribosomal RNA SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis tbuhq 2,5-di-tert-butylhydroquinone TOP 5 terminal oligopyrimidine tract TOR target of rapamycin trna transfer RNA uorf upstream open reading frame UPR unfolded protein response x

11 Acknowledgements I came a seedling. Through you I bear fruits. Leonard S. Jefferson, Ph.D. Scot R. Kimball, Ph.D. Douglas R. Cavener, Ph.D. Robert A. Gabbay, MD, Ph.D. Ralph L. Keil, Ph.D. Ahmed Abbas, Ph.D. Samer al-murrani, Ph.D. Jamie Crispino Stephen Crozier, Ph.D. Kevin Finnerty Rick Horetsky Lynne Hugendubler Christina Leah Banta Kline Neil Kubica, Ph.D. Lydia Kutzler Hau (Susan) Nguyen Sharon Rannels Ali Reiter, Ph.D. Courtney Williamson David Williamson, Ph.D. Laboratory of Douglas Cavener, Ph.D. Kaori Iida, Ph.D. Yulin Li Jeffrey O Neil Vincent Chau, Ph.D. Laboratory of Gary Clawson, Ph.D. Heidi Devlin, Ph.D. Laboratory of James Connor, Ph.D. Stacey Clardy, Ph.D. Bioinformatics Consulting Center Gary Chase, Ph.D. Wenlei Liu, Ph.D. Wei Zhao DNA Microarray Facility Craig Praul, Ph.D. Functional Genomics Core Robert Brucklacher Willard Freeman, Ph.D. Daniel Krissinger Terry Rager General Clinical Research Center Nicholas King Kristen Rice Macromolecular Core Anne Stanley xi

12 Chapter 1. Introduction 1.1. Overview of translation Two cellular processes are anchors for the central dogma of biological information flow from DNA to RNA to protein: transcription (forward or reverse) and translation. Of the two, translation is more capable of fast, immediate responses to both general and local changes in the cellular environment (52, 130). Its temporal quality allows for minutes-to-hours responses to extracellular signals such as hormones and intracellular status such as the protein folding capacity of the endoplasmic reticulum (ER). Its spatial attribute proves necessary for the organism, where graded or confined distribution of protein expression allows for polarity development along an embryo, formation of cellular processes, or mobilization of cells (52, 130). We conceptualize mrna translation as occurring in three stages: initiation, elongation and termination. Biologically, the translation closed-loop model in Saccharomyces cerevisiae proposes that termination, while demarcating the end of the protein being produced, may also facilitate the beginning of the next translational round (210). In eukaryotes, three proteins coordinate the translating ribosome complex decoding of one of the three termination codons (UGA, UAA, UAG) to release the synthesized peptide, and as previously thought, dissociate the ribosome complex (93, 133). Reported interactions among the proteins bound at the 5 and 3 ends of mrnas provide intriguing evidence for the aforementioned model, where circularization of the mrna provides a passageway for translating ribosome complexes to be shuttled from the 3 to 5 end for reinitiation instead of dissociation (210). 1

13 If the translational stages were judged solely on the number of amino acids they add to the final protein product, elongation would be the clear winner. Elongation is a highly conserved event among species that is responsible for the correct interaction between the codon and the decoding aminoacyl trna (93, 133). The 80S ribosome complex provides the context and environment for this interaction, along with a few additional proteins. The 60S subunit of the ribosome complex forms binding pockets to harbor the trna bearing the elongating peptide (P site), and the next decoding aminoacyl-trna (A site). A peptidyl transferase enzymes catalyze the formation of a bond between the elongating peptide and the next amino acid. Shifting of the ribosome complex toward the next 3 codon results in the residency of the recently denuded trna in the E site, and the trna in the A site moving into P. Of the few elongation factors identified, eukaryotic elongation factor 2 (eef2) is regulated by target of rapamycin (TOR), a kinase that also regulates proteins involved in translation initiation (158, 164). An elongation rate of 3-5 amino acids per second underlines the temporal advantage of regulating translation, as new proteins can be synthesized in minutes (121). While elongation is where the bulk of protein synthesis occurs, it depends on initiation to start the chain. The identification so far of 11 different eukaryotic initiation factors (eifs) involved in this first step of mrna translation attests to its importance (52, 71, 82). The implication of intense regulation of this step fits logically with a cell s goal of coordinating its status and metabolic events if protein synthesis were to be altered, it would be most effective and efficient to do so at the first step. 2

14 1.2. Translation initiation The goal of translation initiation is to form the base for elongation by bringing together the ribosome subunits, 40 and 60S, the coding mrna, and the initiating methionyl-trna (met-trna i ). At the end of this stage, the met-trna i would occupy the P site in the 60S ribosome subunit, poised for the catalysis of the first peptide bond and the beginning of translation elongation. The following description of the steps of translation initiation is basic to all rounds of protein synthesis and can occur multiple times sequentially on an mrna, as permitted by its length. We will focus the descriptions to mammalian translation initiation, unless otherwise noted Messenger RNA Prokaryotic mrnas contain a Shine-Dalgarno sequence, a stretch of polypurines 5 to the initiating AUG that binds to the complementary polypyrimidines in the 3 end of the 16S rrna. This essential interaction provides the context for translation initiation (83, 93, 181). The lack of such a marker in eukaryotic mrnas presumably necessitates the involvement and coordinated interactions of the large number of eifs. The unifying features of eukaryotic mrnas that contribute to identification of the correct initiating AUG consist of a 5 cap structure and the Kozak sequence (93). Following transcription in the nucleus, eukaryotic mrnas are processed to lose noncoding introns, gain a 7-methylguanylate structure linked 5-5 with the first nucleotide (m7gpppn, the cap structure), and a stretch of polyadenosines at the 3 end (93, 133). The 5 and 3 additions promote mrna stability by protecting it from exonucleolytic cleavage, and enhance its translation by inducing the formation of protein complexes (30, 39, 133). The initiation factor eif4e binds the 5 cap structure as part of 3

15 the eif4f complex to provide a signpost for recruitment of the 40S ribosome subunit to the mrna (Figure 1.1). The other members of the eif4f complex include eif4a, which provides helicase activity for unwinding of secondary structure in the 5 noncoding region (NCR), and eif4g (93, 133). This last factor serves as a scaffold protein that interacts with eif3 (67) and poly-a binding protein (PABP) (210). A family of eif4e binding proteins (4E-BPs) modulates the interaction between eif4e and eif4g. They are of relatively small molecular weight and contain several Ser/Thr phosphorylation sites. The best characterized of the 4E-BPs, 4E-BP1 sequesters eif4e, when hypophosphorylated, by binding to the same amino acid sequence through which the latter interacts with eif4g (53, 55). This disruption leads to failure of both eif4f formation and translation initiation. Extracellular signals such as hormones and nutritional status can alter the phosphorylation status of 4E-BP1 and its corresponding contact with eif4e (Figure 1.1) (160). Eukaryotic translation initiation most often occurs at the first AUG downstream from the 5 cap (71). In mammals, the distribution of the surrounding nucleotides, as described by Marilyn Kozak, provides additional favorable context for selection of an AUG as the initiating codon (111, 113, 155). The Kozak sequence GCCACCAUGG emphasizes the critical conservation of purine bases at the -3 and +4 positions (111, 113). Since the original studies by Kozak showing relatively higher binding to 80S in reticulocyte lysate by oligonucleotides containing a purine in the -3 and/or G in the +4 positions, understanding of how these conserved bases contribute to a favorable initiation context is still lacking (113). Although base pairing between the mrna and 18S rrna have been reported, a recent analysis indicates that while the purine in position -3 of the 4

16 Kozak sequence is a conserved base, its surrounding neighbors neither are conserved nor occur in correlation with one another (150). These deficient characteristics cast doubt on a conserved mrna-trna interaction at this site (150) Transfer RNA The majority of eukaryotic translation initiation begins with the codon AUG, which codes for a methionine. Through its γ subunit, a GTP-bound eif2 can associate with a met-trna i by virtue of the latter s conserved base-pair A1:U72 (Figure 1.2) (44, 92). This met-trna i eif2 GTP assembly integrates into the pre-initiation complex (PIC) through interaction with the heteromultimeric eif3 (201). Subsequent contact with the 40S ribosome subunit allows location of the initiating AUG. In yeast, preserved interaction among the eif2 subunits (α, β, γ), and the eif5a-assisted hydrolysis of the GTP bound to eif2γ contribute to the correct anticodon-codon base pairing (68, 75, 182, 199). GTP hydrolysis also effects the release of the GDP-bound eif2 from the translation initiation hub. To form a new ternary complex, the GDP must be exchanged for GTP (Figure 1.2). Two features necessitate the involvement of eif2b, a guanine nucleotide exchange protein (101), in this reaction: the ~100x higher affinity for GDP rather than GTP by eif2 and the slow spontaneous rate of GDP release (42, 93). The activity of the heteropentameric eif2b is regulated through several mechanisms, including inhibition by phosphorylated eif2 (168). Phosphorylation of the latter on Ser 51 of its α subunit is a common consequence following various cellular stresses (39). 5

17 Ribosomes In contrast to the prokaryotic 70S ribosomes, the eukaryotic 80S translational apparatus consists of a large and small subunit, whose cores contain the 28S and 18S rrnas, respectively. The rrnas associate with their own contingent of proteins (named by the subunit with which they interact, e.g. L11 is found in the 60S, S6 in the 40S) to form the 60S and 40S ribosome subunits (71, 93, 133). Compared to the other two RNA forms involved in translation initiation, the ribosomes involve the least number of interactions with and regulation by the eifs. Assembly of the 40S subunit with the translating mrna follows the joining of the former to a pre-initiation complex (PIC) that is anchored by the multisubunit protein eif3 (93). The 40S provides the support and mechanism for movement of the met-trna i, brought in from the PIC, along the mrna to the ultimate end of the first anticodon-codon base pairing. In the last step, members of the PIC are released and the 60S subunit joins to form the holo-80s complex, all transpiring at the hydrolysis of two GTPs (93). Ribonuclease protection assays (RPA) have shown the 80S, when at the initiating AUG, covers approximately bases upstream and downstream of the start site (130). Several initiation events can occur sequentially on an mrna as shown by an analysis of ribosomes stacking on a bovine preprolactin mrna, where as many as nine ribosomes can be found on a stretch of 270 nucleotides (211). This analysis also proffered additional support for initiation as a limiting step in translation since one of the four pauses in ribosome loading was detected after initiation (211). (The four pauses occurred at the initiation site, ~ the 75 th codon, correlated with the binding of the signal 6

18 recognition particle to the signal peptide, ~ the 160 th codon and the termination site (211).) Among the protein posse of the 40S subunit, ribosomal protein S6 (rps6) has been most closely linked to regulation during translation initiation. Its phosphorylation by a family of 70 kda protein kinases (p70s6ks) has been correlated with increased protein synthesis and cell proliferation, and increased glucose homeostasis and cell growth (169). Recent studies using a phospho-mutant rps6 knock-in (where all the phosphorylatable Ser were replaced with Ala), or S6K1-/- cells have dismissed the protein s role in selective translation of mrnas containing 5 tract of terminal oligopyrimidine (TOP) (169, 170) (See also section 1.4.1) Heteromultimeric eif3 This colossal translation initiation factor deserves a special address. First identified in the 1970 s this 11-subunit complex contains binding sites for eif1, eif1a, eif2, eif4b, eif4g, eif5a and the 40S ribosome subunit (9, 67, 93, 201). Its size and numerous interactions are hypothesized to coordinate the factors involved in translation initiation into an efficient, yet dynamic, PIC (93), and provide directionality to the scanning process (183) Mechanisms of translation initiation With the aggregation of the three species of RNA and their associated proteins, translation initiation can proceed to locating the initiating AUG. For the majority of mrnas this occurs through scanning of the 40S ribosome along the mrna until it reaches the first AUG (113). As alluded to above, > 90% of eukaryotic translation initiation occurs at this first AUG (93) and results in synthesis of the desired peptide. 7

19 Two instances diverge from this general scheme: the presence of upstream open reading frames (uorfs) and high energy secondary structures. In some mrna species, the target AUG may lie downstream of one to several 5 proximal Agues. Initiation at these upstream AUGs, however, may be largely bypassed if they are not surrounded by the favorable Kozak sequence (112). On the other hand, if they are, translation of these uorfs takes precedence and contributes to regulated expression of the target protein, as in the well-documented case of the yeast GCN4 mrna (73). As the 40S scans along the mrna, eif4a with the assistance of eif4b catalyzes the unwinding of secondary structure in the 5 NCR (71). This action can be inhibited in cases where the secondary structure is stabilized by either trans-acting proteins or high energy of formation (52, 156). An example of the first case is the mrna that codes for the intracellular iron binding protein ferritin, which contains a hairpin structure in its 5 NCR. This iron-regulated element (IRE) is stabilized by IRE-binding proteins (IRPs), whose activities in turn are regulated by cellular iron level. At low intracellular iron levels, IRPs bind and stabilize the IRE hairpin, thus impeding the recruitment of the 40S subunit (141). For mrnas containing high energy and stable secondary structure in their 5 NCR, ribosome scanning may not be the mode of translation initiation. Viral mrnas were among the first known to contain these structures (82). Since their secondary structure poses an insurmountable obstacle to the process of translation initiation proceeding from the 5 cap, their initiation has been hypothesized to happen independent of the ribosome cap-binding event (109, 191). In this cap-independent translation 8

20 initiation model, the 40S subunit along with smattering of initiation factors are directly recruited to the secondary structure, appropriately named the internal ribosomal entry site (IRES). The variation in the initiation factors recruited depends on the viral IRES (109, 191) Upstream regulations of translation initiation Commensurate with the number of proteins involved in translation initiation is the number of signaling pathways and kinases involved in their regulation. Not surprisingly, signaling following variations in extra- or intracellular environment commonly contain a component that feeds into regulation of translation initiation. We will limit this survey to the two pathways most relevant to this thesis: regulation of cap-dependent translation through the target of rapamycin (TOR) and the eif2α kinases TOR kinase Since its discovery, TOR has generated constant interest for its role as a nexus for the integration of inputs (from growth signals, cellular energy and nutrient status) with outputs (in the form of cell growth and proliferation) (Figure 1.3) (69, 160, 196). In league with eif3 and eif4g, TOR is a large protein of approximately 280 kda with several N-terminally located HEAT (Huntington-EF3-A subunit of PP2A-TOR1) repeats that are hypothesized to confer its ability to interact with multiple other proteins (48, 69). Unlike those two initiation factors, TOR also contains Ser/Thr kinase activity at its C- terminus. Proteins identified to interact with TOR include the positive regulators Raptor (regulatory associated protein of TOR) (98) and GβL/mLST8 (G protein β-subunit-like protein), a negative regulatory complex composed of the antifungal macrolide rapamycin 9

21 and the FKBP12 protein (FK506 binding protein of 12 kda), and its kinase substrates (Figure 1.3) (48, 69) Regulators of TOR Research of the past few years has gradually embellished the upstream signaling pathways to TOR (Figure 1.3). Growth signals and cellular energy status are sensed by different mechanisms, but are hypothesized to converge their regulation on the phosphorylation and association of the hamartin/tuberin complex (146). Hamartin and tuberin regulate TOR activity only as a heterodimer; overexpression of either one alone does not have the same effects. Through the GTPase-activating property of tuberin, the complex inactivates the protein Rheb (Ras homolog enriched in brain), an activator of TOR, by converting GTP-Rheb to GDP-Rheb (69). The exact mechanism for the management of TOR by Rheb remains unknown Growth signals Growth factors such as insulin and platelet-derived growth factor (PDGF) regulate TOR through activation of phosphoinositide-3-oh kinase (PI3K) (Figure 1.3) (69). Activated PI3K phosphorylates phosphatidylinositol-3,4-bisphosphate (PIP2) to form phosphatidylinositol-3,4,5-triphosphate (PIP3). The latter compound recruits two kinases 3-phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB) to the plasma membrane. Their proximity allows PDK1 to phosphorylate and activate PKB (48, 69). The current model posits that PKB positively regulates TOR activity by catalyzing the inactivating phosphorylation of tuberin, both directly and through AMPactivated kinase (56). 10

22 Cellular energy status The high cost of energy in maintaining cellular protein synthesis has convinced many researchers of the logic in the co-regulation of cellular energy status and protein production. TOR emerges as a leading candidate for this interaction based on results showing that dephosphorylation of 4E-BP1 and p70s6k1 follows treatments with a glycolytic inhibitor (2-deoxyglucose), an AMP analog (5-aminoimidazole-4- carboxyamide ribonucleoside, AICAR) or conditions that interfere with cellular respiration such as hypoxia (48, 69, 160). Though TOR has been shown to bind ATP, the high concentration required for its half maximal activity (K m ~ 1 mm) and argued lack of sensitivity to small changes in ATP concentrations have prompted the favoring of alternative mechanisms (38, 69). One of these involves the AMP-activated kinase (AMPK) (196). Biochemical studies using AICAR, an activator of AMPK, led to TORdependent dephosphorylation of 4E-BP1 and p70s6k1 (41). In contrast to PKB, AMPK is proposed to phosphorylate and activate the GTPase-activating property of tuberin, hence, ultimately inhibiting the positive regulation of TOR by Rheb (Figure 1.3) (48, 69) Cellular nutrient status In mammalian studies, nutrient status has been heavily focused on the availability of amino acids. Within this confine, the branched-chain amino acids (BCAA), and especially leucine, are the star regulators of TOR. Numerous studies in cell cultures and whole animals have documented a rapamycin-sensitive stimulation or suppression of 4E- BP1 and p70s6k1 phosphorylation following supplementation with or withdrawal of amino acids, respectively (3-5, 7, 177). Still elusive is the mechanism. An original hypothesis a role of tuberin in amino acid signaling to TOR has been questioned with 11

23 recent data showing amino acid regulation of TOR in cells lacking tuberin (186). Deprivation of amino acid decreased intracellular concentrations of BCAA, but did not affect ATP levels (38). Also unaffected by amino acid deprivation was the charging of trnas (38, 149). Yet, experiments using amino acid alcohols, potent inhibitors of aminoacyl trna synthetases, have shown a TOR-dependent decrease of p70s6k1 phosphorylation and activity, implying a requirement for TOR to sense uncharged trnas (28, 78) Effectors of TOR Putative substrates for TOR kinase activity consist of proteins involved in transcription and translation. Belonging in the latter category are eif4b, eif4g, 4E-BPs, p70s6ks, protein phosphatase 2A (PP2A) and eef2 (69). The downstream effect of 4E- BP1 phosphorylation by TOR is most well understood and least controversial of the lot (18). Hyperphosphorylated 4E-BP1 dissociates from the cap binding protein eif4e, allowing the latter s binding to eif4g thus enhancing cap-dependent translation (53, 55). Still unclear are whether TOR directly phosphorylates 4E-BP1 and how the phosphorylation triggers 4E-BP1 dissociation from eif4e. Although TOR has a definitive role in regulating the phosphorylation of Thr 389 on p70s6k1, as treatment with rapamycin leads to rapid and severe dephosphorylation of the latter, there is no consensus on the mechanism (54). The conundrum lies in the lack of understanding about the consequences of the interaction between TOR and Raptor. Raptor forms a constitutive complex with, and presents substrates to TOR for phosphorylation. Yet, under conditions where p70s6k1 is dephosphorylated, no detectable changes in the TOR-Raptor association occurs (69). The fast and complete 12

24 dephosphorylation of p70s6k1 following rapamycin treatment prompted a phosphatase hypothesis, in which PP2A is derepressed by rapamycin-inhibited TOR (48, 69). Also enigmatic with p70s6k1 is the consequences of its phosphorylation. Besides phosphorylating the rps6, p70s6k1 has also been garnering an increasing list of substrates, some coincide with those of TOR, such as eif4b, and even includes TOR itself (27, 69) eif2α kinases At ~ 1/2 the size of TOR, eif2 serves as a seemingly disproportionate center of signal integration through the phosphorylation of Ser 51 on its α subunit. Stresses ranging from global cellular status to specific deficiency can activate any one of the four kinases that phosphorylates eif2α (208). Acutely, each stress activates a specific kinase. However, indication for compensation among them is present for long-term treatments (91) Heme-regulated inhibitor or eif2α kinase 1 (HRI or EIF2K1) Correlation of iron deficiency with decreased protein synthesis has been reported since the 1960 s, while identification of the regulatory mechanism lagged for 30 years until the characterization of HRI (58). This eif2α kinase is a homodimeric heme-binding protein (Figure 1.4). Each of its monomers contains two heme-binding regions that contribute to protein maturation and activity (24). Newly synthesized HRI is stabilized by heat-shock proteins and aided in achieving its active dimer conformation by irreversible binding of heme to the N-terminal binding site. In iron replete condition, Fe 3+ -containing heme binds to the reversible binding site on each monomer and induce disulfide bond formation (24). This hypothetically interferes with ATP binding, resulting 13

25 in an inactive kinase. In low iron condition, heme scarcity leaves the reversible binding site empty, allowing further autophosphorylation and activation of HRI (163). Its action is hypothesized to integrate the availability of iron/heme and hemoglobin synthesis. Of the four kinases, HRI has the most limited tissue distribution, having been detected mainly in erythrocytes, their precursors, and cells of embryonic origin (24, 135). Besides responding to iron deficiency, HRI is also activated in response to heat shock and oxidative stress, caused by chemicals such as arsenite (134, 208). Interestingly, an HRIrelated kinase in Saccharomyces pombe, Hri1p, also phosphorylates eif2α in response to stresses of heat shock, arsenite, or cadmium (217). Deranged expression of HRI does not confer oncogenic potential (24). Knockout of HRI is not lethal and does cause any gross phenotypic abnormalities in unchallenged mice, but does decrease survival in challenged mice (24, 134) Double-stranded RNA-activated protein kinase or eif2α kinase 2 (PKR or EIF2K2) Besides the stimulus implied in its name, PKR is also activated by interferon α or β through transcriptional induction, by Toll-like receptor proteins through phosphorylation, or following arsenite, heat shock or peroxide treatment through association with the PKR-associated activator/pkr-associated protein X (PACT/RAX) (51, 95). Activation of PKR by dsrna occurs in many ways similarly to that of HRI. The 68 kda PKR contains high- and low-affinity dsrna binding domains at its N- terminus (95). Binding of dsrna to these domains causes a conformational change that exposes an ATP-binding site (Figure 1.5). Following dimerization, trans-phosphorylation induces the kinase activity in its C-terminal domain. Regulation of PKR activity by 14

26 dsrna is bimodal. At low concentrations, dsrna binds only the high affinity domain, promoting cooperative binding among the low-affinity domains, and hence dimerization (95). At high concentrations, both the high- and low-affinity domain bind dsrna, hindering dimerization and PKR activation (95). This serves well for the virus s purpose of shutting down production of cellular proteins at the beginning of infection, but eventually promoting its own. PKR is widely expressed and the most promiscuous of the four kinases in interacting with and phosphorylating other proteins including STAT1 and p53, respectively (95, 157). It mediates cell survival by regulating members of the NF-κB family (40, 157). While inhibition of the kinase activity by expression of a dominant negative form or a viral-derived inhibitor led to tumor formation in mice, Pkr knockout experiments did not significantly change the phenotype or tumor incidence (95) PKR-like ER-resident kinase or eif2α kinase 3 (PERK or EIF2K3) PERK was first identified as a protein highly expressed in the pancreas and subsequently linked to regulation of the ER s unfolded protein response (UPR) by sequence comparison to a type-i transmembrane ER-resident protein (66, 179). It is the only one of the four eif2α kinases to span a membrane. PERK senses stress through its N-terminal domain that lies in the ER lumen, while its C-terminal kinase domain resides in the cytoplasm (Figure 1.6) (166). Under basal conditions, the ~ 122 kda PERK monomers are kept from aggregating by the chaperone glucose-regulated protein of 78 kda (GRP78/BiP) (166). An increase in the ER s unfolded protein burden (through ERcalcium depletion by thapsigargin, disturbance of the ER s oxidative environment by dithiothreotol (DTT), or interference with protein glycosylation by tunicamycin) titrates 15

27 GRP78/BiP from PERK, allowing for oligomerization (175). The multimeric PERK complex trans-phosphorylates and acquires eif2α kinase activity (128). In mammals, PERK is ubiquitously expressed and found at the highest level in the pancreas (219). Besides eif2α, a transcription factor involved in promoting cell survival, Nrf2 (nuclear factor, erythroid-derived related factor 2), has been reported to be phosphorylated by PERK (33). Also unique from the other eif2α kinases, global Perk -/- or mutated PERK expression decreases survival at both the cellular and organismal levels (64, 65, 219). Loss of PERK causes bone-associated growth defects, pancreatic β-cell abnormalities, and consequently hyperglycemia from decreased insulin secretion (64, 120, 219) General control nonderepressible 2 or eif2α kinase 4 (GCN2 or EIF2K4) Much of the mechanism for GCN2 function was elucidated in S. cerevisiae as this is the only eif2α kinase present in that organism. The mammalian homolog was only recently identified and contains similar functions and requirements as those of the yeast kinase (11, 61, 131, 220). In contrast to the other three eif2α kinases, the ~ 182 kda GCN2 dimerizes independently of its stimulus (Figure 1.7). The GCN2 dimers are stabilized by heat-shock protein and kept in their inactive state via interactions between the N-terminally located pseudokinase and the C-terminal domains (72). These interactions sequestered the eif2α binding site and the kinase domain, inhibiting the activating autophosphorylation event (72). Binding of uncharged trna to GCN2 disrupts the pseudokinase and C-terminal domain interactions, unveiling the kinase domain and the eif2α binding site for subsequent trans-phosphorylation and activation (209). 16

28 GCN2 contains a C-terminally located sequence that binds to the 28S rrna in the 60S ribosome subunit. This interaction and those with the GCN1/GCN20 complex are required for GCN2 activity, by bringing the kinase into the proper contact with the codon-bound uncharged trna in the A-site of 80S ribosome (129, 172). GCN1 and GCN20 orthologs have been identified in mammals (107). Similar to PERK, GCN2 is ubiquitously expressed. Its activation is not limited to any specific species of uncharged trna. No other substrate has been reported for GCN2 besides eif2α. Global knockout of Gcn2 does not have as a dramatic effect on survival as does Perk, although knockout mice do exhibit stunted growth and preferential metabolic activities upon nutrient deprivation (6, 220). A recent study implicates GCN2 in regulating pathways involved in learning and memory formation in mice (32) Downstream effects of regulation of translation initiation Under basal conditions, mrnas are translated via initiation at their cap structure, a process supported by the cap binding activity of eif4e and assembly of the eif4f complex. Overexpression of eif4e induces transformation of NIH 3T3 mouse embryonic fibroblasts and is found in many tumors (13, 69). In correlation, overexpression of 4E-BP1 in transformed, or expression of a non-phosphorylatable 4E- BP1 mutant in nontransformed cells promotes apoptosis (119). Overexpression of eif4e regulates the translation and subcellular distribution of at least two mrnas involved in tumorigenesis and cell cycle control, ornithine decarboxylase (106, 167), and cyclin D1 (167), respectively. Upstream regulations through the TOR and eif2a kinases pathways also lead to selective mrna translation. 17

29 TOR and translation of mrnas containing 5 terminal oligopyrimidine tract (TOP) With its phosphorylation of 4E-BP1, TOR positively influences cap-dependent mrna translation. In addition, increased TOR activity has been strongly linked with selective translation of 5 TOP mrnas, which code for ribosomal and translation elongation proteins. The effect of TOR on p70s6k1 and the latter s activation of rps6 led many to postulate that rps6 provides the platform for the selection of TOP mrnas. Experiments showing 1) decreased recruitment of a synthetic TOP-containing mrna to polysomes following serum treatment in cells transfected with a dominant negative p70s6k1, and 2) retention of the synthetic TOP-containing mrna in the polysome following rapamycin treatment of cells transfected with a constitutively active p70s6k1 support this hypothesis (87). However, subsequent studies of several endogenous TOPcontaining mrnas, showing their recruitment to polysomes following serum treatment of S6k1 -/- cells, their unaffected translation following decreased rps6 phosphorylation, or their decreased presence in the polysome fraction following amino acid deprivation in S6k1 -/- cells (2, 69, 193), severely crippled this idea. The emerging speculation now focuses on eif4b as TOR delegate, given that this protein assists eif4a in unwinding of mrna secondary structures (69) eif2α kinases and translation of mrnas containing an IRES and/or uorfs Translation of mrnas containing an IRES either persists or flourishes under conditions that are unfavorable for cap-dependent initiation (136, 161, 192). These include 4E-BP1 hypophosphorylation that leads to eif4e sequestration, and eif2α phosphorylation with the subsequent decrease in ternary complex formation (47). Studies 18

30 of viral IRESes show that the highly structured constructs have a high affinity for components of the translation initiation complex, and in some cases do not require all of them to initiate translation (82, 89, 195). In the cases of the cricket paralysis virus, structural study of its IRES implicates that it can conform to the P-site of the 60S ribosome subunit, acting as the initiator trna (85). The list of cellular mrnas containing an IRES, as well as their trans-acting factors are growing, despite an undefined common mechanism of action (109, 161, 191). The first and most solid example of decreased ternary complex s derepression of mrna translation is that of yeast GCN4 (198). This mrna contains four uorfs and codes for a transcription factor responsible for the expression of proteins involved in amino acid biosynthesis (73). The model for GCN4 translation proposes that in nutrient replete cells, translation initiation occurs at the first uorf (73). At the termination of uorf1 translation, while the 60S subunit dissociates, the 40S remains on the mrna and continues scanning. Within a favorable time frame, short enough for the 40S to remain bound and long enough for it to recruit a new ternary complex, translation initiation can occur at a downstream uorf (73). The termination of this second round of translation would purportedly leave the 40S too close to the GCN4 ORF for reinitiation. Under amino acid deprivation conditions, ternary complex would become scarce, thus lengthening the 40S subunit s scanning after translation of the first uorf (73). This prompts bypassing of the remaining uorfs and reinitiation at the GCN4 ORF (182). A similar mechanism of action has been invoked for the mammalian Atf4 mrna (203). In mammals, among the ATF4-regulated genes is Gadd34 (growth arrest and DNA damage-inducible), whose protein product modulates the activity of protein 19

31 phosphatase 1 (PP1) (19, 147). PP1 dephosphorylates eif2α and hence, completes a negative-feedback loop (124, 147, 148) Clinical correlates In accordance with the role of translation initiation in regulating cellular protein synthesis in response to metabolic and other stress signals, dysregulation at any of its stages underlies many pathological conditions. The dysregulation can occur at the mrna level via changes in the 5 NCR, at the trna level via dysfunction in ternary complex formation, or at the rrna level via TOR signaling. Beyond the identified diseases, studies in various model systems continue to offer both potential causes (152, 154, 162, 171, 180, 188, 194) and cures (15, 86) Diseases associated with mrna The majority of cases in this category involve mutations, alternative splicings or dysregulation of trans-acting factors that produce an altered 5 NCR environment. In hereditary thrombocythemia, mutations in uorfs of the thrombopoietin mrna favor translation reinitiation at the usually suppressed target ORF (22, 112). Increased thrombopoietin production leads to increased platelet synthesis and ultimately abnormal blood clotting. Polymorphisms or mutations that altered the context of the Kozak sequence follows a similar concept of altered ribosome scanning, and has been implicated in many conditions (112). A mutation in the upstream AUG of the mrna coding for the vasopressin precursor precipitates translation initiation at an internal AUG (112). The resulting product carries an incomplete signal peptide sequence, barring it from correct folding in the ER. This leads to neurohypophyseal diabetes insipidus, a disorder characterized by excessive production of dilute urine and corresponding fluid imbibing. 20

32 Alternative splicings occur in mrnas coding for both pro-oncogenic and tumor suppressor proteins. The tumor suppressor gene BRCA1 codes for two mrna isoforms, the shorter α form found in normal tissue and the longer β form found in cancerous mammary glands (156). The long 5 NCR of the β form contains upstream AUGs and forms stable secondary structures. Both features are hypothesized to contribute to the reduced translation of BRCA1 protein. Another example of tumor suppressor protein down regulation is hyaluronidase. Cell lines derived from squamous cell carcinomas contain an intron-retaining hyaluronidase mrna that adds uorfs 5 to the target ORF and hence suppresses its translation (112). Two examples of mrnas that code for prooncogenic proteins are glioma-associated oncogene (Gli1) and mouse double minute 2 (Mdm2). In the first case, more efficient processing of the Gli1 mrna in basal cell carcinomas removes an intron that would have introduced uorfs, thus derepressing Gli1 translation (112). Expression of an Mdm2 isoform with a shorter 5 UTR in Burkitt s lymphoma cells leads to increased Mdm2 production (112, 156). This leads to increased Mdm2 and tumor suppressor protein p53 association, and degradation of the latter. Finally, several diseases have their base in deranged regulation of mrna transacting factors. In chronic myeloid leukemia, high expression of the heterogeneous nuclear ribonucleoprotein (hnrnp) E2 binds the 5 NCR of the mrna coding for the CCAAT/enhancer-binding protein α (C/EBPα), a transcription factor that induces cell cycle arrest and inhibits proliferation (22). In contrast to this is the complete loss or altered expression of a mutated fragile-x-mental retardation protein (FMRP) that causes the fragile-x-mental-retardation syndrome. FMRP binds, relocates to the postsynapse and regulates translation of mrnas involved in synaptic activity (17, 31, 76, 189). The 21

33 last example involves alteration on a cis-regulatory element. In hereditary hyperferritinemia-cataract syndrome (HHCS), mutations in the IRE of L-ferritin decrease its affinity for the IRPs (22, 156). The increased ferritin proteins aggregate and crystallize in the lens leading to cataract formation and, in severe cases, blindness Diseases associated with factors involved in regulation of ternary complex formation As intimated, of the four known eif2α kinases the absence of PERK has the most profound effects on an organism s survival. Indeed, the only human eif2α-kinaserelated disease known to date, the Wolcott-Rallison Syndrome (WRS), involves truncation of, or mutations in, the kinase domain of PERK (36, 176). Less than 20 WRS cases are identified worldwide and the oldest patient recorded was 35 years old. Common features of WRS include early-onset insulin-dependent diabetes, most likely the result of increased pancreatic β-cell death, and growth retardation, secondary to abnormal growth plate development (36, 176). The individuals also have issues involving functions of other organs and mental development. The severity of the phenotype may correlate with the degree of PERK inactivation. Seventy seven different mutations in the subunits of eif2b, most associated with the ε subunit, have been identified among individuals with the demyelinating syndrome childhood ataxia with central nervous system hypomyelination/leukoencephalopathy with vanishing white matter (CACH/VWM) (50). The outstanding feature of CACH/VWM is a fever- or trauma-induced, progressive loss of brain white matter, to be replaced by cerebrospinal fluid-filled cysts (22, 50). The onset of disease correlates with the degree of decreased eif2b activity. 22

34 Diseases associated with factors involved in regulation of TOR Although rapamycin is used as an immunosuppressant and is in phases II-III clinical trials as an antitumor drug (86), no direct connection between TOR and human diseases has been identified. In contrast, several upstream regulators of TOR have been implicated. Mutations in the gene coding for the regulatory subunit of AMPK, AMPK γ2, reduces its activity and causes uncontrolled growth of cardiac myocytes in familial hypertrophic cardiomyopathy and Wolff-Parkinson-White syndrome (79). Mutations in the phosphatase and tensin homolog (PTEN, which dephosphorylates PIP3 and inhibits PKB activation), hamartin and tuberin have all been associated with a group of syndromes whose main feature includes benign hamartomatous tumor formation (22, 79) Thesis objectives The introduction provides an overwhelming web of knowledge for the translation initiation process that is poised to keep growing. Two major unmined areas, however, pertain to the interactions among the upstream regulators of translation initiation, particularly those between TOR and eif2α kinases, and the differentiation among the downstream effects of mrna translation. Our overall hypothesis is that interactions among the upstream regulation of translation initiation occur to modulate a program of downstream protein expression both general and specific to an activating stress. We pursue this through two aims. 1. Assess the effect of the regulation of ternary complex formation on TOR signaling. 2. Compare the downstream regulation of mrna translation by GCN2 and PERK. 23

35 4E-BP1 eif4e (A) n eif4a P P P P eif4g (A) n 24 Figure 1.1. Interactions between the mrna being translated and the eukaryotic initiation factors (eifs) that form the translation initiation complex. eif4e binds the 5 - m7gpppn cap structure of the mrna being translated. Hypophosphorylated 4E-BP1 inhibits the interaction between eif4e and another member of the translation initiation complex, eif4g. eif4a provides helicase activity to unwind the mrna. (See section for a more detailed description.) (A)n poly-a tail; eif eukaryotic initiation factor; 4E-BP1 eif4 binding protein. P phosphorylation.

36 met β α + + γ eif2b GDP 25 met-trna i eif2 GTP ternary complex Figure 1.2. Formation of the translation initiation ternary complex. eif2, met-trna i and GTP interact to form the ternary complex and deliver the initiating trna to the pre-initiation complex. Hydrolysis of the GTP during translation initiation necessitates the involvement of the guanine nucleotide exchange factor, eif2b, to begin another cycle. (See section for a more detailed description.) eif2 eukaryotic initiation factor 2; GDP guanine nucleoside diphosphate; GTP guanine nucleoside triphosphate.

37 Amino acids? Insulin/IGF IR Rheb TSC1 PIP 3 PIP 2? TSC2 Akt PDK1 IRS1 PI3K Raptor GβL mtor Rapamycin PI3K AMPK PI3K/Akt 4E-BP1 S6K1/2 Low Energy ( AMP/ATP) eif4e Cell Proliferation (cell growth and cell cycle progression) 26 Figure 1.3. Upstream regulators and downstream effectors of mtor. Growth hormones activate mtor activity through the PI3K/Akt pathway. Cellular energy status regulates mtor activity through the AMP-activated kinase. Both of these pathways converge on the TSC1/2 complex upstream of mtor. The exact mechanism through which amino acids activate mtor is unclear. mtor activity is further influenced by other positive and negative regulatory proteins. (See section and its subsections for a more detailed description.) 4E-BP1 eukaryotic initiation factor 4E binding protein; Akt/PKB protein kinase B; AMPK AMP-activated kinase; eif4e eukaryotic initiation factor 4E; GβL - G protein β-subunit-like protein ; IGF insulin-like growth factor; IR insulin receptor; IRS1 insulin receptor substrate 1; mtor mammalian target of rapamycin; PDK1 3 -phosphoinositide-dependent kinase 1; PI3K phosphoinositide-3-oh kinase; PIP 2 phosphatidylinositol-3,4-bisphosphate; PIP 3 phosphatidylinositol-3,4,5-triphosphate; Raptor regulatory associated protein of TOR; Rheb Ras homolog enriched in brain; S6K1/2 ribosomal protein S6 kinase 1/2; TSC1 tuberous sclerosis complex 1 (hamartin); TSC2 tuberous sclerosis complex 2 (tuberin). Adapted from (49).

38 Mature-Competent Inactive Kinase Hsp90 & Hsc70 Heme Deficiency R ATP N N SH SH KI KI N N S S Heme KI R R KI Inactive Kinase P P R P P P P P P PPase N N Heme-Reversible Active Autokinase Heme Deficiency ATP P KI SH SH KI P Active Kinase P P P P P P 27 Figure 1.4. A model of heme-regulated inhibitor (HRI) kinase activation. Following synthesis, the HRI homodimer is bound to two irreversible hemes (red hexagon), two reversible hemes (R-labeled red hexagon) and chaperone proteins (Hsp90, Hsc70). In heme-deficient conditions, the reversible hemes dissociate from the kinase insert (KI) sites and autophosphorylation occurs. High heme levels induce binding of heme to the KI sites and subsequent disufide bond formation, thus inactivating the kinase. Low heme levels induce further autophosphorylation and maintain the active kinase state. (See section for a more detailed description.) KI kinase insert; N amino terminal domain; P phosphorylation; pink oval HRI subunit; R reversible heme; red hexagon heme; SH sulfhydryl group; S-S disulfide bond. Adapted from (24).

39 dsrna Binding Dimerization Activation P P eif2 ATP ATP ATP S P P eif2 P 28 K R Figure 1.5. A model of double-stranded RNA-activated kinase (PKR) activation. A PKR subunit is kept inactivated by interaction between the kinase (K) and RNA-binding (R) domains. Binding of dsrna (hatched rectangle) to the dsrna binding domain exposes the kinase domain and promotes dimerization. The dimerized protein transphosphorylates, turning itself into an active eif2 kinase. (See section for a more detailed description.) eif2 eukaryotic initiation factor 2; K kinase domain; P phosphorylation; R dsrna binding domain; S spacer domain. Adapted from (95).

40 Endoplastic Reticulum Endoplastic Reticulum BiP BiP BiP Stress R R R Malfolded Protein K K K P P eif2 eif2 P 29 Lumen Cytoplasm Figure 1.6. A model of PKR-like ER-resident kinase (PERK) activation. PERK is an ER transmembrane protein with its regulatory domain in the ER lumen and its kinase domain in the cytoplasm. Under unstressed condition, PERK remains in its non-aggregated form, with its monomer stabilized by BiP, an ER chaperone protein. Stresses, such as the presence of unfolded proteins, cause BiP to be released from PERK, allowing oligomerization of the latter. The PERK oligomers transphosphorylate and give rise to an active eif2 kinase. (See section for a more detailed description.) BiP/Grp78 glucose-regulated protein of 78 kda; K kinase domain; R regulatory domain. Adapted from (166).

41 N eif2 N N ΨPK ΨPK HSP90 ΨPK eif2 Uncharged trna P P PK PK PK C Autophosphorylation HisRS HisRS Inactive GCN2 dimer HSP90 C C Active GCN2 dimer 30 Figure 1.7. A model of general control nonderepressible 2 (GCN2) kinase activation. A GCN2 dimer is maintained in its inactive form through stabilization by HSP90, a chaperone protein, and interactions among its protein domains. The binding of uncharged trna to GCN2 causes the release of HSP90, disruption of the interdomain interactions and autophosphorylation. These events lead to the exposure of the kinase domain. (See section for a more detailed description.) C carboxy-terminal domain; eif2 eukaryotic initiation factor 2; HisRS trna-binding domain; HSP90 heat-shock protein of 90 kda; N amino terminal domain; PK phosphokinase domain; ψpk pseudo phosphokinase domain. Adapted from (72).

42 2.1. Animals Chapter 2. Materials and Methods All animals received water and food (Harkland, TX) ad libitum and were maintained according to the Institutional Animal Care and Use Committee-approved protocol at The Pennsylvania State University College of Medicine. C57Bl/6 mice, g in weight, were purchased from Jackson s laboratory. Global Gcn2-/- and wild-type littermate mice, 3-6 months old, were generated according to a previously published protocol (220). Alb/Cre Perk-/- and wild-type littermate mice, also 3-6 months old, were generated as previously described (219) Liver perfusion We followed previously published protocols (45, 197) for single-pass perfusion with modifications. On the day prior to perfusion, red blood cells (RBCs) were collected from bovine blood (Groff s Meats, Elizabethtown, PA) by centrifugation and washed with 0.9% NaCl/10x rat plasma concentration of leucine. In studies involving leucine deprivation, the wash solution contained 10x valine instead. For experiments where protein synthetic rate was measured, RBCs were washed with 0.9% NaCl/5 mm leucine. On the day of perfusion, buffer A, containing modified Ringer II buffer and glucose (see Appendix A), was gassed with 95% O 2 /5% CO 2 for 30 min. Buffer B was made by combining buffer A, amino acids at 10x the concentration of fasted rat plasma, and albumin (Albumin bovine fraction V; MP Biomedicals, LLC, Aurora, OH) and filtered through a 3.0 µm nitrocellulose filter (Millipore). RBCs were washed again with a 1:1 mix of buffer A and 0.9% NaCl/20x plasma concentration of leucine or valine, and filtered through glass wool. The perfusate contained a 2:1 ratio of buffer B and washed 31

43 RBCs. For amino acid deprivation perfusions, the indicated amino acid was omitted from Buffer B, and hence, the perfusate. All perfusions were done at 37 C for 35 min total. SDS-PAGE and Western blots samples were generated by homogenizing perfused liver samples in 7 volumes of homogenization buffer (20 mm HEPES, ph 7.4; 2 mm EGTA; 50 mm NaF; 100 mm KCl; 0.2 mm EDTA; 50 mm β-glycerophosphate; 1 mm DTT; 0.1 mm PMSF; 1 mm benzamidine; 0.5 mm NaVO 4 ; 10 µl/ml Sigma protease inhibitor cocktail). An aliquot of the supernatant following a 1, 000 x g, 3 min centrifugation at 4 C was added to an equal volume of 2x sample buffer (0.125 M Tris- HCl, ph 6.8; 25% v/v glycerol; 2.5% SDS; 2.5% v/v β-mercaptoethanol; 0.2% bromophenol blue), boiled at > 95 C for 4 min and store at 70 C. For measuring eif2b activity, perfused liver samples were dounce homogenized in 4 volumes of homogenization buffer (45 mm HEPES, ph 7.4; mm MgOAc; mm EDTA; 95 mm KOAc; 10% glycerol; 2 mm digitonin; 3 µm microcystin). The homogenate was centrifuged at 10, 000 x g, 4 C for 10 min, and the supernatant used in the assay. Samples for polysome profiles were generated by dounce homogenizing one gram of perfused liver sample in 3 volumes homogenization buffer (50 mm HEPES, ph 7.4; 250 mm KCl; 5 mm MgCl 2 ; 250 mm sucrose; 100 µg/ml cycloheximide). The homogenate was centrifuged at 3, 000 x g, 4 C for 15 min. Per ml of supernatant, 100 µl each of 10%, w/v Triton X-100 and 13%, w/v sodium deoxycholate were added. 32

44 2.3. Guanine nucleotide exchange activity assay The guanine nucleotide exchange activity of eif2b was measured following published protocols (35, 101). The radioactive exchange complex was made up immediately prior to the study by incubating at 30 C a mixture of 14 µl buffer (62.5 mm MOPS, ph 7.4; 125 mm KCl; 1.25 mm DTT; 2.5 mm MgOAc; 0.25 mg/ml BSA), 1.4 µg purified rat eif2, 1.5 µl [8, 5-3 H]GDP (PerkinElmer Life and Analytical Sciences, Boston, MA), with H 2 O added to bring the final volume to 21 µl. After 10 min, 14 µl 6 mm MgOAc were added and the complex was cooled on ice. The assay reaction mix containing 97 µl H 2 O, 140 µl non-radioactive GDP (52.1 mm MOPS, ph 7.4; 0.22 mm GDP; 104 mm KCl; 1.04 mm DTT; 2.08 mm MgOAc; 0.21 mg/ml bovine serum albumin), and 25 µl of supernatant was pre-incubated at 30 C for 1 min. The reaction was started with the addition of 35 µl of the 3 H-GDP eif2 Mg 2+ radioactive exchange complex and incubated at 30 C. Aliquots were removed from the assay reaction mix into 2.5 ml ice-cold wash buffer (50 mm MOPS, ph 7.4; 2 mm MgOAc; 100 mm KCl; 1 mm DTT) at 0, 30, 60 and 90 sec and vacuum filtered through 0.45 µm cellulose nitrate membrane filters (Whatman International, Ltd., Maidstone, England). The membranes were washed twice with 2.5 ml of wash buffer, dissolved in Filtron-X (National Diagnostics, Atlanta, GA) and counted in a scintillation counter (LS 6500 Multi-purpose scintillation counter, Beckman). The guanine nucleotide exchange activity was calculated as followed: count t0 (dpm) count t (dpm) GDP exchanged (pmol) = , 3 H-GDP specific activity (dpm/pmol) 33

45 where t = 30, 60 or 90 seconds. The slope of the GDP-exchanged-versus-time graph gives GE activity as pmol/min SDS-PA gel electrophoresis and Western blotting Protein samples were resolved on SDS-PAG, transferred onto 0.45 µm polyvinylidene fluoride (PVDF) membranes (Pall Life Sciences) and blocked with 5% non-fat dry milk. Primary antibody incubation was done overnight, at 4 C with the indicated dilution (Appendix B). Secondary antibody incubation was done at room temperature for one hour with either 1:10, 000 dilution of horseradish peroxidaseconjugated goat anti-rabbit or anti-mouse antibody (Bethyl) as appropriate. Membranes were developed with enhanced chemiluminescence (ECL) or ECL plus reagents (Amersham Biosciences, Piscataway, NJ). Images were captured using GeneGnome software (SynGene, Frederick, MD) and quantitated using GeneTools software (SynGene) Sucrose density gradients Discontinuous, 9-step 20% (10 mm HEPES, ph 7.4; 250 mm KCl; 5 mm MgCl 2 ; 0.5 mm EDTA; 20% w/w sucrose) to 47% (10 mm HEPES, ph 7.4; 250 mm KCl; 5 mm MgCl 2 ; 0.5 mm EDTA; 47% w/w sucrose) sucrose density gradients were used to separate mrnas based on the number of bound ribosomes. One ml of detergentcontaining supernatant (from perfused liver homogenate) was loaded onto the 20-47% sucrose gradients and centrifuged at 28,000 rpm (Beckmann SW-28), 4 C, for 3 h 38 min for polysome profiles or 22,500 rpm (Beckmann JS-24.38), 4 C, for 19 h 9 min for subunit profiles. Gradient profiles were visualized using an ISCO UV detection unit with a 254 nm filter and fractionated using an ISCO gradient pump (Teledyne Isco, Inc., 34

46 Lincoln, NE). The settings for the ISCO pump and UV detection system were 3 ml/min flow rate, 2.0 sensitivity and 60 cm/h chart speed. 35

47 Chapter 3. Amino acid regulation of TOR targets is dependent on eif2α kinase activity 3.1. Rationale In mammals, amino acid availability influences two steps in translation initiation: the assembly of the met-trna i eif2 GTP complex through GCN2-dependent phosphorylation of eif2α, and the function of 4E-BP1 and p70s6k1 through phosphorylation by TOR (43, 88, 99, 159). Many studies have documented the effects of essential amino acid deprivation on eif2α phosphorylation and translation initiation (100, 177, 202, 204). Not less in volume is the evidence for the role of amino acids, particularly leucine, in regulating phosphorylation of downstream targets of TOR and eif4f complex assembly (3, 5, 7, 177). Signaling by amino acids through these two pathways leads to alterations in the pattern of mrnas selected for translation (7, 84, 106). In S. cerevisiae, a connection between the GCN2 and TOR pathways was suggested by data showing derepression of GCN4 mrna translation following rapamycin treatment (114). In the yeast model, TOR indirectly inhibits GCN2 dephosphorylation at Ser 577 by promoting the binding of the phosphatase Sit4 to its regulatory and associated proteins (26, 48, 165). Phosphorylated GCN2 has decreased affinity for uncharged trna, leading to decreased phosphorylation of eif2α and nonderepression of GCN4 mrna translation (26). In mammals, interactions between these two pathways have not been studied extensively. This deficit relates to a dearth in understanding of how the signaling of supplementation or deprivation of amino acids is relayed to TOR in mammals. A 36

48 response similar to that of the stringent response in bacteria has been suggested, but the model remains to be tested (62). Several papers have proposed a role for uncharged trnas as the intracellular signal regulating TOR, and thus also a role for GCN2 (62, 78). Feeding experiments from both our laboratory (unpublished data) and that of Anthony et al. (6) provided strong evidence for this proposition. In those studies, 4E-BP1 and p70s6k1 in liver of animals fed amino acid-deficient diets exhibited decreased phosphorylation, but only when GCN2 was present. Recently, a model for pre-initiation complex assembly offered insight into how the GCN2 and TOR pathways may interact (74). At the center of this model is the protein eif3, and its intrinsic ability to associate with most of the remaining eifs, including eif2 and eif4e. Holz et al. (74) proposed that at the beginning of translation, TOR is recruited to eif3 within the translation initiation complex to phosphorylate 4E- BP1 and p70s6k1. Data from previous studies bolster this picture. Wang et al. (205) demonstrated that 4E-BP1 phosphorylation by TOR is dependent on the former binding to its substrate, eif4e. Together, these studies provide a spatial connection that may enable the coordination of inputs from several regulatory pathways. Based on these data, we hypothesize that during translation initiation complex assembly, regulation of translation initiation through ternary complex formation can influence the regulation of TOR targets, 4E-BP1 and p70s6k1. A corollary of this is that any stress that affects translation initiation may affect the phosphorylation of 4E-BP1 and p70s6k1. Finally, treatments that attenuate the translation initiation block, i.e. inhibition of translation elongation, may reverse the effects on 4E-BP1 and p70s6k1. 37

49 3.2. Materials and Methods Cell culture Gcn2 +/+ and -/- mouse embryonic fibroblasts (MEFs; provided by Dr. David Ron) were maintained at 37 C, in high glucose Dulbeco s modified Eagle s medium (DMEM; Invitrogen Corporation, Grand Island, NY) containing 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (Invitrogen). In all experiments, cells were plated overnight and used on the day of experiment at ~ 50-70% confluence. Histidine or leucine deprivation was accomplished by incubating cells for 2 h in the corresponding amino acid-deficient medium (Atlanta Biologicals, Norcross, GA), without serum. Control cells were incubated in serum-free DMEM. Histidine or leucine re-addition, to a concentration 1x that of regular DMEM, was done for 30 min. For inhibition of TOR, cells were pretreated for 15 min with EtOH or 20 ng/ml of rapamycin dissolved in EtOH, then for 2 h in the EtOH- or rapamycin-containing amino acidcomplete or deficient medium. To inhibit proteosome activity, cells were treated for 2 h with the indicated medium containing the vehicle DMSO, 5 µm of clasto-lactacystin β- lactone (CLBL; Boston BioChem), or 1 µm MG-132 (Calbiochem, La Jolla, CA). To activate PERK or HRI, cells were pre-treated for 30 min with 1 µm thapsigargin (Sigma) or 100 µm NaAsO 2 (Sigma), respectively, then for 2 h with the combined inhibitor and indicated medium. For inhibition of elongation, cells were treated for 2 h with the indicated medium containing either the vehicle EtOH, 1 µm cycloheximide (A. G. Scientific, Inc., San Diego, CA), or 100 µg/ml puromycin (Calbiochem). Histidinol 38

50 (Sigma) or leucinol (Sigma) was added to the indicated medium to a final concentration of 5 mm and used to treat cells for 2 h. SDS-PAGE and Western blot samples were generated by harvesting cells directly in 1x sample buffer (1:1 dilution of 2x sample buffer in water), boiled for 5 min, and loaded by equal volume. For polysome analysis, cells were harvested in buffer (50 mm HEPES, ph 7.4; 75 mm KCl; 5 mm MgCl 2 ; 250 mm sucrose; 1/10 volume 10% Triton X-100 and 13% sodium deoxycholate; 1 mg/ml cycloheximide) and the samples rocked for 10 min at 4 C. Supernatant from a 3, 000 x g, 15 min, 4 C centrifugation was used for the detergent-compatible protein assay (BioRad) Sucrose density gradient Discontinuous, 9-step 20-47% sucrose gradients were made as described in section 2.5, except the KCl concentration was changed to 75 mm. An equal amount of protein was loaded onto each gradient and centrifuged at 41, 000 rpm (Beckman SW41), 4 C, for 1 h 50 min. Gradient profiles were visualized as described in section 2.5 with these settings for the ISCO pump and UV detection system: 2 ml/min flow rate, 0.5 sensitivity and 150 cm/h chart speed Measurement of cellular amino acid concentrations For measurement of cellular amino acid concentrations, cells were washed twice in cold phosphate buffered saline, harvested in 3% perchloric acid and centrifuged at 1, 000 x g for three min. The supernatant was neutralized with 1 M K 2 HPO 4, mixed and centrifuged at 1, 000 x g for three min. The supernatant was subjected to amino acid 39

51 analysis by HPLC (126) (Nicholas King and Kristin Rice, General Clinical Research Center, PSU-COM) Statistical analysis Statistical analyses were done using the InStat software. The use of t-test or oneway ANOVA with the corresponding post-tests is noted in the figure legends Results Deprivation of different essential amino acids disparately affects translation initiation and regulation of GCN2 and TOR targets in perfused mouse liver. Perfusion of C57Bl/6 mouse livers with medium containing complete 10x amino acids or deficient of the nonessential amino acid glycine had similar effects on mrna translation (Figure 3.1 A and B). In contrast, perfusion without one of the essential amino acids, leucine, methionine or tryptophan, affected translation initiation, as evident by the increased 80S monomer content (Figure 3.1 A and B). Surprisingly, exclusion of the essential amino acid histidine from the perfusate also did not effect a change in the polysome profile (Figure 3.1 A and B). Essential amino acid deprivation has been shown in various model systems to activate GCN2 and promote subsequent phosphorylation of eif2α (6, 91). Lack of leucine, methionine or tryptophan in the perfusate led to eif2α phosphorylation, while that of glycine and histidine did not (Figure 3.2 A). Furthermore, perfusion with medium lacking methionine or tryptophan caused decreased eif2b GE activity, although the effect by leucine deprivation did not reach statistical significance in this study (Figure 3.2 B). In contrast, glycine or histidine deprivation either increased or did not affect eif2b activity, respectively (Figure 3.2 B). The increase in eif2 phosphorylation and inhibition 40

52 of eif2b activity correspond well with the changes in polysome profiles described above. In previous studies using liver perfusion, histidine deprivation with histidinol treatment led to polysome disaggregation, eif2α phosphorylation and inhibition of eif2b activity (100, 220). One fascinating possible explanation of the lack of such effects in this study may involve the different pathways of histidine metabolism and their trigger, a topic beyond the scope of this work (140). The disparity among the amino acid effects is evident in the regulation of TOR targets 4E-BP1 and p70s6k1. Only leucine deprivation led to their decreased phosphorylation, as shown by the reduction of the higher phosphorylated forms of 4E- BP1 and p70s6k1 (Figure 3.3). This suggests that the inhibition of translation initiation in the cases of methionine or tryptophan deprivation, exhibited in Figure 3.1 as increased 80S monomer content, resulted mainly from regulation of eif2α and eif2b instead of 4EBP-1 or p70s6k1. This agrees with previously published results (102) Regulation of TOR targets following amino acid deprivation depends on the presence of GCN2. Data from feeding studies have shown similar effects on eif2α phosphorylation (107), eif2b activity (107), and 4E-BP1 and p70s6k1 phosphorylation (unpublished data; (6)) in liver samples of rats fed a leu-deficient diet. The effects on TOR targets in these studies, however, may be influenced by other factors, since a GCN2-dependency for the decreased insulin secretion and phosphorylation of 4E-BP1 and p70s6k1 in animals fed a leu-deficient diet was observed (unpublished data). To examine the interaction between the GCN2 and TOR signaling pathways independently of the systemic effects of hormones and other regulatory factors, we used Gcn2 +/+ and -/- 41

53 mouse embryonic fibroblasts (MEFs). Unlike in the perfused liver, eif2α phosphorylation in the wild-type Gcn2 cells increased significantly following either histidine or leucine deprivation (Figure 3.4 A, B; compare lane 2 vs. 1 and 8 vs. 7). As expected, this response was not seen in the absence of GCN2 (Figure 3.4 A, B; compare lane 5 vs. 4 and 11 vs. 10). Similar to the perfused liver and meal feeding study results, only leucine deprivation led to hypophosphorylation of 4E-BP1 and p70s6k1 (Figure 3.4 C-F; compare lane 2 vs. 1 and 8 vs. 7). The decrease in phosphorylated 4E-BP1 and p70s6k1 was abolished in MEFs lacking GCN2 (Figure 3.4 D, F; compare lane 11 vs. 10), providing strong evidence that regulation of these TOR targets does not depend on systemic hormonal effects. Unexpectedly, histidine deprivation, while having no effect on TOR targets in the Gcn2 +/+ MEFs, increased their phosphorylation in the knockout cells (Figure 3.4 C, E; compare lane 5 vs. 4). Re-addition of the deprived amino acid to the medium reversed the effects on phosphorylation of eif2α (Figure 3.4 A, B; lanes 3, 9), 4E-BP1 (Figure 3.4 C, D; lanes 9), and p70s6k1 (Figure 3.4 E, F; lane 9) Amino acid deprivation-induced regulation of 4E-BP1 and p70s6k1 in Gcn2 -/- MEFs is TOR-dependent. Evaluation using phopho-specific antibodies against TOR-regulated sites (Thr 36/45 in 4E-BP1 and Thr 389 in p70s6k1) suggested that the regulation following leucine deprivation in Gcn2 +/+ (Figure 3.4 D, F compare lane 11 vs. 8) and histidine deprivation in Gcn2 -/- (Figure 3.4 C, E compare lane 5 vs. 2) MEFs is TOR-dependent. To confirm the role of TOR in the regulation of 4E-BP1 and p70s6k1 phosphorylation in Gcn2 -/- MEFs following amino acid deprivation, we added rapamycin to the treatment. Rapamycin did not affect eif2α phosphorylation (Figure 3.5 A, lanes 2 vs. 1 and 6 vs. 5). 42

54 Regardless of amino acid or GCN2 status, rapamycin decreased 4E-BP1 and p70s6k1 hyperphosphorylation to a similar level in all conditions (Figure 3.5 B and C, lanes 2-4 and 6-8). This strongly suggests that the phosphorylation of 4E-BP1 and p70s6k1 following histidine or leucine deprivation in Gcn2 -/- MEFs was the result of TOR regulated activities and not that of an inhibited phosphatase. Although, this does not rule out that the TOR regulated activities include inhibition of a phosphatase Amino acid deprivation inhibits translation initiation in Gcn2 +/+ and elongation in -/- MEFs. To specify the effect of amino acid deprivation on the stages of translation, we used sucrose density gradient to profile the distribution of mrna-ribosome association under each condition. As depicted in the top, center panel of Figure 3.6 A, mrnas in a sample were resolved along a 20-47% sucrose density gradient based on their density, derived from the number of associated ribosomes. The nonpolysomal peak contained protein, trna and free mrna. The 40, 60 and 80S peaks, parts of the subpolysomal region, contained mrnas associated with each or both of the respective ribosomal subunits. The broad polysomal peak contained mrnas bound to increasing number of holo-80s ribosomes. Either histidine or leucine deprivation increased the content of the 80S monosome as compared to serum-deprived controls in wild-type MEFs, suggesting an inhibition of translation initiation (Fig. 3.6 A). Conversely, in Gcn2 -/- cells, histidine or leucine deprivation led to a decrease in the 80S monosome peak as compared to the control (Fig. 3.6 B), suggestive of inhibition of translation elongation. Notably, re-addition of the 43

55 deprived amino acid returned the monosome content to a similar level as that of the condition s respective control (Fig. 3.6 A and B) Intracellular branched-chain amino acid (BCAA) concentrations do not explain the effects on 4E-BP1 and p70s6k1 in Gcn2 -/- MEFs following amino acid deprivation. Previous work showed that treatment with the translation elongation inhibitor cycloheximide increased phosphorylation of 4E-BP1 and p70s6k1 in wild-type Chinese hamster ovary (CHO) cells and L6 myoblasts deprived of amino acids (12, 178). Beugnet et al. (12) attributed the hyperphosphorylation of 4E-BP1 and p70s6k1 following cycloheximide treatment to increased cellular autophagy and intracellular BCAA. Similarly, maintenance and recovery of protein synthesis following amino acid deprivation have been shown to require proteolysis (49, 200). Together, these studies suggest that a maintenance of 4E-BP1 and p70s6k1 phosphorylation and protein synthesis is achieved by increased intracellular BCAA. Thus, the phosphorylation of TOR targets seen here in Gcn2 -/- MEFs following histidine or leucine deprivation can be attributed to a change in intracellular amino acid concentration. Contrary to these previous reports, however, intracellular BCAA levels and proteosome function did not correlate with the effects on 4E-BP1 and p70s6k1 in this study. Measurement of cellular BCAA concentration showed decreased cellular leucine in samples treated with leucine-deprived medium, but no increase following any of the treatment conditions (Figure 3.7). Treatment of MEFs with clasto-lactacystin β-lactone (CLBL), an inhibitor of the 20S proteosome, did not affect the phosphorylation pattern of eif2α (Figure 3.8 A, compare lane 2 vs. 1 and 6 vs. 5). This is in contrast to previous data (90), 4E-BP1 (Figure 3.8 B, lanes 2-4), or p70s6k1 (Figure 3.8 C, lanes 2-4) in 44

56 Gcn2 +/+ cells, either by itself or in combination with amino acid deprivation. Moreover, in Gcn2 -/- cells the hyper or maintained level of phosphorylation of the two latter proteins following histidine or leucine deprivation, respectively, was not affected by CLBL treatment (Figure 3.8 B, C; compare lane 7 and 8 vs. 6). (Preliminary data using another proteosome inhibitor, MG-132, suggested similar effects (Appendix C)). Together, these data suggested that the regulation of 4E-BP1 and p70s6k1 following amino acid-deprivation in Gcn2 -/- MEFs was not dependent on increased intracellular amino acid substrates or proteosome activity Inhibition of translation initiation restores the regulation of TOR targets in Gcn2 - /- to the same as that in +/+ MEFs. Holz et al. (74) recently proposed a model for the involvement of TOR and its targets during translation initiation complex assembly. Based on this model, eif3 serves as a scaffold for TOR to phosphorylate 4E-BP1 and p70s6k1 during translation initiation. In addition to this newly proposed role, eif3 also has been shown to interact with the 40S ribosome subunit and a majority of the remaining protein factors involved in initiation, including those in the eif4f complex and the met-trna i eif2 GTP ternary complex (9, 67, 93, 201). According to current models, upon recognition of the initiation codon, hydrolysis of the GTP in the ternary complex occurs, leading to release of the initiation factors from the 40S ribosome. The release of eif3 from the 40S subunit would enable it to reprise its binding with TOR and its targets, enabling continued phosphorylation of 4E-BP1 and p70s6k1. Thus, the release of eif3 is dependent on recognition of the initiating AUG, which in turns depends on formation of the ternary complex and eif2α. 45

57 To test whether or not inhibition of translation initiation influences the regulation of 4E-BP1 and p70s6k1, we pretreated MEFs with either thapsigargin or arsenite. Thapsigargin, an activator of ER stress and the eif2α kinase PERK, caused an increase in eif2α phosphorylation in both Gcn2 +/+ and -/- MEFs (Figure 3.9 A). Interestingly, thapsigargin also increased phosphorylation of 4E-BP1 and p70s6k1 in the Gcn2 +/+ MEFs (Figure 3.9 B and C, compare lane 2 vs. 1). In wild-type cells, combined thapsigargin and leucine-deprivation treatment effected dephosphorylation of 4E-BP1 and p70s6k1 as compared to treatment with thapsigargin and complete amino acids (Figure 3.9 B and C; compare lane 4 vs. 2). This is similar to that seen in treatment with amino acid-deprivation alone (Figure 3.4 D and F; compare lane 8 vs. 7). In knockout cells, PERK activation restored the dephosphorylation of 4E-BP1 and p70s6k1 seen in Gcn2 +/+ cells following leucine deprivation (Figure 3.9 B and C; compare lane 8 vs. 6. For p70s6k1, 8 vs. 6 two-tailed t-test p = ). This is contrary to that seen in treatment with amino acid-deprivation alone (Figure 3.4 D and F; compare lane 11 vs. 10). Additionally, PERK activation ablated the hyperphosphorylation of these proteins in the knockout cells following histidine deprivation (Figure 3.9 B and C; compare lane 7 vs. 6). Polysome profiles reflected the block in translation initiation caused by thapsigargin. Its addition to the amino acid deprivation treatment led to increased 80S monomer content in both the Gcn2 +/+ and -/- MEFs (Figure 3.10). The combination treatment of arsenite, an activator of the eif2α kinase HRI, and amino acid-deprivation treatment showed similar results for leucine but not histidine. Arsenite treatment caused increased eif2α phosphorylation in all conditions except for histidine deprivation in the knockout cells (Figure 3.11 A). Like thapsigargin, combined 46

58 arsenite and leucine deprivation treatment caused hypophosphorylation of 4E-BP1 and p70s6k1 in knockout cells, similar to the responses in wild type cells (Figure 3.11 B and C; compare lane 8 vs. 6. For 4E-BP1, 8 vs. 6 two-tailed t-test p = For p70s6k1, 8 vs. 6 two-tailed t-test p = ). Unlike thapsigargin, arsenite and histidine deprivation treatment did not affect the hyperphosphorylation of 4E-BP1 and p70s6k1 in the Gcn2 -/- MEFs (Figure 3.11 B and C; compare lane 7 vs. 6). This corresponded with the lower eif2α phosphorylation seen. Though unexpected, this result highlighted the importance of eif2α phosphorylation and thus ternary complex availability in the regulation of TOR targets Inhibition of translation elongation promotes hyperphosphorylation of TOR targets. In total, the data from the preceding section support a model in which dephosphorylation of TOR targets following leucine deprivation in Gcn2 +/+ MEFs depends on an inhibition of translation initiation. Furthermore, the lack of an initiation inhibition in Gcn2 -/- cells presumably allows for inhibition of elongation following amino acid deprivation, a response that leads to hyperphosphorylation of TOR targets. The corollary experiment to those using thapsigargin and arsenite would be treating the MEFs with a combination of an elongation inhibitor and amino acid deprivation. Previous results have shown that treatment with cycloheximide, an elongation inhibitor, alone increased 4E-BP1 and p70s6k1 phosphorylation in L6 myoblasts and CHO cells (12, 178). Combined cycloheximide and amino acid-deprivation treatment showed the expected effects on TOR targets and a surprising one on eif2α. Elongation inhibition 47

59 seemed to negate activation of GCN2 by uncharged trnas, as manifested by a lack in increased eif2α phosphorylation following histidine or leucine deprivation in Gcn2 +/+ cells (Figure 3.12 A; compare lanes 3 and 4 vs. 2). The lack in eif2α response correlated with an absence of dephosphorylation of TOR targets following leucine deprivation (Figure 3.12 B and C; compare lane 4 vs. 2). Treatment with cycloheximide actually increased 4E-BP1 and p70s6k1 phosphorylation regardless of GCN2 or amino acid status (Figure 3.12 B and C). Preliminary data using another elongation inhibitor, puromycin, showed similar results (Appendix D) Amino acid alcohols do not regulate GCN2 and TOR targets similarly as does amino acid deprivation. Amino acid alcohols, particularly histidinol, inhibit trna synthetases and have been known and used in place of amino acid deprivation to inhibit protein synthesis at the translation initiation stage (60, 204, 207). In one of the few papers to link detection of uncharged trna to regulation of a TOR substrate, Iiboshi et al. (78) presented evidence for repression of p70s6k1 activity by treating Jurkat cells with various L-amino acid alcohols, or maintaining golden hamster kidney cells, containing a temperature-sensitive trna synthetase mutant, at an inhibitory temperature. They proposed a role for an uncharged trna-sensing protein such as GCN2 in the regulation of TOR targets. Results from treatment of wild-type and knockout MEFs with amino acid alcohols from this study both corroborate and challenge this idea. Treatment of Gcn2 +/+ and -/- MEFs with either histidinol or leucinol affected TOR targets similarly, but differed on GCN2-dependency. Histidinol treatment caused a slight decrease in 4E-BP1 and p70s6k1 phosphorylation in Gcn2 +/+ (Figure 3.13 B and C; 48

60 compare lane 2 vs. 1) that was not present in -/- cells (Figure 3.13 B and C; compare lane 6 vs. 5). In contrast, the effect of leucinol on the two TOR targets were both more pronounced (Figure 3.14 B and C; compare lane 2 vs. 1) and GCN2-independent (Figure 3.14 B and C; compare lane 6 vs. 5). Adding to this perplexity are the results from the combined amino acid alcohol and amino acid deprivation treatment. The combined treatment of histidinol and histidine deprivation differed from treatment with deprivation alone in that marked hyperphosphorylation of 4E-BP1 and p70s6k1 occurred in both Gcn2 +/+ and -/- cells (Figure 3.13 B and C; compare lane 3 vs. 1 and 7 vs. 5). Combined histidinol and leucine deprivation displayed the same pattern seen with leucine deprivation alone. Dephosphorylation of 4E-BP1 and p70s6k1 was present in wild-type but not knockout cells (Figure 3.13 B and C; compare lane 4 vs. 1 and 8 vs. 5). On the other hand, leucinol seemed to have the dominant effect on suppression of phosphorylation of TOR targets under any condition (Figure 3.14 B and C; compare lanes 2-4 vs. 1 and 6, 7 vs. 5), except when in combination with leucine deprivation in knockout cells (Figure 3.14 B and C; compare lane 8 vs. 5). The one consistency seemed to be that in whatever treatment combination, in either genotype, the effects by leucine deprivation take precedence, i.e. the results from the combined treatment reflected the results from treatment with leucine deprivation alone Discussion Translation initiation in eukaryotes is a complexly but beautifully coordinated opus. The large number of proteins involved intimates a necessity for the formation of multi-protein complexes that are stable enough to foster efficiency by reducing 49

61 asynchronous assembly, but dynamic enough to enable regulation by allowing initiation factor interactions. Once again, experiments in the yeast model system have provided the first and most insights into these orchestral events. From these works, eif3, a protein known so far to contain 11 subunits, serves as the scaffold that supplies the stability and dynamism. In cells under normal conditions, eif3 interacts with the 40S ribosomal subunit, the cap-binding complex and the ternary complex (via eif2) (9, 67, 93, 201). Identification of the initiating AUG induces hydrolysis of GTP in the ternary complex and release of most of the initiation factors from the translating mrna and 40S ribosome (93). The recent model presented by Holtz et al. (74) incorporated regulation of TOR targets temporally and spatially closer to formation of the translation initiation complex, particularly the ternary complex and eif2. Their model posits the binding of TOR to eif3, allowing the former to phosphorylate 4E-BP1 and p70s6k1, also bound to eif3. The latter two proteins are released from the complex upon being phosphorylated. The data presented in this paper expand upon these models to include the effect of translation initiation state on regulation of TOR targets. In untreated wild-type cells, translation initiation and complex assembly occur as described above (Figure 3.15 A). Deprivation of essential amino acid induces eif2α phosphorylation by GCN2 and decreases ternary complex formation. Without the binding of the ternary complex and the hydrolysis of its GTP, the onset of dissociation of translation initiation factors does not occur. This impedes the recycling of eif3 to the start of the pathway where it would be available to recruit more 4E-BP1 and p70s6k1 for phosphorylation by TOR (Figure 3.15 B). Decreased phosphorylation of 4E-BP1 and p70s6k1 in liver samples from rodents fed a leucine-deficient diet, or perfused with a medium lacking leucine, or MEFs 50

62 treated with leucine-deficient medium correspond with this proposition. The maintenance of 4E-BP1 and p70s6k1 phosphorylation level in histidine-deprived cells, however, would indicate that this effect on signaling is either specific or more effective for leucine deprivation. If the signaling from amino acid deprivation and eif2α phosphorylation were disrupted, as in Gcn2 -/- cells, ternary complex formation and the subsequent GTP hydrolysis and initiation factor release would not be affected. Polysome profiling suggests an inhibition in elongation rather than initiation. Thus, in amino acid-deprived Gcn2 -/- cells, initiation complex assembly and disassembly, and interaction between TOR and its targets would occur as described above (Figure 3.15 C). The effect of amino acid deprivation, however, would be manifested as the ribosomes translate along the mrna, encounter uncharged trna and stall. Results from both meal feeding (6) and MEF experiments showed a maintenance of 4E-BP1 and p70s6k1 phosphorylation level following leucine deprivation as compared to complete amino acid treatment in the Gcn2 -/- MEFs. Interestingly, the effect of histidine deprivation on phosphorylation of 4E-BP1 and p70s6k1, although different in magnitude, was the same in direction when comparing results from Gcn2 +/+ and -/- MEFs. This hyperphosphorylation of 4E-BP1 and p70s6k1 also occurred following treatment with elongation inhibitors (12, 178), suggesting histidine deprivation activated a similar response mechanism in Gcn2 -/- MEFs. In addition to the basal level of translation initiation complex assembly and phosphorylation of 4E-BP1 and p70s6k1, the inhibition of elongation generates a signal to increase recruitment or recycling of TOR (Figure 3.15 C). Involvement of TOR is 51

63 evident in the lower phosphorylation state of its targets following rapamycin treatment. Previous reports have implicated that the TOR recruitment signal may be changes in intracellular BCAA concentration (12, 178). However, results from this study showed no change in intracellular BCAA concentration under amino acid-deprivation conditions. In addition, inhibition of the proteosome activity did not affect the phosphorylation changes seen with amino acid-deprivation treatments. Based on the presently proposed model, if inhibition of translation initiation via eif2α phosphorylation were accomplished through activation of any of the other three kinases, the effects of amino acid deprivation on 4E-BP1 and p70s6k1 in Gcn2 -/- should be similar to those in +/+ MEFs. In Gcn2 -/- MEFs, treatment with thapsigargin in addition to amino acid deprivation indeed recapitulated the phosphorylation effect on 4E-BP1 and p70s6k1 seen in the +/+ cells. Combined treatment with arsenite, a hypoxia-inducing chemical shown to activate HRI, also recapitulated the effect. The exception to this is the NaAsO 2 /histidine-deprivation combined treatment, wherein phosphorylation level decreased for eif2α and increased for 4E-BP1 and p70s6k1 as compared to their respective control. While the results are unexpected, the pattern fits and even strengthens the model, asserting that increased eif2α phosphorylation is necessary for regulation of the TOR target proteins following amino acid deprivation. Thus, we propose that translation initiation status and its attendant complex assembly events provide a platform for modulating TOR phosphorylation of 4E-BP1 and p70s6k1. This implicates an integrated stress response between two major pathways regulating mrna translation. 52

64 53

65 Ser 51 Total ** 2.0 * * ** ** ** 0.0 +AA -Gly -His -Leu -Met -Trp 0.0 +AA -Gly -His -Leu -Met -Trp 54 A. B. +AA -Gly -His -Leu -Met -Trp Figure 3.2. Regulation of eif2α and eif2b in C57Bl/6 mouse livers perfused with different amino acid conditions. C57Bl/6 mouse livers were perfused for 35 min with perfusate containing complete 10x amino acids (+AA) or perfusate containing 10x amino acids minus glycine (-Gly), histidine (-His), leucine (-Leu), methionine (-Met), or tryptophan (-Trp). A. Phosphorylation of eif2α. The upper panels show representative Western blots of phosphorylated and total eif2α. The lower panel shows quantitation of Western blots band density graphed as fraction of the ratio of phosphorylated over total eif2α in +AA samples. B. Guanine nucleotide exchange activity of eif2b graphed as fraction of the +AA samples. n = One-way ANOVA p < , Dunnett s post-test *p < 0.05, **p < 0.01 versus +AA. eif2α phosphorylation (fraction of +AA) eif2b activity (fraction of +AA)

66 A. B. +AA -Gly -His -Leu -Met -Trp +AA -Gly -His -Leu -Met -Trp Thr 389 γ β α δ γ β α p = ** +AA -Gly -His -Leu -Met -Trp AA -Gly -His -Leu -Met -Trp 55 Figure 3.3. Regulation of 4E-BP1 and p70s6k1 in C57Bl/6 mouse livers perfused with different amino acid conditions. C57Bl/6 mouse livers were perfused for 35 min with perfusate containing complete 10x amino acids (+AA) or perfusate containing 10x amino acids minus glycine (-Gly), histidine (-His), leucine (-Leu), methionine (-Met), or tryptophan (-Trp). A. Phosphorylation of 4E-BP1. The upper panel shows representative Western blots of 4E-BP1 total resolved into different phosphorylated isoforms, with γ being the most phosphorylated form. The lower panel shows quantitation of band densities graphed as the ratio of γ over total relative to +AA samples. One-way ANOVA p = Dunnett s post-test **p < 0.01 versus +AA. B. Phosphorylation of p70s6k1. The upper panels show representative Western blots of p70s6k1 phosphorylated at Thr 389 and p70s6k1 total resolved into different phosphorylated isoforms, with δ being the most phosphorylated form. The lower panel shows quantitation of band densities graphed as the ratio of γδ over total relative to +AA samples. ANOVA p = n = E-BP1γ phosphorylation (fraction of +AA) p70s6k1δγ phosphorylation (fraction of +AA)

67 Figure 3.4. Regulation of GCN2 and TOR targets following histidine or leucine deprivation in MEFs. Gcn2 +/+ and -/- MEFs treated for 2 h with serum-free DMEM (control) or serum-free DMEM lacking histidine (A, C, E) or leucine (B, D, F). Re-addition of amino acid (-/+) was for 30 min. A, B. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of eif2α Ser 51 over total relative to the control condition of the respective genotype. C, D. Phosphorylated 4E-BP1 at TOR regulated sites, Thr 36/45 (upper panels) and total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated (lower panels). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of 4E-BP1γ over total relative to the control condition of the respective genotype. E, F. Phosphorylated p70s6k1 at a TOR regulated site, Thr 389 (upper panel) and total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated (lower panel). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of p70s6k1δγ over total relative to the control condition of the respective genotype. Amino acid deprivation experiments were done three times in 2-3 replicates. Amino acid re-addition experiments were done twice in triplicates. Statistical significance was determined using t-test: *p < 0.05, ***p < versus wild type control; # p < 0.05, ## p < 0.01 versus knockout control. 56

68 Thr 36/45 Gcn2 +/+ -/- His + - -/ /+ Gcn2 +/+ -/- Leu + - -/ / *** Ser 51 Total Avg * Ser 51 Total SD γ β α Thr 36/45 γ β α ## 1.23 Avg *** SD # 1.25 Thr 389 δ γ β α Avg * Thr 389 δ γ β α SD Avg SD Avg SD Avg SD Figure 3.4. p70s6k1 hyperphosphorylation 4E-BP1 hyperphosphorylation eif2α phosphorylation A. p70s6k1 hyperphosphorylation 4E-BP1 hyperphosphorylation eif2α phosphorylation B. D. F. C. E.

69 Gcn2 +/+ -/- Rapamycin (20 ng/ml) His Leu Ser 51 Total γ β α δ γ β α 58 A. B. C. Figure 3.5. Regulation of GCN2 and TOR targets following combined treatment with rapamycin and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were pretreated for 15 min with 20 ng/ml rapamycin, then for 2 h with rapamycin-containing serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). B. 4E-BP1 total resolved into different phosphorylated forms, with γ being most phosphorylated. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. Experiments were done twice in triplicates.

70 Gcn2 -/- Gcn2 +/+ Serum-free media Nonpolysome 40S 60S 80S Serum-free media Abs 254 bound ribosomes -his -/+his -his -/+his -leu -/+leu -leu -/+leu 59 A. B % Figure 3.6. Polysome profiles of amino acid-deprived Gcn2 +/+ and -/- MEFs. Gcn2 +/+ and -/- MEFs were treated for 2 h with serum-free DMEM or serum-free DMEM lacking histidine or leucine. Re-addition of AA (-/+) was for 30 min. Supernatant of the cell homogenate was resolved on a 20-47% sucrose density gradient. Continuous monitoring of absorbance at 254 nm generated the polysome profiles. Notations of the various peaks along the profile are depicted in the top center panel in A. (See section for more details.) Black arrows denote the 80S peak in each profile. A. Gcn2 +/+ MEFs. B. Gcn2 -/- MEFs. Representative profiles from three independent experiments.

71 a a 1 1 b Gcn2 +/+ Gcn2 -/- 2 +AA -his -leu B. C Gcn2 +/+ Gcn2 -/ Gcn2 +/+ Gcn2 -/- 60 Figure 3.7. Intracellular branched-chain amino acids concentration following histidine or leucine deprivation in MEFs. Gcn2 +/+ or -/- MEFs were treated for 2 h with serum-free DMEM or serum-free DMEM lacking his or leu. The cells were washed with PBS to remove media, harvested in 3% perchloric acid, and supernatant neutralized with 1 M K 2 HPO 4. The final supernatant was submitted for intracellular amino acid analyses by HPLC. (See section for more details.) The concentration of each amino acid was normalized against the intracellular glycine concentration. Experiments were done three times in triplicates. Statistical significance was determined using one-way ANOVA and Tukey s post-test to compare among all three conditions within the same genotype. A. Intracellular leucine concentration. Different letters or numbers denote statistically significant differences. One-way ANOVA, p < 0.01 for both groups. B. Intracellular isoleucine concentration. One-way ANOVA, p > C. Intracellular valine concentration. One-way ANOVA, p > Intracellular Ile concentration (fraction of +AA) Intracellular Val concentration (fraction of +AA) Intracellular Leu concentration (fraction of +AA) A.

72 Gcn2 +/+ -/- CLBL (5 µm) His Leu A. eif2α phosphorylation Ser 51 Total B. 4E-BP1 hyperphosphorylation γ β α C. p70s6k1 hyperphosphorylation δ γ β α 61 Figure 3.8. Regulation of GCN2 and TOR targets following combined treatment with clasto-lactacystin β-lactone and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were treated for 2 h with a combination of 5 µm clasto-lactacystin β-lactone (CLBL) and serum-free DMEM or serum-free DMEM lacking histidine or leucine. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. Experiments were done two times in triplicates.

73 Figure 3.9. Regulation of GCN2 and TOR targets following combined treatment with thapsigargin and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were pretreated for 30 min with 1 µm thapsigargin, then for 2 h with thapsigargin-containing serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of eif2α Ser 51 over total relative to the no thapsigargin control of the respective genotype. B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of 4E-BP1γ over total relative to the no thapsigargin control of the respective genotype. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of p70s6k1δγ over total relative to the no thapsigargin control of the respective genotype. Experiments were done three times in 2-3 replicates. Statistical significance was determined using one-way ANOVA and Tukey s post-test, comparing among all four conditions within the same genotype cells. Different superscripts denote statistically significant differences, p <

74 Thapsigargin (1 µm) His Leu Ser 51 eif2α phosphorylation Total Avg 1.00 a 3.18 b 3.13 b 2.85 b SD B. 4E-BP1 hyperphosphorylation γ β α Avg 1.00 a 1.57 b,c 1.68 b 1.11 a,c , SD C. p70s6k1 hyperphosphorylation δ γ β α Avg 1.00 a 1.92 b 2.25 b 1.10 a SD Figure 3.9. Gcn2 +/+ -/- A.

75 A. B. Gcn2 +/+ Gcn2 -/- Serum-free media Serum-free media -his -his -leu -leu Figure Polysome profiles of combined thapsigargin-treated, amino acid-deprived Gcn2 +/+ and -/- MEFs. Gcn2 +/+ and -/- MEFs were pretreated for 30 min with 1 µm thapsigargin, then for 2 h with thapsigargin-containing serum-free DMEM or serum-free DMEM lacking his or leu. Supernatant of the cell homogenate was resolved on a 20-47% sucrose density gradient. Continuous monitoring of absorbance at 254 nm generated the polysome profiles. See Figure 3.6 A for notations of the various peaks along the profile. Black arrows denote the 80S peak in each profile. A. Gcn2 +/+ MEFs treated with thapsigargin and indicated media. B. Gcn2 -/- MEFs treated with thapsigargin and indicated media. Representative profiles from two independent experiments. 64

76 Figure Regulation of GCN2 and TOR targets following combined treatment with arsenite and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were pretreated for 30 min with 100 µm NaAsO 2 (arsenite), then for 2 h with NaAsO 2 -containing serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of eif2α Ser 51 over total relative to the no NaAsO 2 control of the respective genotype. B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of 4E-BP1γ over total relative to the no NaAsO 2 control of the respective genotype. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of p70s6k1δγ over total relative to the no NaAsO 2 control of the respective genotype. Experiments were done three times in 2-3 replicates. Statistical significance was determined using one-way ANOVA and Tukey s post-test, comparing among all four conditions within the same genotype cells. Different superscripts denote statistically significant differences, p <

77 Gcn2 +/+ -/- NaAsO2 (100 µm) His Leu A. eif2α phosphorylation Ser 51 Total Avg 1.00 a 2.17 b 2.63 b 2.76 b , SD B. 4E-BP1 hyperphosphorylation γ β α Avg 1.00 a 0.70 a 1.00 a 0.47 c , SD C. p70s6k1 hyperphosphorylation δ γ β α Avg 1.00 a 1.12 a,b 1.90 b 0.57 c , , SD Figure 3.11.

78 Figure Regulation of GCN2 and TOR targets following combined treatment with cycloheximide and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were treated for 2 h with a combination of 1 µm cycloheximide and serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of eif2α Ser 51 over total relative to the no cycloheximide control of the respective genotype. B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of 4E-BP1γ over total relative to the no cycloheximide control of the respective genotype. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. The numeric values at the bottom represent the average and standard deviation of quantitated band densities expressed as the ratio of p70s6k1δγ over total relative to the no cycloheximide control of the respective genotype. Experiments were done three times in duplicates. Statistical significance was determined using one-way ANOVA and Tukey s post-test, comparing among all four conditions within the same genotype cells. Different superscripts denote statistically significant differences, p <

79 His Leu A. eif2α phosphorylation Ser 51 Total Avg 1.00 a 1.08 a 1.20 a 1.09 a SD B. 4E-BP1 hyperphosphorylation γ β α Avg 1.00 a 1.68 b 1.74 b 1.61 b SD p70s6k1 hyperphosphorylation δ γ β α Avg 1.00 a 3.35 b 3.63 b 3.03 b SD Gcn2 +/+ -/- Cycloheximide (1 µm) C. Figure 3.12.

80 Leu Ser 51 eif2α phosphorylation Total B. 4E-BP1 hyperphosphorylation γ β α C. p70s6k1 hyperphosphorylation δ γ β α 69 Gcn2 +/+ -/- Histidinol (5 mm) His A. Figure Regulation of GCN2 and TOR targets following combined treatment with histidinol and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were treated for 2 h with a combination of 5 mm histidinol and serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. Experiments were done two times in triplicates.

81 Leucinol (5 mm) His Leu A. eif2α phosphorylation Ser 51 Total B. 4E-BP1 hyperphosphorylation γ β α C. p70s6k1 hyperphosphorylation δ γ β α 70 Gcn2 +/+ -/- Figure Regulation of GCN2 and TOR targets following combined treatment with leucinol and amino acid deprivation in MEFs. Gcn2 +/+ and -/- MEFs were treated for 2 h with a combination of 5 mm leucinol and serum-free DMEM or serum-free DMEM lacking his or leu. A. Phosphorylated eif2α at Ser 51 (upper panel) and total eif2α (lower panel). B. Total 4E-BP1 resolved into different phosphorylated forms, with γ being most phosphorylated. C. Total p70s6k1 resolved into different phosphorylated forms, with δ being most phosphorylated. Experiments were done two times in triplicates.

82 A. Figure A model for the regulation of TOR targets by translation initiation state. A. Current model of interactions among translation initiation factors. B. Proposed model of regulation of interaction among translation initiation factors during eif2-kinase-activating stresses. C. Proposed model of regulation of interaction among translation initiation factors in the absence of eif2 kinase. See section 3.4 for more detailed description. 1 eif1; 2 eif2; 3 eif3; 2B eif2b; 4E eif4e; 4G eif4g; BP1 4E-BP1. 71